ESTIMATING CELLULAR POPULATIONS

Abstract

An immunoassay for assisting in the diagnosis of sepsis or severe infection in a patient/subject, the assay comprising the steps of: (i) optionally contacting a test sample comprising neutrophils from the patient with an agent that permeabilises or solubilizes neutrophils; (ii) simultaneously with (i) or sequentially, contacting the sample with a binding agent that binds specifically to CD64 in the sample and forms a CD64-binding agent complex a; (iii) simultaneously with (i) and/or (ii) or sequentially, contacting the sample with a second binding agent that binds specifically to a neutrophil number marker (NNM) in the sample and forms a neutrophil marker-binding agent complex b; (iv) employ the amount of complex a and complex b to determine the relative level of CD46 and of NNM in the sample.

Claims

1. An immunoassay for evaluating test sample neutrophil activation, the assay comprising the steps of: (i) contacting the sample with an agent that permeabilises or solubilises neutrophils to release intracellular CD64 and allow for the detection of total, intracellular and extracellular, CD64 in the sample; (ii) simultaneously with (i) or sequentially, contacting the sample with a binding agent that binds specifically to CD64 in the sample and forms CD64-binding agent complex a; (iii) employ the amount of complex a to determine the level of total CD64 in the sample and directly or indirectly score the sample as comprising control or supranormal levels of neutrophil activation.

2. The immunoassay of claim 1, wherein step (ii) further comprises simultaneously with (i) or sequentially, contacting the sample with a neutrophil number marker (NNM) binding agent that binds specifically to a NNM in the sample and forms a NNM-binding agent complex b; and step (iii) comprises employing the amount of complex a and/or complex b to determine the relative level of total CD64 and/or NNM in the sample and directly or indirectly score the sample as comprising control or supranormal levels of neutrophil activation.

3. (canceled)

4. The immunoassay of claim 1, wherein the test sample is a whole blood cell sample; or wherein the test sample is from a neonatal subject.

5. (canceled)

6. The immunoassay of claim 35, wherein step (iii) includes comparing the level of total CD64 in the test sample with respective total CD64 levels predetermined from a control healthy sample.

7. The immunoassay of claim 2, wherein step (iii) includes determining a ratio of total CD64 and total NNM from the test sample.

8. The assay of claim 35, wherein step (iii) includes scoring sepsis or severe infection when total CD64 levels are above the predetermined mean plus 2 or more standard deviations of healthy control populations.

9. The assay of claim 36, wherein step (iii) includes: a) scoring sepsis or severe infection when NNM levels are above the predetermined mean NNM level plus 3 or more standard deviations of healthy control populations; b) scoring sepsis or severe infection when total CD64 relative to total NNM levels from the test sample are elevated compared to a predetermined multiple of the line of best fit for total CD64 versus total NNM of a selected control population; or c) scoring the test sample as comprising one or two or three of: (i) elevated total CD64 above the mean plus 2 or more standard deviations relative to a control population; (ii) elevated total NNM above the mean plus 3 or more standard deviations relative to a control population; and (iii) elevated total CD64 relative to total NNM.

10-13. (canceled)

14. The immunoassay of claim 2, comprising scoring the subject as having supranormal neutrophil activation if the relative level of total CD64 and of NNM in the sample exceeds a cutoff level predetermined as a function of the amount of the NNM in the test sample.

15. The immunoassay of claim 2, wherein the relative level of total CD64 and of NNM in the sample is internally corrected for the number of neutrophils in the sample determined using the level of NNM in the sample.

16. The immunoassay of claim 2, wherein the NNM is selected from: neutrophil elastase (NE), lactoferrin, myeloperoxidase and human neutrophil lipocalin.

17. The immunoassay of claim 1, wherein the assay is selected from a: point-of-care assay, enzyme-linked immunosorbent (ELISA)-type, flow cytometry, bead array, lateral flow, cartridge, microfluidic and immunochromatographic based method.

18. (canceled)

19. The immunoassay of claim 1, wherein the binding agent is an antibody or an antigen-binding fragment or derivative thereof, or an antigen-binding construct such as an affimer, aptamer or a ligand or binding part thereof.

20. The immunoassay of claim 1, wherein the binding agent is immobilised on a support.

21. The immunoassay of claim 2, wherein in step (ii), the sample is contacted with the binding agents of step (ii) and (iii) by applying the sample to a sample portion of an immunoassay device wherein the device sample portion is operably connected to spaced capture portions of the device and whereby the components of the sample flow from the device sample portion to and through the device capture portions, and wherein one capture portion comprises the binding agent which specifically binds to CD64 in the sample such that the CD64 is captured by the binding agent to form a binding agent-CD64 complex a in the capture portion, and wherein a second capture portion comprises the binding agent which specifically binds to NNM in the sample such that the NNM captured by the binding agent to form a binding agent-neutrophil number marker complex b in the capture portion.

22-25. (canceled)

26. The immunoassay of claim 35, wherein the sepsis is neonatal sepsis.

27. A kit or component of a diagnostic comprising (i) an immunoassay device comprising a microchannel or porous membrane operably connected to a sample portion comprising or in use comprising an agent that permeabilises or solubilises neutrophils to release intracellular CD64 and allow for the detection of total (intracellular and extracellular) CD64 in the sample, two or more capture portions, and one or more of the following; a conjugate (detection marker) portion, a sucker portion, a suitable control portion and a RBC cell lysis, dilution or solubilisation agent portion, and (ii) a binding agent that binds specifically to CD64 in the sample and forms a CD64-binding agent complex a.

28-34. (canceled)

35. The immunoassay of claim 1 for evaluating test sample neutrophil activation for assisting in the diagnosis or prognosis of sepsis or severe infection in a subject, wherein step (iii) of claim 1 is used to directly or indirectly score the sample or the subject as not indicated for sepsis or severe infection, or as indicated for sepsis or severe infection.

36. The immunoassay of claim 2 for evaluating test sample neutrophil activation for assisting in the diagnosis or prognosis of sepsis or severe infection in a subject, wherein step (iii) of claim 2 is used to directly or indirectly score the sample and thus the subject as not indicated for sepsis or severe infection or as indicated for sepsis or severe infection.

37. An immunoassay for evaluating test sample neutrophil activation suitable for assisting in the diagnosis of sepsis or severe infection, the assay comprising the steps of: (i) contacting the sample with an agent that permeabilises or solubilises neutrophils; (ii) simultaneously with (i) or sequentially, contacting the sample with a binding agent that binds specifically to CD64 in the sample and forms CD64-binding agent complex a; (iii) simultaneously with (i) and/or (ii) or sequentially, contacting the sample with a second binding agent that binds specifically to a neutrophil number marker (NNM) in the sample and forms neutrophil marker-binding agent complex b; (iv) employ the amount of complex a and complex b to determine the relative level of CD46 and of NNM in the sample; and (v) directly or indirectly score the sample as comprising control (eg. healthy) or supranormal levels of neutrophil CD64/activation.

38. The kit or diagnostic of claim 27, further comprising a neutrophil number marker (NNM) binding agent that binds specifically to a neutrophil number marker (NNM) in the sample and forms a NNM-binding agent complex b and wherein the binding agents are either immobilised to separate capture portions and/or contained within conjugate portions.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0123] FIG. 1 illustrates the large number of candidate Sepsis Biomarkers evaluated in the literature.

[0124] FIG. 2 illustrates that Visitect CD4 Test developed by some of the inventors, at Burnet institute (produced under license by Omega Diagnostics, UK). In the CD4 T-cell test, monocytes and macrophages are depleted from the whole blood sample (30 μl) by interaction with RosetteSep reagent (StemCell Technologies, Vancouver, Canada), causing their agglutination with red blood cells in the sample pad (well “A”). The remaining blood fraction containing plasma and white blood cells apart from monocytes/macrophages flows towards the test zone and interacts with detergent (Triton X-100), lysing the white blood cells and solubilising cell-associated full-length CD4 on T-cells in the sample. The sample then flows past the test line, being a binding reagent (monoclonal antibody) directed against the cytoplasmic domain of CD4, found only in cell-associated CD4 and not in the fraction of soluble CD4 in plasma. The captured CD4 is then detected with colloidal gold complexed with a second monoclonal antibody, directed against the extracellular domain of CD4, initiated by addition of buffer to the gold conjugate pad (well “B”). The intensity of the test line is therefore representative of the total amount of T-cell associated CD4 in the sample, and by inference the number of CD4 T-cells because the amount of CD4 molecules per cell is substantially constant. That is, the amount of CD4 is directly proportional to the number of CD4 T-cells. The test signal is then compared visually or by instrument reader versus one or more reference lines representing clinically relevant levels of CD4 T-cells, in the figure the example of 350 T-cells per μl which is recommended by WHO for prioritisation of antiretroviral therapy in HIV infection,

[0125] FIG. 3 illustrates the principles of the CD4 T-cell test and SepsiTest nCD64i test. In the condition of HIV infection progressing towards acquired immunodeficiency syndrome (AIDS), the number of CD4 T-cells may decline, while the amount of CD4 on each remaining cell remains constant. The amount of CD4 is therefore proportional to the number of CD4 T-cells present in the same. In the condition of invasive infection or sepsis, the number of neutrophils may increase, decrease or stay the same, but the level of expression of CD64 (shown as blue molecules on the surface of the cells) is substantially increased. The number of neutrophils can be measured in the same way as the number of T-cells using an antigen that is associated with the neutrophils, such as but not limited to neutrophil elastase. The amount of CD64 can be measured in the same way as CD4, and either cell-associated or both cell-associated and soluble CD64 may be measured using methods well known in the art including methods for exclusively cell-associated transmembrane proteins (U.S. Pat. No. 8,409,818 and other territories). However in the case of CD64 the amount per cell will be either low in healthy individuals, or elevated in the case of severe infection or sepsis, and this difference will be detected as an increase in the ratio between CD64 and the neutrophil-specific protein (neutrophil elastase in this example).

[0126] FIG. 4 is a graphical representation of neutrophil elastase, CD64 and neutrophil counts among healthy individuals. 4A shows the good correlation (R2=0 89609) between ELISA Neutrophil Elastase (Neutrophil specific marker) and Neutrophil (Granulocyte) count 4B shows good correlation (R2=0.86146) between CD64/NE ratio (nCD64 index) and Granulocyte count, but a surprising higher level of CD64 index at lower neutrophil counts. 4C shows good correlation (R2=0.9151) for CD64 versus granulocyte count in Whole Blood Lysate, with the unexpected observation that CD64 amounts are higher in samples with lower neutrophil (granulocyte) count, leading to the observed variable relationship seen in FIG. 4B.

[0127] FIG. 5 is a graphical representation showing estimation of a potential diagnostic cutoff for nCD64 index using CD64 and NE values. The total amount of NE and CD64 was measured in monocyte-depleted whole blood from healthy adult volunteers, and the nCD64 index ratio calculated. The results show a highly significant correlation (P=0.0166) and again show the variable relationship with higher nCD64i at lower neutrophil counts. The dashed red lines show the lower and upper 95% confidence intervals for the relationship, and it is predicted that samples with nCD64i falling above the upper 95% confidence interval for the measured NE concentration would have a strong suspicion of sepsis.

[0128] FIG. 4 illustrates a format for lateral flow immunochromatographic POC test for sepsis measuring the neutrophil CD64 index. Visitect CD4 test (A), expanded view and schematic of proposed nCD64 index test (B, red boxes), and Axxin AX-2 reader (C). For visual interpretation an intensity for the CD64 line greater than the NE line might be considered to be diagnostic, however with the use of a reader such as the Axxin AX-2 reader (FIG. 6C) the cutoff would be adjusted according to the total amount of neutrophils (NE quantity), and the appropriate cutoff calculated for the instant patient sample.

[0129] FIG. 7 shows results of ELISA for NE and CD64 in whole blood for healthy subjects. NE and CD64 were assessed via ELISA in whole blood for healthy subjects (n=30), and shows that in healthy subjects, the total amount of CD64 in whole blood is closely correlated with the total amount of NE in whole blood (R2=0.74).

[0130] FIG. 8 shows the results of ELISA for NE and CD64 in whole blood for sepsis patients. NE and CD64 were assessed via ELISA in whole blood for sepsis patients (red markers, n=11) together with the healthy subjects as shown in FIG. 7 (blue markers).

[0131] FIG. 9 shows the same results as FIGS. 7 and 8 but highlights the four different classifications of sepsis patient samples according to the cutoffs based on the mean plus 2 SD of CD64 and mean plus 3 SD of NE. By these criteria, one patient is positive only for NE, 5 patients are positive for both NE and CD64, 4 patients are positive for CD64 only, while one patient is negative for both NE and CD64 with very low values for both markers, and suggesting that the patient may have low levels of neutrophils.

[0132] FIG. 10 shows the analysis of CD64 versus NE for low values of NE. The X axis shows total CD64 as ng/mL and the X axis shows the total NE as μg/mL.

[0133] FIG. 11 shows the analysis of CD64 versus NE for low values of NE. The X axis shows total CD64 as ng/mL and the X axis shows the total NE as μg/mL.

[0134] FIGS. 12A-C show the gate for healthy levels of CD64 versus NE.

[0135] FIG. 13 illustrates the results of a comparison of Leuko64 and the subject assay (in ELASA format) to identify sepsis patients, Leuko64 showed highly significantly higher values for sepsis patients than for healthy controls, but using the manufacturer recommended cutoff of 1.2, only 8/11 sepsis patients were identified with this test, consistent with many reports in the literature that show around 70-80% sensitivity of this method. Similarly, detection of total whole blood CD64 alone by ELISA showed highly significantly higher values for sepsis patients than for healthy controls, but using the cutoff of mean plus 2 SD (209 ng/ml), only 9/11 sepsis patients were identified with this test.

[0136] FIG. 14 shows the results of this analysis (FIG. 13) for patient SEP009, the patient with the lowest levels of NE in FIG. 13, in panel A, it is shown that the neutrophils from this patient have high levels of surface expression of CD64 (red line) consistent with very high relative CD64 expression in Leuko64 (panel B), but even higher levels of CD64 per cell when intracellular CD64 is detected as well (blue line). However the low level of NE (panel D) and presumed low neutrophil counts for this patient result in a total CD64 result that is below the cutoff based on CD64 alone (panel C), but detectable using the algorithm approach illustrated in FIGS. 8-12 and described herein. This result also suggests that for patients with low levels of NE, the Leuko64 approach or other methods may be of additional utility given the very high Leuko64 result for this patient.

[0137] FIG. 15 shows the same analysis for patient SEP010, the patient with elevated levels of CD64 but just below the cutoff in FIG. 9 derived from the mean plus 2 SD of CD64 from healthy subjects. In this patient, there was minimal surface staining of CD64 (panel A, comparing red line and isotype control for surface staining) consistent with low relative CD64 expression in Leuko64 (panel B), but there were high levels of CD64 detected when intracellular CD64 was stained as well (panel A blue line). Consistent with this observation, patient SEP010 had modest levels of total CD64 by ELISA, although still just below the cutoff for total CD64 based on mean plus 2SD (panel C), whereas the Leuko64 result of 0.7 was well below the manufacturer cutoff of 1.2 for that test (panel B). However, when NE is examined the patient SEP010 is seen to have very highly elevated levels of NE, providing unequivocal diagnosis of sepsis using the cutoff derived from the mean plus 3SD of NE, and also using the cutoff derived from the trendline equation.

[0138] FIG. 16 shows the same analysis for patient SEP011, a patient with elevated levels of both CD64 and NE. In this patient, there was significant surface staining of both intracellular and surface CD64 (panel A, comparing red line and isotype control for surface staining, blue line and isotype control for total staining) consistent with high relative CD64 expression in Leuko64 (panel B), well above the manufacturer cutoff. Patient SEP011 had highly elevated levels of total CD64 by ELISA (panel C), and also highly elevated levels of NE (panel D), providing unequivocal diagnosis of sepsis using the cutoff derived from the mean plus 3SD of NE, AND the cutoff derived from the mean plus 2SD of CD64. AND also using the cutoff derived from the trendline equation.

[0139] FIG. 17 is an illustration, modified from van der Poel (2011), showing how in healthy individuals, low levels of CD64 (FcγR1) are expressed on the cell surface, and these are saturated by monomeric IgG which does not result in cell signaling or antigen complex internalization (FIG. 17A). Upon initial stimulation of the neutrophil by interferon gamma or other stimuli associated with bacterial infection or sepsis, additional CD64 is synthesized and translocated to the surface of the neutrophil and associated with membrane microdomains, together allowing for binding of multimeric Ig in immune complexes which leads to cell signaling (FIG. 17B).

[0140] FIG. 18 illustrates activated neutrophils having different levels of surface CD64, even though they have the same elevated level of total CD64. On the left, a “healthy” neutrophil is shown schematically with low levels of CD64, expressed on the surface. These cells may also contain small amounts of pre-forme d, intracellular CD64. In sepsis, “activated” neutrophils are shown on the right, each with equally large amounts of CD64, significantly increased compared to healthy neutrophils, but with distribution on the surface (similar to the patient sample SEP09, FIG. 14), or evenly distributed between the surface and intracellular (similar to the patient sample SEP011, FIG. 16), or predominantly distributed to the intracellular compartment (similar to patient sample SEP010, FIG. 15). As such, any method that allows the detection of both cell surface and intracellular CD64 is likely to allow improved diagnosis of sepsis.

DISCUSSION OF EMBODIMENTS

[0141] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present disclosure. Practitioners are particularly directed to Wild D. “The Immunoassay Handbook” Nature Publishing Group, 4.sup.th Edition, 2013 and Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press. Inc., Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986, for definitions and terms of the art and other methods known to the person skilled in the art. Immunoassays can be done in any convenient format known in the art.

[0142] Antibodies are often used in immunoassays because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production is derived by fusing an immortal cell line and lymphocytes sensitized against the antigen of interest or can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; European Journal of immunology 6: 511-519, 1976 or more recent references Sambrook, Molecular Cloning: A. Laboratory Manual, 3rd Edition, CSHLP, CSH, NY, 2001). The DNA that encodes antibodies can be manipulated in vitro and introduced back into lymphoid cell lines, thus allowing the production of genetically-engineered antibodies. In the last decade, the use of transient mammalian expression systems for the production of native complex proteins has increased (see S. Geisse, B. Voedisch Methods Mol. Biol., 899 (2012), pp. 203-219) boosted by the publication of efficient transfection protocols and the availability of suspension cell lines growing at high density. For transient expression, mainly the HEK-293 and CHO-K1 cell lines have been applied. Both cell lines can be adapted to suspension culture, and sub clones are available which grow in chemically defined medium at high cell densities. With its ease of transfection, high expression yield and native human glycosylation, the human embryonic kidney 293 cell line may be used.

[0143] Antibodies (and their smaller formats such as scFv and Fab fragments can be produced in any cell type known in the art. Cell-free protein synthesis, also termed in vitro translation, facilitates the production of a given target protein by utilizing the translational machinery without using the living cell. Cell-free systems have been successfully used for the high-throughput production of protein libraries as well as for the high-yield synthesis of selected target proteins. In particular, the use of linear DNA templates contributes to the ease and speed of cell-free translation systems, since no time-consuming cloning steps are required prior to protein production. Antibodies of a given target protein takes approximately one to two days, whereas cell-based expression, including the cloning procedure and cell-transformation, may take up to two weeks. Phage display technology is the most commonly used technique for the in vitro selection and evolution of antibody fragments.

[0144] Alternatives to antibodies as specific binding agent are described in the literature and are recognised in the field for their potential to improve inter alia, assay reproducibility and stability. A review of antibody alternatives is provided by McLeod et al. The Scientist February 2016 incorporated herein, and includes aptamers and affimers. Any such binding agent may be employed in the present assays and kits without undue experimentation.

[0145] The presence of complexes may be evaluated using ELISA-type procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site or “sandwich” assays of the non-competitive types, as well as in the traditional competitive binding assays. The skilled person will appreciate that the selection and implementation of a known label system involves only routine experimentation.

[0146] Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an binding agent is immobilized on a solid or semi-solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an binding agent-antigen complex, a second binding agent specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of binding agent-antigen-labelled binding agent. Any unreacted material is washed away, and the presence of the marker is determined by observation of a signal produced by the detectable marker (reporter molecule). The results may be qualitative or quantitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of marker. Variations on the forward assay include a simultaneous assay in which both sample and labelled binding agent are added simultaneously to the bound binding agent. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is generally a biological sample comprising biological fluid, and is most conveniently a whole blood sample such as capillary or venous blood that may be treated with an anticoagulant.

[0147] In a typical forward sandwich assay, a first binding agent having specificity for a marker is either covalently or passively bound to a solid or semi-solid support. The support is typically glass or a polymer, the most commonly used polymers being nitrocellulose, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene or mixture or derivatives of these. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the polymer-binding agent complex to the solid surface which is then washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (eg. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37° C. including 25° C.) to allow binding of any subunit present in the binding agent. Following the incubation period, the binding agent subunit solid phase is washed and incubated with a second binding agent specific for a portion of the antigen. The second binding agent is linked to a detectable marker which is used to indicate the binding of the second binding agent to the antigen.

[0148] An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific binding agent which may or may not be labelled with a detectable marker. Depending on the amount of target and the strength of the signal from the detectable marker, a bound target may be detectable by direct labelling with the binding agent. Alternatively, a second labelled binding agent, specific to the first binding agent is exposed to the target-first binding agent complex to form a target-first binding agent-second binding agent tertiary complex. The complex is detected by the signal emitted by the reporter molecule. A significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide or electrophoretic tag, so as to facilitate identification of the amino acid sequence of each library member. These improved bead-based methods are described in International Publication No. WO 93/06121.

[0149] In other embodiments, the method is a liquid phase method. In one example of a liquid phase immunoassay (see for example U.S. Pat. No. 6,632,603) the sample is contacted with an agent capable of binding a marker and a detector agent comprising a visually detectable agent such as colloidal gold or silver labelled. The test sample is applied by flowing onto a defined zone of an insoluble porous support film having a pore size impassable to a complex formed between the marker and its target, and if present, with a binding substance and a detector substance, but passable to the binding substance and detector substance while remaining uncomplexed in the absence of the desired target. If the target is present in the test specimen, the detector substance binds with the target and the binding substance to form a visually inspectable complex on the surface of the porous support film. After application of the test sample to the porous support, the surface of the porous support is visually inspected for colour to determine the presence and quantity or the absence of the marker being assayed.

[0150] In another assay, magnetic antibodies that bind to markers are used to tag markers and a high Tc superconducting quantum interference device is used to measure the amount of target protein. A liposome immunomigration, liquid-phase competition strip immunoassay is, for example, described in Glorio-Paulet et al J Agric Food Chem 48 (5):1678-1682, 2000.

[0151] General formats and protocols for the conduct of various formals of ELISA are disclosed in the art and are known to those of skill in the field of diagnostics. For example, reference may be made to Chapter 11 of Ausubel (Ed) Current Protocols in Molecular Biology, 5th Edition, John Wiley & Sons, Inc, N Y, 2002. Rundström, G et al. describe lateral Flow immunoassay using Europium (III) Chelate Microparticles and Time-Resolved Fluorescence for eosinophils and neutrophils in Whole Blood. Clinical Chemistry 53, 342-348 (2007), incorporated herein.

[0152] Various methods are available for depleting monocytes or red blood cells from blood, if required. In some embodiments, monocytes in the sample are depleted by contacting the sample with anti-CD14 or other relevant antibodies bound to a solid or semi-solid support. In another embodiment, red blood cells are depleted in the sample by contacting the sample with anti-glycophorin A antibodies bound to a solid or semi-solid support.

[0153] In some embodiments, a chromatographic device is provided comprising material which has a pore size which allows or facilitates capillary flow of the components of the method. In some embodiments, the device comprises portions comprising material of different pore size, or non-porous material, the material being contiguous with the first material and designed to receive a sample or receive or store components of the method. In some embodiments, the portions of the chromatographic device are separate, contiguous or overlapping or designed to come together in use.

[0154] In some embodiments, the sample pad is chromatographically connected to a test portion of the device, the test portion comprising a binding agent such as an antibody or an antibody binding fragment thereof, an antigen-binding constructs such as an affimers or ligands or parts thereof. In an illustrative embodiment, the subject is a mammal and the test portion comprises an antibody or affimer which, under the appropriate conditions, recognizes and binds CD64. In an illustrative embodiment, the subject is a mammal and a second test portion comprises an antibody or affimer which, under the appropriate conditions, recognizes and binds a neutrophil specific marker, such as NE.

[0155] In some embodiments, the sample pad is chromatographically connected to a test portion of the device, the test portion comprising an antibody or an antibody binding fragment thereof, affimers, aptamers or ligands or parts thereof. In an illustrative embodiment, the subject is a mammal and the test portion comprises an antibody which, under the appropriate conditions, recognizes and binds a neutrophil specific marker.

[0156] In some embodiments, when chromatographically active portions of the sample to be tested move from the sample pad towards and through the test portion, NE and CD64 are captured onto test or control portions of the device and the remainder of the sample flowing from the sample pad is uncaptured. In some embodiments, the uncaptured components of the test sample are collected chromatographically into an absorbent pad, which is positioned in any orientation with respect to the test portion.

[0157] Components of the subject sample, such as red blood cells or particular white blood cells may be retained in the sample pad, for example, by selecting a pad of suitable mesh or pore size and/or by the inclusion of specific reagents such as antibodies or lectins to bind and retain these components. For example monocytes may be retained using anti-CD14 antibodies. Anti-glycophorin antibodies may be used to retain/remove red blood cells.

[0158] In some embodiments, once the test portion of the immunochromatographic device has been exposed to CD64 and neutrophil specific marker in the subject sample, the method proceeds by allowing contact between the test portions and a detection binding agent. In some embodiments, the detection marker/s stored in a separate portion of the kit.

[0159] In some embodiments, the detection marker comprises a visually detectable reporter molecule and a positive result may be essentially immediately observed in the test and/or control portions of the immunochromatographic device.

[0160] Permeabilising agents and solutions may also cause leukocyte cell lysis, fixation or solubilisation and the term is used broadly herein to encompass these events, if preferred. Permeabilization is performed to facilitate access to cell markers within the cell (internal).

[0161] Permeabilization of the cells can be performed by any suitable method known in the art. These methods include, but are not limited to, exposure to a detergent (such as CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-PD-maltoside, lauryl sulfate, glycodeoxycholic acid, n-lauroylsarcosine, saponin, and Triton X-100). Other permeabilising methods comprise the use of certain peptides or toxins that render membranes permeable. Permeabilization may also be performed by addition of an organic alcohol to the cells. Selection of an appropriate permeabilising agent and buffers if required can easily be performed by one of ordinary skill in the art. Permeabilization can occur concurrently with a fixation step. Fixing solutions include, for example, Cytofix/Cytoperm (BD Biosciences). Commonly used cell fixatives include, but not limited to formaldehyde, paraformaldehyde, glutaraldehyde, acetic acid, picric acid, methanol, ethanol, and acetone. Illustrative fixation buffer suitable for whole blood samples is Phosilow Lyse/Fix Buffer (BD Biosciences). Fixatives have been used for detection of both surface and intracellular antigens. Some fixatives include alcohol and formaldehyde/paraformaldehyde. Illustrative fixatives are described in U.S. Pat. Nos. 5,422,277 and 5,597,688.

[0162] In other embodiments, the detection marker may be detected using further detection protocols and devices such as will be well known to those of ordinary skill in the art. For example, colloidal metal or metal oxide particles or colloidal non-metal particles or dyes or colored latex are conveniently used.

[0163] In early experiments described in example t a diagnostic gate or cutoff was determined by assessing the ratio of CD64 to NNM.

[0164] In one embodiment the assay comprises: [0165] (i) optionally contacting the sample with an agent that lyses or solubilises neutrophils; [0166] (ii) contacting the sample with a binding agent that binds specifically to CD64 in the sample and forms a CD64-binding agent complex, and a second binding agent that binds specifically to a neutrophil marker in the sample and forms a neutrophil marker-binding agent complex, and [0167] (iii) measuring and determining the relative amount of each complex from step (ii) to obtain a modified CD64 index (ratio of CD64 to NNM) indicating or representing the average amount of CD64 per neutrophil in the sample.

[0168] In another early embodiment, the CD64 index from step (iii) is corrected for the number of neutrophils in the sample determined by the amount of the neutrophil marker-binding agent complex measured in step (iii).

[0169] In one embodiment, the assay further comprises diagnosing the subject including a neonatal subject as having sepsis including neonatal sepsis or having a risk of developing sepsis including neonatal sepsis if the CD64 index exceeds a cutoff level above which supranormal amounts of neutrophil CD64 are indicated in the sample. Cutoff levels can be modified depending upon, inter alia, the age of the subject.

[0170] In one embodiment, the CD64 index from step (iii) is compared with or plotted against the number of neutrophils in the sample determined by the amount of the neutrophil marker-binding agent complex measured in step (iii) and the cutoff nCD64 index is corrected for the number of neutrophils in the sample. As shown in FIG. 4C there is a strong correlation between total CD64 and neutrophil counts in healthy donors. However, in samples with low neutrophil counts the total amount of CD46 expressed is in fact higher. This means, surprisingly, that the cutoff nCD4 ratio indicating sepsis or risk of sepsis should not, for increased accuracy, be a constant value, but instead should be corrected on a case by case basis for the total number of neutrophils in the sample (eg., neutrophils expressed as cells/volume). As the value of the index may be a function of the neutrophil count greater accuracy is obtained by making an adjustment of the cutoff, which was not possible when using flow cytometry alone as without a separate reference method, there is no way of getting an absolute neutrophil count by Flow cytometry. In one embodiment, a simple algorithm is embedded or available via an instrument reader to set the cutoff value for any given value of neutrophil count. For example, in one embodiment, the cutoff value is above the 95% confidence interval of the best line of fit for the CD64/NE ratio, as shown in FIG. 38. This is illustrated in FIG. 5.

[0171] In one embodiment, the binding agent is immobilised on a support. Suitable supports are well known in the an, as described herein.

[0172] In some embodiments, the assay is performed wherein, in step (ii), the sample is contacted with the binding agents of step (ii) by applying the sample to a sample portion of an immunochromatographic or microfluidic device wherein the device sample portion is operably connected to spaced capture portions of the device and whereby the components of the sample flow from the device sample portion to and through the device capture portions, and wherein one capture portion comprises the binding agent which specifically binds to CD64 in the sample such that the CD64 is captured by the binding agent to form a binding agent-CD64 complex in the capture portion, and wherein a second capture portion comprises the binding agent which specifically binds to neutrophil markers in the sample such that the neutrophil marker or the binding part thereof is captured by the binding agent to form a binding agent-neutrophil marker complex in the capture portion.

[0173] In one embodiment, the amount of CD64 complex and the neutrophil-marker binding agent complex is detected using binding agents such as antibody or antigen-binding fragments that bind to CD64 or the neutrophil marker respectively and directly or indirectly provide a detectable signal that can be quantified visually or photometrically including fluorometrically (by instrument reader).

[0174] In relation to this embodiment, the binding agents may be conjugated to a detectable marker or microparticles comprising a detectable marker, that provide a detectable signal. Illustrative particles and methods are described in Rundström, G et al. Lateral Flow immunoassay Using Europium (III) Chelate Microparticles and Time-Resolved Fluorescence for Eosinophils and Neutrophils in Whole Blood. Clinical Chemistry 53, 342-348 (2007).

[0175] In one embodiment, the capture portion is a test line.

[0176] In one embodiment, the ratio of the visual or photometrically/instrument calculated signal from the CD64 test line and the visual or photometrically/instrument calculated signal from the neutrophil marker test line provides the modified neutrophil CD64 index indicating the amount of CD64 per neutrophil in the sample.

[0177] Immunochromatographic test kits are expressly contemplated and another aspect the present specification provides a kit comprising (i) a chromatographic device comprising a porous membrane operably connected to a sample portion, two or more capture (test) portions, and optionally one or more of the following, a conjugate (detection marker) portion, a sucker portion, a suitable control portion and a cell lysis or solubilisation portion, and (ii) a binding agent that binds specifically to CD64 in the sample and forms a CD64-binding agent complex, and a second binding agent that binds specifically to neutrophil markers (NNM) in the sample and forms a neutrophil marker-binding agent complex wherein the binding agents are either immobilised to separate capture portions and/or contained within conjugate portions and (iii) optionally instructions for using the device to determine a modified neutrophil CD64 index and/or assessment of sepsis.

[0178] In one embodiment, the kit is suitable for reverse or lateral flow immunochromatographic formats.

[0179] In one embodiment, the binding agent is an antibody or antigen-binding fragment or a derivative thereof, ligand or receptor binding part thereof, or an antigen-binding construct such as an affimer.

[0180] Illustrative binding agents include antigen-binding construct comprising a protein scaffold which is linked to one or more epitope-binding domains wherein the antigen-binding construct has at least two antigen binding sites at least one of which is from an epitope binding domain and at least one of which is from a paired VH/VL domain.

[0181] In one embodiment the neutrophil specific marker is selected from the group comprising neutrophil elastase, lactoferrin, myeloperoxidase and human neutrophil lipocalin, or equivalent cell marker whose amount on neutrophils effectively correlates with neutrophil cell number.

[0182] In one embodiment the neutrophil specific marker is selected from the group comprising neutrophil elastase, lactoferrin, and human neutrophil lipocalin, or equivalent cell marker whose amount on neutrophils effectively correlates with neutrophil cell number.

[0183] The level of linear or reproducible non-linear correlation needed for the marker to serve as an accurate indication of neutrophil numbers will vary depending upon the marker and the assay employed. Generally a correlation of not less than about 70% will be effective. Levels of 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% 87%, 88%, 89%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% are contemplated.

[0184] As illustrated in Example 2 the specification teaches that NNMs can provide a useful correction for the number of neutrophils, without a strong absolute correlation with neutrophil number. This is because the utility of the NNM is not necessarily dependent on being well correlated with neutrophil numbers, but rather being correlated with neutrophil CD64 levels and associated with neutrophil number.

[0185] Accordingly, in some embodiments, a NNM biomarker may have a positive correlation with neutrophil numbers of less than about 70%, but a correlation with neutrophil-specific CD64 of not less than about 70%, and this will be effective.

[0186] By “about” is meant a measurement, quantity, level, activity, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference measurement, quantity, level, activity, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0187] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0188] In one embodiment, the present specification enables an immunoassay suitable for point of care use performed on a whole blood sample obtained from a subject, or the whole blood sample substantially depleted of one or more of red blood cells, monocytes and macrophages, for determining a modified neutrophil CD4 index.

[0189] As used herein the phrase “substantially depleted” means at least 70%, 71%, 72%, 73%, 74%, 75% 76%, 77%, 7%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86% 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% free of the cell, such as monocytes and macrophages.

[0190] In some embodiments, the subject is a human and includes neonatal humans. The present invention extends, however, to primates, livestock animals, laboratory test animals, companion animals and avian species as well as non-mammalian animals such as reptiles. The assay has applications, therefore in human, livestock, veterinary and wild-life therapy and diagnosis.

[0191] Whole blood refers to an essentially unprocessed sample of blood from a subject, generally anticoagulant has been added when the blood is obtained by venous collection, or it may or may not be omitted if the blood is obtained by fingerprick or fingerstick collection and used without delay at point of care.

[0192] Numbered clauses describing embodiments of the assay and kits described inter alia in example 1 and FIGS. 1 to 6 as also provided as follows: [0193] 1. An immunoassay suitable for point of care use performed on whole blood sample obtained from a subject, or the whole blood sample substantially depleted of one or more of red blood cells, monocytes and macrophages, for determining a modified neutrophil CD64 index, the assay comprising. [0194] (ii) optionally contacting the sample with an agent that lyses or solubilises neutrophils; [0195] (iii) contacting the sample with a binding agent that binds specifically to CD64 in the sample and forms a CD64-binding agent complex, and a second binding agent that binds specifically to neutrophil markers in the sample and forms a neutrophil marker-binding agent complex; and [0196] (iv) measure and determine the relative amount of each complex front step (ii) to obtain a modified CD64 index (ratio) indicating the average amount of CD64 per neutrophil in the sample [0197] 2. The assay of clause 1 further comprising diagnosing the subject as having supranormal amounts of neutrophil CD64 if the CD64 index exceeds a cutoff level indicative of supranormal amounts of neutrophil CD64 in the sample, wherein said cutoff level is determined as a function of the amount of the neutrophil marker-binding agent complex measured in step (iii). [0198] 3. The assay of clause 1 further comprising diagnosing the subject as having sepsis or having a risk of developing sepsis if the CD64 index exceeds a cutoff level indicative of supranormal amounts of neutrophil CD64 in the sample, wherein said cutoff level is determined as a function of the amount of the neutrophil marker-binding agent complex measured in step (iii). [0199] 4. The assay of clause 1 wherein the CD64 index is corrected for the number of neutrophils in the sample determined by the amount of the neutrophil marker-binding agent complex measured in step (iii). [0200] 5. The assay of any one of clause 1 to 4 wherein the assay is an enzyme-linked immunosorbent (ELISA)-type, flow cytometry or immunochromatographic method. [0201] 6. The assay of any one of clause 1 to 5 wherein the binding agent is an antibody or an antigen-binding fragment thereof, an antigen-binding construct such as an affimer, or a ligand or binding part thereof. [0202] 7. The assay of clause 5 or 6 wherein the assay is an enzyme-linked immunosorbent (ELISA)-type or chromatographic method and the binding agent is immobilised on a support. [0203] 8. The assay of any one of clause 1 to 7 wherein, in step (ii), the sample is contacted with the binding agents of step (ii) by applying the sample to a sample portion of an immunochromatographic device wherein the device sample portion is operably connected to spaced capture portions of the device and whereby the components of the sample flow from the device sample portion to and through the device capture portions, and wherein one capture portion comprises the binding agent which specifically binds to CD64 in the sample such that the CD64 is captured by the binding agent to form a binding agent-CD64 complex in the capture portion, and wherein a second capture portion comprises the binding agent which specifically binds to neutrophils in the sample such that the neutrophil or the binding part thereof is captured by the binding agent to form a binding agent-neutrophil marker complex in the capture portion. [0204] 9. The assay of any one of clause 1 to 8 wherein the amount of CD64 complex and the amount of neutrophil-marker binding agent complex is detected using binding agents such as an antibody or antigen-binding fragment, ligand or affimer that bind to CD64 or the neutrophil marker respectively and directly or indirectly provide a detectable signal that can be quantified visually or photometrically including fluorometrically (by instrument reader). [0205] 10. The assay of clause 9 wherein the binding agents are conjugated to a detectable marker or microparticles comprising a detectable marker, that provide a detectable signal. [0206] 11. The assay of any one of clause 8 to 10 wherein the capture portion is a test line. [0207] 12. The assay of clause 11 wherein the ratio of the visual or photometrically calculated signal from the CD64 test line and the visual or photometrically calculated signal from the neutrophil marker test line provides the modified neutrophil CD64 index indicating the amount of CD64 per neutrophil in the sample. [0208] 13. The assay of any one of clause 1 to 12 wherein the neutrophil specific marker is selected from the group comprising neutrophil elastate, lactoferrin, and human neutrophil lipocalin, or equivalent cell marker whose amount on neutrophils correlates with neutrophil cell number. [0209] 14. The assay of any one of clause 1 to 12 wherein monocytes and macrophages are substantially depleted by, for example, red cell agglutination. [0210] 15. A kit comprising (i) a chromatographic device comprising a porous membrane operably connected to a sample portion, two or more capture (test) portions, and optionally one or more of the following; a conjugate (detection marker) portion, a sucker portion, a suitable control portion and a cell lysis or solubilisation portion, and (ii) a binding agent that binds specifically to CD64 in the sample and forms a CD64-binding agent complex, and a second binding agent that binds specifically to neutrophils in the sample and forms a neutrophil marker-binding agent complex wherein the binding agents are either immobilised to separate capture portions and/or contained within conjugate portions and (iii) optionally instructions for using the device to determine a modified neutrophil CD64 index and/or assessment of sepsis. [0211] 16. The kit of clause 14 suitable for reverse or lateral flow immunogaphic formats. [0212] 17. The kit of clause 14 or 15 wherein the binding agent is an antigen binding construct such as an affimer, a ligand or an antibody or antigen-binding fragment thereof. [0213] 18. The kit of clause 17 wherein the binding agent is an affimer. [0214] 19. The kit of any one of clause 14 to 17 wherein the neutrophil specific marker is selected from the group comprising neutrophil elastase, lactoferrin, and human neutrophil lipocalin, or equivalent cell marker whose amount on neutrophils correlates with neutrophil cell number. [0215] 20. The kit of any one of clause 15 to 19 for use in the assay of any one of clause 1 to 1.

[0216] The following kits/reagents are illustrative of the reagents used in the development of the assays described herein.

[0217] ELISA kits used:

Item Description: Human FCGR1A/CD64 ELISA kit

Catalogue Number: LS-F4795

Company Name: LS Bio

Item Description: PMN Elastase Human ELISA Kit

[0218] Catalog Number: ab119553

Company Name: Abcam

[0219] Antibodies and recombinant proteins used:
Item Description. Neutrophil Elastase, Recombinant Protein

Catalog Number: MBS957715

Company Name: MyBioSource

Item Description: Supply Human Neutrophil Elastase

Catalog Number: INHE

Company Name: Cardinal Bioresearch

[0220] Item Description: Anti-Neutrophil Elastase antibody Mouse Monoclonal
Catalog Number: ab41179

Company Name: Abcam

[0221] Item Description: Anti-Neutrophil Elastase antibody Rabbit Polyclonal
Catalog Number: ab126154

Company Name: Abeam

[0222] Item Description: Anti-Neutrophil Elastase antibody (Biotin)
Catalog Number: ab79962

Company Name: Abcam

Item Description: Recombinant Human CD64

Catalog Number 1257-FC

Company Name: R&D Systems

[0223] Item Description: Anti-CD64 antibody [10.1] (Biotin)
Catalog Number: ab27928

Company Name: Abcam

[0224] Item Description: Anti-CD64 antibody Rabbit Polyclonal
Catalog Number: ab95244

Company Name: Abeam

[0225] Item Description: Anti-CD64 antibody [3D3] Mouse Monoclonal
Catalog Number: ab140779

[0226] The embodiments of the kits, steps and assays are further identified with the following illustrative examples.

Example 1

[0227] Development of an alternative to Flow cytometric based neutrophil activation assay useful for point of care (POC). Assessing the immunoassay cutoff using the ratio of CD64 to NNM.

[0228] Recognising that a POC test for measuring neutrophil activation (nCD64i) would have great clinical utility, a novel method was devised as described herein that would allow the measurement of an analogue or equivalent of the Flow Cytometry nCD64i value, using an algorithm and the values for a combination of markers that are independently suited to measurement at POC.

[0229] In some respects, the present approach draws upon some of the inventors experience in development of a lateral flow, POC test for measuring CD4 T-cells (U.S. Pat. No. 8,409,818 and other territories).

[0230] From the inventors experience with the CD4 POC test, it was understood that the amount of CD4 per cell is constant and therefore the amount of CD4 is directly proportional to the number of CD4 T-cells. In contrast, the amount of CD64 on neutrophils is variable (being elevated in sepsis), and the nCD64i value measured by Flow Cytometry represents the average amount of CD64 protein per neutrophil (and is sometimes expressed as Mean Fluorescence Index). Therefore nCD64i can be represented as the numerical fraction (total CD4 in blood depleted of monocytes and macrophages)/(total number of neutrophils) or CD64/neutrophil. In the Flow Cytometer, this is measured as fluorescence intensity for each individual cell and then averaged over the entire number of neutrophils sampled (typically 50,000 cells or 100,000 cells).

[0231] The inventor/s predicted that if they were able to measure the total amount of CD64 in whole blood, after removal of monocytes and macrophages, for example, using the same method shown in the example of the CD4 test, then that would give the numerator of the CD64/neutrophil fraction. This measurement of CD64 could be achieved by methods such as enzyme-linked immunosorbent assay (ELISA-type assays seldom use enzymes but are still called ELISA's) using simple laboratory facilities, or more preferably by POC methods such as lateral flow immuonochromatography following the example of CD4 T-cells in the Visitect CD4 T-cell test.

[0232] The inventor/s further recognised that the number of neutrophils, representing the denominator of the CD64/neutrophil fraction, could be measured using the same method as the CD4 T-cell test, based on a protein that demonstrates a constant or predictable level of expression for each neutrophil, such that the amount of such protein is proportional to the number of neutrophils. As one example, the neutrophil-specific protein neutrophil elastase (NE) may be measured, but other proteins such as lactoferrin and myeloperoxidase may also be used, among others. Illustrative lateral flow methods and a radioimmunoassay method for the detection and enumeration of neutrophils together with eosinophils has been reported in the literature, using the proteins eosinophil protein X (EPX) and human neutrophil lipocalin (HNL), and showing acceptable levels of agreement with traditional cell counting methods (Rundström, G et al. Lateral Flow immunoassay Using Europium (III) Chelate Microparticles and Time-Resolved Fluorescence for Eosinophils and Neutrophils in Whole Blood. Clinical Chemistry 53, 342-348 (2007)).

[0233] Prior art methods for detecting NE are used on plasma or cell culture, where they detect the NE that has been released due to degranulation of neutrophils. In contrast, in the present assay, total NE is measured in whole blood including that inside intact neutrophils. The amount in plasma is much lower and essentially irrelevant compared to the intracellular amount.

[0234] NE has been considered as a marker for sepsis itself but only the plasma/serum amount, since the whole blood amount serves instead as a marker of neutrophil count NE is released in response to infection but the balance between release and turnover is considered too variable to make this useful as a marker for sepsis.

[0235] It was therefore predicted that by using immunoassays such as a lateral flow immunochromatographic immunoassay for parallel measurement (in the same assay rum at the same time) of both the total amount of neutrophil CD64 and the absolute number of neutrophils (for example using neutrophil elastase as the marker) in whole blood, the simple arithmetic fraction of CD64/NE could be obtained and would provide an alternative, point of care (POC) friendly measurement of the nCD64 index or (also called the nCD64 ratio). The principle of the tests for enumeration of CD4 T-cell numbers (Visitect CD4) and the herein described POC friendly nCD64i test (which may be called the SepsiTest) is shown in FIG. 3.

[0236] To test the prediction that the simple arithmetic fraction of CD64/NE could be obtained as an alternative measurement of the nCD64 index, ELISAs were used to measure the total amount of Neurophil Elastase (NE) and CD64 in whole blood from healthy volunteers. For these initial experiments (FIG. 4), the monocytes and macrophages were not first removed as the total number of monocytes and macrophages is much smaller than the number of neutrophils and so the effect of these cells is not expected to be a problem, however in the final test it is expected that monocytes and macrophages will be substantially/optionally depleted.

[0237] The experimental data (see FIG. 4) using ELISAs show strong correlation between the total amount of the neutrophil specific protein, neutrophil elastase (NE) and neutrophil (granulocyte) counts in healthy donors (FIG. 4A: p=0.0002, R2=0.896).

[0238] Similarly, there is strong correlation between total CD64 and neutrophil (granulocytes) counts in healthy donors (FIG. 4C, p=0.0003, R2=0.915. However, the surprising observation was made that the amount of CD64 per cell in healthy individuals is not constant, but instead is affected by the total number of neutrophil/granulocytes. That is, in samples with low levels of neutrophils (approaching the level of neutropenia), the total amount of CD64 is in fact higher, representing a higher amount of CD64 per cell even in these healthy individuals. To our knowledge this has not previously been observed.

[0239] The ratio between CD64 and NE therefore provides the nCD64 index, shown in FIG. 4B. Because the relative amount of CD64 is higher in samples with lower total neutrophil counts, a similar relationship is seen in the calculated values for the nCD64 index, with a higher nCD64i value for samples with low neutrophil counts (R2=0.861). This novel observation is likely to have strong clinical significance in the development of more effective sepsis tests based on CD64 expression, because it suggests that the cutoff ratio for nCD64i should not be a constant value, as recommended in the Leuko64 Flow Cytometry kit but subject to individual optimisation in each laboratory, [Beckman Coulter—see US Patent publication no. 2013/0230867 in the name of Trillium Diagnostics LLC-this kit comprises a cocktail of three monoclonal antibodies with specificities to CD64 (FITC conjugated) and CD163 (phycoerythrin conjugated) and a proprietary fluorescent bead suspension used for instrument calibration and the standardization of the leukocyte CD64 and CD163 expression on human blood leukocytes. The kit also contain a red cell lysing solution concentrate].

[0240] Rather, the cut off should be a variable value depending on the total neutrophil count in each instant sample. An algorithm could be constructed to set the cutoff value for any given value of neutrophil count using methods well known in the art, for example setting the cutoff as above the 95% confidence interval of the line of best fit for the CD64/NE ratio shown in FIG. 3B. An example of this calculation is shown in FIG. 5, where the nCD64 index is calculated for a larger set of healthy control samples following monocyte depletion, and the lower and upper 95% confidence interval for the estimated nCD64 index at different amount of NE (representing neutrophil count) is shown as dotted red lines. Any value of nCD64i that is higher than the upper illustrative 95% confidence interval would therefore have a high suspicion of representing sepsis.

[0241] In some embodiments, the cutoff is set above any value between about 90% and 99%. The choice of cutoff would be determined by expanded clinical studies and receiver operating characteristics (ROC) analysis.

[0242] Correction of the cutoff value in light of the observed variation in CD64 levels depending upon the neutrophil concentration may be made by various approaches known in the art. In one embodiment, the correction factor is applied to the CD64 index, such as by making it a semi-log ratio or multiplying it by an inverse of the neutrophil count). Alternatively, the cutoff numerical value is adjusted to have the same effect.

[0243] The methods for transforming the individual ELISA assays for CD64 and NE into POC tests such as lateral flow immunoassays are well known in the art, and the Visitect CD4 test and the neutrophil/eosinophil test described by Rundström et al (Rundström, G et al. Lateral Flow Immunoassay Using Europium (III) Chelate Microparticles and Time-Resolved Fluorescence for Eosinophils and Neutrophils in Whole Blood. Clinical Chemistry 53, 342-349 (2007)) are relevant examples incorporated herein in their entirety.

[0244] For the purpose of using lateral flow immunochromatography or other POC methods to calculate the nCD64 index, it is anticipated that a simple arrangement such as that shown schematically in FIG. 6 world be effective. In this predictive example, the test would have a similar format to the Visitect CD4 T-cell test, including the method for substantial removal of red blood cells, monocytes and macrophages incorporated in that test. However in place of the test line for CD4 and the reference line representing a cutoff of 350 CD4 T-cells, in one embodiment, there will be 2 test lines with one measuring the amount of CD64 and the other measuring the amount of NE or other neutrophil-specific marker. To determine a cut off, the arithmetic ratio between the measured intensity of the two lines would then be calculated, either by visual inspection or by instrument reader such as the Axxin AX-2 instrument (see for example US publication no. 2013/0162981 incorporated by reference in its entirety,) and the cutoff for the individual sample would be calculated from the measured amount of NE or other neutrophil-specific marker. The measured nCD64i would then be compared to the instant NE-dependent cutoff, such as the upper 95% confidence interval for healthy samples, and a decision on “likely sepsis” or “not likely sepsis” would be reported.

Example 2

[0245] Further developments are described and in particular the utility of total CD64 and total NNM levels (i.e., internal and external levels) and different cutoff determinations in the diagnosis of sepsis.

[0246] In (FIG. 4C) it is illustrated that the amount of CD64 compared to NE (the nCD64i measured by ELISA) was higher in samples with lower neutrophil/granulocyte counts, with the novel suggestion of the need for a variable cutoff for CD64i as a function of NE value to improve the use of CD64 in diagnosis of sepsis (see also FIG. 5). Notably, the results in that experiment were obtained using whole blood that had been depleted of monocytes/macrophages using magnetic beads.

[0247] In further work, these observations have been extended and a simplified method is described that requires only the use of NE and CD64 values from whole blood, without the depletion of monocytes/macrophages. Monocyte/macrophage depletion is an optional step especially for samples with low values of NE where the relative contribution of monocyte/macrophage CD64 would be higher than in samples with higher values of NE, for example in patient samples having NE values of less than about 1.0 or less than about 0.5 μg/ml NE in whole blood.

[0248] In the improved method, rather than calculating the nCD64i (CD64 per neutrophil) and comparing that number with the absolute value of NE (FIG. 5), the absolute value of CD64 is used in comparison with the absolute value of NE. That is, the method is derived from the work described in example 1 but provides additional insights into the system and additional strategies for using CD64 and NNM together as a sensitive diagnostic in a wide range of immunoassays. Rather than relating the amount of NNM to the absolute neutrophil/granulocyte count, the close correlation between CD64 and NNM in healthy subjects is used to define the threshold “healthy” level of CD64 for any given level of NNM.

[0249] FIG. 7 shows results of ELISA for NE and CD64 in whole blood for healthy subjects (n=30), and shows that in healthy subjects, the total amount of CD64 in whole blood is closely correlated with the total amount of NE in whole blood (R2=0.74). This is likely to reflect the correlation of NE with neutrophil/granulocyte counts, but for the purpose of our assay this correlation of NE and neutrophil/granulocyte count is not necessary—it is the NE concentration per se that is important (or other neutrophil number marker (NNM) that may substitute for NE). Consistent with our observations in FIG. 4C but novel compared to the known art, the level of CD64 reached a plateau when NE reached around 5 μg/ml, suggesting a feedback mechanism that may limit the expression of CD64 when NE levels (and presumably neutrophil counts) are in the higher range of normal, or elevated above normal, in healthy subjects. From this figure it is predicted that sepsis patients would have elevated levels of CD64 relative to the amount of NE present in the respective patient's sample, and a presumptive cutoff for CD64 of the mean plus 2 standard deviations of the healthy samples (209 ng/ml) could be contemplated for samples with average or higher levels of NE.

[0250] FIG. 8 shows the results of ELISA for NE and CD64 in whole blood for sepsis patients (red markers, n=11) together with the healthy subjects as shown in FIG. 7 (blue markers). It is seen that indeed 9/11 sepsis patients have highly elevated levels of CD4 above the mean plus 2 SD. Unexpectedly however, 6/11 sepsis patients were also found to have NE levels higher than the mean plus 3 SD of healthy subjects (17.6 μg/ml), which suggests an additional cutoff based on NE levels for sepsis. This additional NE cutoff unequivocally identifies an additional sample that was just below the CD64 cutoff of 209 ng/ml.

[0251] FIG. 9 shows the same results but highlighting the four different classifications of sepsis patient samples according to the cutoffs based on the mean plus 2 SD of CD64 and mean plus 3 SD of NE. By these criteria, one patient is positive only for NE, 5 patients are positive for both NE and CD64, 4 patients are positive for CD64 only, while one patient is negative for both NE and CD64, with very low values for both markers, and suggesting that the patient may have low levels of neutrophils. This result highlights that the combination of NE and CD64 is more sensitive for the diagnosis of sepsis than the use of NE or CD64 alone, probably related to the novel observation that with increasing supranormal levels of NE, the induced expression of CD64 in sepsis may be somewhat restricted by a feedback mechanism as observed in healthy subjects. While these results suggest that the simple cutoffs for NE and CD64 would provide a highly sensitive method for diagnosis of sepsis in patients with normal or elevated levels of NE in whole blood and by inference, normal or elevated neutrophil counts, there are many patients with sepsis or at high risk of sepsis who have low neutrophil counts (neutropenia), for example many neonates and patients who have undergone chemotherapy or radiation therapy for cancer treatment.

[0252] For these neutropenic patients, advantage can be taken of the close correlation of NE versus CD64 in healthy subjects, even when the NE level is low. As shown in FIG. 10, when the analysis of NE versus CD64 is limited to those healthy subjects with NE of ≤0.25 μg/ml, a very strong correlation is seen (R2=0.88), and the single sepsis patient not identified using the NE and CD64 cutoffs of FIGS. 8 and 9 (red marker) is seen to have a higher level of CD64 relative to NE than the healthy subjects (blue markers). This is further illustrated in FIG. 11, where an alternative cutoff for CD64 is calculated as a multiple of the trendline for CD64 versus NE in healthy subjects. In this case, the multiple is 1.6 times the trendline and the trendline is described by the equation y=5.6953x2+45.769x where y is CD64 (ng/ml) and x is NE (μg/ml). It will be obvious that this cutoff may be extrapolated until it reaches the same level of CD64 described by the simple cutoff of mean plus 2SD as in FIGS. 8 and 9, or higher levels as may be desired to improve the specificity of the assay for example mean plus 3 SD or higher.

[0253] On this basis, the combination of NE and CD64 markers may be used to diagnose sepsis using the algorithm of sepsis being indicated when one or more of the following conditions are met: CD64>mean plus 2 SD of healthy subjects, OR NE>mean plus 3 SD of healthy subjects, OR CD64>1.6 times the level of CD64 predicted by the trendline for healthy subjects with levels of NE below 2.5 μg/m or a higher amount such as 5 μg/ml.

[0254] However a more simple algorithm is suggested by the observation that the CD64 cutoffs can also be described by a multiple of the polynomial trendline for NE versus CD64 in healthy subjects, as shown in FIG. 12A where the multiple is 1.6 and the trendline is described by the equation y=−0.0137x4+0.6094x3−9.857x2+65.776x, where y is CD64 (ng/ml) and x is NE (μg/ml). In this example, the red shaded area defines a “gate” of values that represent “healthy” (non-septic) levels of CD64 and NE, with an upper level of NE representing the mean plus 3 SD of healthy subjects. FIG. 12B shows the sepsis patients superimposed on this model, with 11/11 (100%) sepsis patients identified correctly.

[0255] An even more simple algorithm is achieved by further extrapolation of the cutoff derived by a multiple of the trendline for CD64 versus NE. As shown in FIG. 12C, extrapolation of the cutoff where the multiple is 1.6 and the trendline is described by the equation y=−0.0137x4+0.6094x3−9.857x2+65.776x, where y is CD64 (ng/ml) and x is NE (μg/ml) gives a similar “gate” of healthy levels of CD64 versus NE to that obtained in FIG. 12B. Therefore, sepsis would be indicated in any subject where CD64 (ng/ml)>1.6×(−0.0137x4+0.6094x3−9.857x2+65.776x), where x is NE (μg/ml). It would be obvious to one skilled in the art that any combination or modification of these various cutoff calculations could be used to achieve the sensitive and specific diagnosis of sepsis using the values of NE and CD64 derived from whole blood, and the actual values used in the trendline equation and the choice of multiple could be determined using any appropriate set of samples from healthy subjects and CD64 combined with NE or other neutrophil specific markers and any alternative method as well as ELISA for the measurement of these respective analytes.

Example 3

[0256] To better understand the utility of the herein described assay, sepsis patients and a subset of the healthy subjects were examined using the commercial Leuko64 assay, which measures the cell surface expression of CD64 on neutrophils by Flow cytometry.

[0257] The Leuko64 kit [see for example Beckman Coulter—see US Patent publication no 2013/0230867 in the name of Trillium Diagnostics LLC] comprises a cocktail of three monoclonal antibodies with specificities to surface CD64 (FITC conjugated) and surface CD163 (phycoerythrin conjugated) and a proprietary fluorescent bead suspension used for instrument calibration and the standardization of the leukocyte CD64 and CD163 expression on human blood leukocytes. The kit also contain a red cell lysing solution concentrate. Variants of this kit are available such as the Accellix CD64 cartridge and reader (LeukoDx)

[0258] As shown in FIG. 13, Leuko64 showed highly significantly higher values for sepsis patients than for healthy controls, but using the Leuko64 kit manufacturer recommended cutoff of 1.2, only 8/11 sepsis patients were identified with this test, consistent with many reports in the literature that show around 70-80% sensitivity of this method. Similarly, detection of total whole blood CD64 alone by ELISA showed highly significantly higher values for sepsis patients than for healthy controls, but using the cutoff of mean plus 2 SD (209 ng/ml), only 9/11 sepsis patients were identified with this test. The total CD64 and NE values are determined by comparison with reference standards (standard curve) provided with commercially available kits and tested in each assay run, and then adjusted for the sample dilution used in the assays.

[0259] Individual sepsis patient samples were then examined in more detail by comparing the amount of CD64 detected by Leuko64 (surface CD64 only) versus an standard flow cytometry method in which cells were stained in the same way as for Leuko64 (surface CD64 only) or after the cells were first permeabilised using a neutrophil lysis solution comprises a detergent to allow detection of both cell surface and intracellular CD64.

[0260] FIG. 14 shows the results of this analysis for patient SEP009, the patient with the lowest levels of NE in our earlier analysis. In panel A, it is shown that the neutrophils from this patient have high levels of surface expression of CD64 (red line—middle peak) consistent with very high relative CD64 expression in Leuko64 (panel B), but even higher levels of CD64 per cell when intracellular CD64 is detected as well (blue line—total—peak on right hand side). However the low level of NE (panel D) and presumed low neutrophil counts for this patient result in a total CD64 result that is below the cutoff based on CD64 alone (panel C, but detectable using the algorithm approach (FIGS. 8-12). This result also suggests that for patients with low levels of NE, the Leuko64 approach or other methods may be of additional utility given the very high Leuko64 result for this patient.

[0261] FIG. 15 shows the same analysis for patient SEP010, the patient with elevated levels of CD64 but just below the cutoff in FIG. 9 derived from the mean plus 2 SD of CD64 from healthy subjects. In this patient, there was minimal surface staining of CD64 (panel A, comparing red line and isotype control for surface staining) consistent with low relative CD64 expression in Leuko64 (panel B), but there were high levels of CD64 detected when intracellular CD64 was stained as well (panel A blue line, right hand peak). Consistent with this observation, patient SEP010 had modest levels of total CD64 by ELISA, although still just below the cutoff for total CD64 based on mean plus 2SD (panel C), whereas the Leuko4 result of 0.7 was well below the manufacturer cutoff of 1.2 for that test (panel B). However, when NE is examined the patient SEP010 is seen to have very highly elevated levels of NE, providing unequivocal diagnosis of sepsis using the cutoff derived from the mean plus 3SD of NE, and also using the cutoff derived from the trendline equation.

[0262] FIG. 16 shows the same analysis for patient SEP011, a patient with elevated levels of both CD64 and NE. In this patient, there was significant surface staining of both intracellular and surface CD64 (panel A, comparing red line and isotype control for surface staining, blue line (middle peak) and isotype control for total staining) consistent with high relative CD64 expression in Leuko64 (panel B), well above the manufacturer cutoff. Patient SEP011 had highly elevated levels of total CD64 by ELISA (panel C), and also highly elevated levels of NE (panel D), providing unequivocal diagnosis of sepsis using the cutoff derived from the mean plus 3SD of NE, AND the cutoff derived from the mean plus 2SD of CD64, AND also using the cutoff derived from the trendline equation.

[0263] The results in FIGS. 13, 14, 15 and 16 demonstrate that a further advantage of the present method is the detection of total CD64, using ELISA or other methods and solubilized whole blood or permeabilised intact cells, rather than only cell surface CD64 as conventionally performed using Leuko64 or other methods.

[0264] The likely biological explanation for this novel observation is that only a variable proportion of CD64 is present on the surface of neutrophils at any given time. This is illustrated schematically in FIGS. 17 and 18. In FIG. 17, modified from van der Poel 2011, in healthy individuals, low levels of CD64 (FcγR1) are expressed on the cell surface, and these are saturated by monomeric IgG which does not result in cell signaling or antigen complex internalization (FIG. 17A). Upon initial stimulation of the neutrophil by interferon gamma or other stimuli associated with bacterial infection or sepsis, additional CD64 is synthesized and translocated to the surface of the neutrophil and associated with membrane microdomains, together allowing for binding of multimeric Ig in immune complexes which leads to cell signaling (FIG. 17B).

[0265] However a further function of CD64 is internalization of the antigens in such immune complexes, to allow for antigen processing and presentation. As shown in FIG. 18, this may result in activated neutrophils having different levels of surface CD64, even though they have the same elevated level of total CD64. On the left, a “healthy” neutrophil is shown schematically with low levels of CD64, expressed on the surface. These cells may also contain small amounts of pro-formed, intracellular CD64. In sepsis, “activated” neutrophils are shown on the right, each with equally large amounts of CD64, significantly increased compared to healthy neutrophils, but with distribution on the surface (similar to the patient sample SEP09, FIG. 14), or evenly distributed between the surface and intracellular (similar to the patient sample SEP011, FIG. 16), or predominantly distributed to the intracellular compartment (similar to patient sample SEP010, FIG. 15). As such, any method that allows the detection of both cell surface and intracellular CD64 will allow for improved diagnosis of sepsis.

[0266] It is also noted that the present method can be used to similarly detect soluble CD64 that has been shed from the surface of cells into the plasma fraction, as well as NE which has been released from activated neutrophils (sometimes known as neutrophil extracellular traps or NETs), which may be a further advantage of our method of whole blood analysis of CD64 and NE. In the known art, soluble NE and CD64 have been measured in either the serum or plasma fraction, but NOT in the whole blood fraction that would incorporate both intact cells and cells in various stages of activation and degranulation.

[0267] Accordingly, the present disclosure provides for the use of permeabilization/lysis/fixation of neutrophils to allow for total CD64 staining to improve the sensitivity of flow cytometry and other methods for the detection of CD64 as a biomarker for sepsis.

[0268] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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