METHOD FOR DETECTION OF CD16B

Abstract

The invention provides methods of determining the neutrophil level in a sample by determining the level of CD16b, including the intracellular form of CD16b in a sample, such as a blood sample. The methods may further comprise the determination of lactate levels Also provided are associated diagnostic and therapeutic methods. Further provided are kits and devices for performing the methods, particularly lateral flow kits and devices.

Claims

1. A method of determining the neutrophil level in a sample by determining the level of CD16b, wherein the CD16b comprises the intracellular form of CD16b.

2. A method for predicting, diagnosing, excluding and/or monitoring neutropenia, neutropenic sepsis and/or non-neutropenic sepsis in a subject, the method comprising determining the level of CD16b, in a sample, wherein the CD16b comprises the intracellular form of CD16b.

3. The method according to any preceding claim, wherein the CD16b comprises, consists essentially of, or consists of the membrane-anchored form of CD16b and the intracellular form of CD16b.

4. The method according to any preceding claim, wherein (i) the method further comprises a step of removing at least a proportion, and preferably most or all, of the soluble form of CD16b from the sample prior to determining the level of CD16b; or (ii) the method is performed on a sample from which at least a proportion, and preferably most or all, of the soluble form of CD16b has previously been removed.

5. The method of claim 4, wherein the step of removing at least a proportion of the soluble form of CD16b is a filtration step, wherein the filtration step preferably yields: (i) a filtered sample comprising white blood cells, wherein the filtered sample is preferably depleted of the soluble components of the sample; and (ii) a solution comprising soluble components of the sample, preferably the soluble form of CD16b; wherein the solution is preferably white blood cell-free but preferably contains red blood cells.

6. The method of claim 4 or 5, wherein the step of removing at least a proportion of the soluble form of CD16b from the sample of claim 4 or the filtration step of claim 5, is carried out under conditions that keep at least a large proportion, and preferably substantially all, of the neutrophils present in the sample intact and preferably alive.

7. The method according to any preceding claim, wherein the level of CD16b is determined using one or more CD16b detection moieties, preferably labelled binding reagents, preferably one or more CD16b-binding antibodies.

8. The method of claim 7, wherein the CD16b-binding antibodies are polyclonal antibodies or monoclonal antibodies.

9. The method according to any preceding claim, wherein the neutrophil level is determined to be above or below one or more predetermined thresholds, preferably wherein: (i) a neutrophil level below a predetermined threshold is indicative of neutropenia, neutropenic sepsis, a significant risk of developing neutropenia, and/or a significant risk of developing neutropenic sepsis; (ii) a neutrophil level above a predetermined threshold is indicative of the absence, or an insignificant risk, of the presence or development of neutropenia and/or neutropenic sepsis; (iii) a neutrophil level above a predetermined threshold is indicative of (non-neutropenic) sepsis or a significant risk of developing (non-neutropenic) sepsis; or (iv) a neutrophil level below a predetermined threshold is indicative of the absence, or an insignificant risk, of the presence or development of (non-neutropenic) sepsis.

10. The method according to claim 9, wherein one or more thresholds are used, wherein the one or more thresholds are selected from: (a) less than 1000 neutrophil cells/μL blood, which is indicative of neutropenia, neutropenic sepsis, a significant risk of developing neutropenia and/or a significant risk of developing neutropenic sepsis; (b) 1000-1500 neutrophil cells/μL blood, which is indicative of a need to further monitor the subject for the development neutropenia and/or neutropenic sepsis; (c) more than 1500 neutrophil cells/μL blood, which is indicative of the absence, or an insignificant risk, of the presence or development of neutropenia and/or neutropenic sepsis; (d) more than 7500 neutrophil cells/μL blood, which is indicative of (non-neutropenic) sepsis, or a significant risk of developing (non-neutropenic) sepsis; and/or (e) less than 7500 neutrophil cells/μL blood, which is indicative of the absence of (non-neutropenic) sepsis, or an insignificant risk of presence or development of (non-neutropenic) sepsis.

11. The method according to any preceding claim, wherein the sample is from a subject who (i) has been exposed to radiation; (ii) has received or is receiving a drug capable of causing neutropenia, such as an anti-psychotic drug or a thyroid drug; (iii) is suffering from HIV, hepatitis and/or an autoimmune disorder such as rheumatoid arthritis; (iv) has cancer and/or has received or is receiving chemotherapy and/or radiotherapy; (v) is displaying or experiencing symptoms of an infection; and/or (vi) has recently undergone surgery.

12. The method according to any preceding claim, wherein the method comprises a step of cell lysis prior to determining the level of CD16b, preferably using a surfactant, which may be ionic or non-ionic, such as Triton X-100.

13. The method of claim 12, wherein the step of cell lysis is carried out: (i) after a step of removing at least a proportion of the soluble CD16b; or (ii) on a sample from which at least a proportion of the soluble CD16b has previously been removed.

14. The method of any preceding claim, wherein the level of CD16b is determined via a lateral flow assay.

15. The method according to any preceding claim, wherein the method further comprises detecting one or more different markers indicative of the presence of (neutropenic or non-neutropenic) sepsis in the sample.

16. The method of claim 15, wherein the method comprises a step of detecting lactate, and/or determining the level of lactate, in the sample.

17. The method of claim 16, wherein the step of detecting lactate, and/or determining the level of lactate is carried out: (i) before determining the level of CD16b; (ii) before any step of cell lysis; (iii) on a solution comprising soluble components of the sample, such as a solution in accordance with claim 5 (ii); and/or (iv) via a lateral flow assay.

18. The method according to any preceding claim, wherein the method comprises: (a) a step of filtering the sample to yield: (i) a filtered sample comprising white blood cells, wherein the filtered sample is preferably depleted of the soluble components of the sample; and (ii) a solution comprising soluble components of the sample; wherein the solution is preferably white blood cell-free; (b) a step of cell lysis of the filtered sample from (i); (c) a step of determining the level of CD16b in the sample from (b), optionally on a first lateral flow strip; and optionally (d) a step of detecting lactate or determining the lactate level in (ii), optionally on a second lateral flow strip, wherein step (d), if present, may be simultaneous or sequential with step (b) or (c).

19. The method according to any one of claims 16 to 18, wherein lactate is detected and/or the level of lactate is determined using lactate dehydrogenase and a reagent, preferably wherein the reagent is a coloured reagent, more preferably wherein the reagent changes the colour upon the action of lactate dehydrogenase in the presence of lactate.

20. The method according to any preceding claim wherein at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more samples are taken from the subject at different times and the levels of CD16b and optionally one or more further markers, such as lactate, are determined, preferably wherein the samples are taken every 6 to 24 hours, such as daily, or every 2, 3, 4, 5, 6, 7 or 14 days.

21. The method according to any preceding claim, wherein the sample is taken form the subject no more than 24 hours prior to determining the level of CD16b and optionally one or more further markers, such as lactate, preferably no more than 4, 3, 2 or 1 hours or 30, 25, 20, 15 or 10 minutes prior to determining the level of CD16b and optionally one or more further markers, such as lactate.

22. The method according to any preceding claim, wherein a prediction of impending neutropenia and/or neutropenic sepsis, or of (non-neutropenic) sepsis, or a diagnosis of neutropenia and/or neutropenic sepsis, or of (non-neutropenic) sepsis, results in a decision to treat and/or treatment of the subject with an antibacterial agent, preferably broad-spectrum antibiotics.

23. A method of predicting responsiveness of a subject to treatment with an antibiotic and/or selecting a subject for treatment with an antibiotic comprising performing the method of any preceding claim and predicting responsiveness and/or selecting the subject for treatment where neutropenia and/or neutropenic sepsis, or (non-neutropenic) sepsis, is predicted or diagnosed.

24. A method of treating or preventing neutropenia and/or neutropenic sepsis, or (non-neutropenic) sepsis, comprising administering an antibiotic to the subject suffering from neutropenia and/or neutropenic sepsis, or (non-neutropenic) sepsis, wherein the subject displays, in a sample, an altered level of at least CD16b and optionally also an altered level of lactate and/or wherein the subject has been selected for treatment by performing the method of any preceding claim.

25. An antibiotic for use in a method of treating or preventing neutropenia and/or neutropenic sepsis, or (non-neutropenic) sepsis, wherein the subject displays, in a sample, an altered level of at least CD16b and optionally also an altered level of lactate; and/or has been selected for treatment by performing the method of any one of any preceding claim.

26. The method of any one of claims 22-24 or the antibiotic for use of claim 25, wherein the antibiotic is administered intravenously and/or is selected from macrolides (e.g. azithromycin, clarithromycin), cephalosporins (e.g. cefuroxime, cefpodoxime, cefdinir), ketolides (e.g. telithromycin), fluoroquinolones (e.g. moxifloxacin, gemifloxacin, levofloxacin), doxycycline, trimethoprim/sulfamethoxazole and amoxicillin/clavunate.

27. A testing device, testing kit or testing composition of matter comprising: a. a sample receiving zone to which a sample from a subject is added; b. a conjugate zone comprising at least one labelled detection moiety which binds to CD16b; c. a solid support defining a liquid flow path for the sample and comprising (i) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that also binds to CD16b thereby immobilising the CD16b at the test line to produce a signal via the labelled detection moiety also bound to the CD16b; or (ii) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that binds to the CD16b-bound labelled detection moiety of step (b) thereby immobilising it at the test line producing a signal; and optionally further comprising: d. at least one labelled control binding reagent that binds to a binding partner immobilised at a control line downstream of the test line for the CD16b and thus confirms that the test has completed successfully; and optionally further comprising: e. an absorbent material downstream of the test (and control, where present) lines to absorb excess sample.

28. The testing device, testing kit or testing composition of matter of claim 27, wherein the solid support comprises a chromatographic medium and/or a capillary flow device.

29. The testing device, testing kit or testing composition of matter of claim 27 or claim 28 which is a test strip.

30. The testing device, testing kit or testing composition of matter of any one of claims 27-29, wherein the sample from the subject is a filtered sample comprising cells in accordance with claim 5 (i).

31. A testing device, testing kit or testing composition of matter comprising: a. a first test strip comprising a first sample application zone to which a sample from a subject is added, wherein the first sample application zone comprises a filter that traps the white blood cells; b. a second test strip comprising: i. a second sample application zone to which the filter with trapped white blood cells is added; ii. a conjugate zone comprising at least one labelled detection moiety which binds to CD16b; iii. a solid support defining a liquid flow path for the sample and comprising (i) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that also binds to CD16b thereby immobilising the CD16b at the test line to produce a signal via the labelled detection moiety also bound to the CD16b; or (ii) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that binds to the CD16b-bound labelled detection moiety of step ii. thereby immobilising it at the test line producing a signal; and optionally further comprising: iv. at least one labelled control binding reagent that binds to a binding partner immobilised at a control line downstream of the test line for the CD16b and thus confirms that the test has completed successfully; and optionally further comprising: v. an absorbent material downstream of the test (and control, where present) lines to absorb excess sample.

32. A system or test kit for diagnosing or monitoring a subject, comprising: a. a testing device for determining levels of at least CD16b in a sample, preferably according to any one of claims 27-31; b. a processor; and c. a storage medium comprising a computer application that, when executed by the processor, is configured to: i. access and/or calculate the determined levels of at least CD16b in the sample on the testing device; ii. calculate a test score from the levels of the at least CD16b in the sample, optionally including a comparison of the levels with one or more thresholds and/or CD16b levels determined at one or more earlier time points, to thereby predict or diagnose neutropenia and/or neutropenic sepsis, or (non-neutropenic) sepsis; and iii. output from the processor the predicted or diagnostic result for the subject.

33. The method according to any one of claims 1-24 or claim 26, wherein the method comprises the use of a lateral flow device kit or composition, as defined in any one of claims 27-31.

34. The method according to any one of claims 1-24, comprising the steps of: i. adding a sample to a first sample application zone of a first test strip to trap any neutrophils on a filter comprised within the first test strip; ii. transferring the filter with trapped neutrophils to a second sample application zone of a second test strip; iii. adding to the filter a lysis reagent to lyse the neutrophils; and iv. determining the level of CD16b.

35. The method of claim 33, wherein the step of determining the level of CD16b comprises: i. contacting a solution of the lysed neutrophils with a conjugate zone comprising at least one labelled detection moiety which binds to CD16b; ii. contacting the mixture of the lysed neutrophils and the at least one labelled detection moiety which binds to CD16b with: (i) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that also binds to CD16b thereby immobilising the CD16b at the test line to produce a signal via the labelled detection moiety also bound to the CD16b; or (ii) a test line for at least CD16b, the test line comprising an immobilised further detection moiety that binds to the CD16b-bound labelled detection moiety of step ii. thereby immobilising it at the test line producing a signal.

36. The method of claim 34 or claim 35, wherein the lysis reagent is a surfactant, which may be ionic or non-ionic, such as Triton X-100.

37. The method of any one of claims 34-36, wherein the labelled detection moiety which binds to CD16b is an anti-CD16b antibody conjugated to a gold particle.

38. The method of any one of claims 34-37, wherein the further detection moiety is an anti-CD16b antibody, preferably a polyclonal antibody.

39. The method of any one of claims 34-38, wherein the step of adding the sample to the first application zone is followed by a step of cell wash.

40. The method of any one of claims 34-39, wherein the filter is a membrane suitable for trapping neutrophils, such as a microporous membrane.

41. The method of any one of claims 34-40, wherein transferring the filter with trapped neutrophils is done manually or automatically.

42. The method of any one of claims 34-41, wherein the method further comprises detecting lactate, and/or determining the level of lactate, in the sample.

Description

DESCRIPTION OF THE FIGURES

[0259] The invention will now be described by way of example with respect to the accompanying drawings in which:

[0260] FIG. 1 is a bar graph to represent the change in test line measurement (cube units) for AF1597 deposited onto CN95 at 1 mg/mL (PBS+1% sucrose) with AF1597 conjugated to gold with varying concentrations of analyte spiked into plasma;

[0261] FIG. 2 is a diagram to represent how histopaque may be used to separate blood into (i) plasma with cells and (ii) erythrocytes;

[0262] FIG. 3 is a bar graph to represent the change in cube units for AF1597 deposited onto CN95 at 1 mg/mL (PBS+1% sucrose) with AF1597 conjugated to gold with buffy coat (BC), cells and washed cells collected from histopaque processed EDTA blood with and without the addition of Triton x100, water and cell extraction buffer (CEB) as lysis buffers;

[0263] FIG. 4. Is a bar graph to represent the change in test line measurement (cube units) for AF1597 deposited onto CN95 at 1 mg/mL (PBS+1% sucrose) with AF1597 conjugated to gold with washed cells collected from histopaque processed EDTA blood (1109) with and without the addition of Triton x100 and the corresponding cell count by flow cytometry;

[0264] FIG. 5 is a bar graph to represent the change in test line measurement (cube units) for AF1597 deposited onto CN95 at 1 mg/mL (PBS+1% sucrose) with AF1597 conjugated to gold with washed cells collected from histopaque processed EDTA blood donors 1113 and 1114, with and without the addition of Triton x100 and the corresponding cell count by flow cytometry;

[0265] FIG. 6 is a bar graph to represent the change in test line measurement (cube units) for AF1597 deposited onto CN95 at 1 mg/mL (PBS+1% sucrose) with AF1597 conjugated to gold with washed cells collected from histopaque processed EDTA blood donors 1117 and 1118, with and without the addition of Triton x100 and the corresponding cell count by flow cytometry;

[0266] FIG. 7 is a scatter graph to represent the correlation between CD16b measurement (cube units, y-axis) obtained for lysed cells and the corresponding cell count (x-axis) obtained by flow cytometry for five donors, wherein y=0.0124x+8.8677 and R.sup.2=0.861; FIG. 8 is a diagram of a typical lateral flow device configuration;

[0267] FIG. 8 is a schematic of a typical lateral flow devide.

[0268] FIG. 9 is a picture to show the aggregation of 40 nm gold with RP02 antibody conjugated at 15 μg/ml in different buffers and different pHs.

[0269] FIG. 10 is a scheme to demonstrate how the biotin system works.

[0270] FIG. 11 is a graph to demonstrate the change of cube units for different concentrations of isolated cells from two blood donors with increasing lysis incubation time (0, 1, 3 and 5 minutes shown from left to right for each assay) in wet assay system with RP02 on CN95 NC and AF1597 AuC.

[0271] FIG. 12 is a proposed format for the lactate assay and the overall enzymatic reaction which produces the purple Formazan product.

[0272] FIG. 13 is a graph to demonstrate the change in cube units for varying concentrations of Sodium Lactate in triplicate with dried dye mix on the conjugate pad (8951) at a 10 minute read time.

[0273] FIG. 14 is a photo to demonstrate the results for 10 μL of whole blood with either 0 or 10 mM spiked Na-L-Lactate chased with 60 μL PBS at either 5 or 10 minutes.

[0274] FIG. 15 is a graph to show the correlation between cell-associated (intracellular and membrane-anchored) CD16b and neutrophil levels.

[0275] FIG. 16 is a graph to show the correlation between soluble CD16b and neutrophil levels.

[0276] FIG. 17 is a graph to demonstrate the change in cube units for varying concentrations of CD16b in a wet assay format with a 10 minute read time.

[0277] The invention will be further understood with reference to the following experimental Examples.

EXAMPLES

1. Materials and Methods

Reagents and Equipment

[0278]

TABLE-US-00001 Material Supplier Product Code FC gamma RIIIB/CD16b antibody Novus-Biologicals NBP2-12148 (MM0272-5L11) α-CD16b Fc gamma RIIIB/CD16b Novus-Biologicals MM0272- Antibody 5L11(Mab) NBP2-12148 α-CD16b Human Fcy RIIIB/CD16b R&D Systems MAB1597 Antibody (Monoclonal Mouse IgG2 b) Clone #245514 α-CD16b Human Fcy RIIIA/B (CD16b) R&D Systems AF 1597 Antibody (Polyclonal Goat IgG) CD16b -Std Material Recombinant R&D Systems 9948-FC-100 Human Fcy RIIIB/CD16b (aa18-200) α- Human CD16b Mouse MAB Stratech Scientific Ltd 11046-MM01 α- Human CD16b Rabbit Antibody Stratech Scientific Ltd 11046 -RP02 Goat- α-Mouse (10 mg/mL) Lampire Biological 7455507 Laboratories Ltd. Goat- α-Rabbit (10 mg/mL) Lampire Biological 7455607 Laboratories Ltd. CD16b-Std Material Recombinant Human Fcy R&D Systems 9948-FC-100 RIIIB/CD16b (aa18-200) Recombinant Human Fc gamma R&D Systems 1597-FC-050 RIIIB/CD16b Protein Recombinant Human Fey RIIIA/CD16a R&D Systems 8894-FC protein (Gly17-Gln208) Human CD16b/FCGR3B Protein Stratech Scientific Ltd 11046-H08H (His Tag, NA2 allotype) (Met 1-Ser 200) BSA Sigma Aldrich 1002740634 Fatty acid Free BSA Sigma Aldrich A7030 Rabbit-α-Goat (10 mg/mL) Lampire Biological 7457307 Laboratories Ltd. Cell Extraction Buffer PTR-5X Abcam ab253215 # P3138 Tween 20 Sigma Aldrich P1379 PBS (tablets) Sigma Aldrich P4417 PBST (PBS + 0.1% Tween 20) Mologic N/A PBSTB(PBST + 1% BSA) Mologic N/A PBSE(PBS + EDTA 1.8 mg/mL} Mologic N/A 20 mM MES pH 5.5 Mologic N/A Gold Drying Buffer (20 mM Taps pH 8.5 + Mologic N/A 5% Sucrose + 3% BSA + I % Tween 20) 200 mg/mL aq. BSA Mologic N/A 40 nm Gold (OD 5, OD 4.76) BBI Solutions EMGC40 1M Tris pH 9.4 Mologic N/A 20 mM MES pH 6.5 Mologic N/A 20 mM MES pH 6.7 Mologic N/A 20 mM BES pH 6.9 Mologic N/A 20 mM TES pH 7.1 Mologic N/A 20 mM TES pH 7.5 Mologic N/A 20 mM TES pH 7.8 Mologic N/A 20 mM TAPS pH 8.5 Mologic N/A 20 mM Borate pH 8.5 Mologic N/A 20 mM Borate pH 9.0 Mologic N/A 20 mM Borate pH 9.3 Mologic N/A 1M NaCl Mologic N/A 5%, 50% Sucrose Mologic N/A α -Mouse IgG AP Conjugate Sigma A4102 pNPP substrate BioPanda pNPP-001 Vivaspin 500 Sartorius VS0121 Biotinylation Lightning Link Kit Expedeon 701-0000 Polystr eptavidin R BioTez K558-M Streptavidin - HRP Conjugate R&D Systems Duo Set 893975 TMB Substrate Bio Panda TMB-S-004 Stop Reagent Bio Panda STP-001 TBST (ELISA Wash Solution) Mologic SOP 321vl Absorbent Pad/sink pad - 22 mm Ahlstrom-Munksj 535400669224 Backing Card - 60 mm Unisart 33393619 Conjugate Pad - 17 mm Ahlstrom Grade 8951 Conjugate Pad- 17 mm Ahlstrom Grade 8980 CN180 Sartorius Stedtim 500752766 CN140-25 mm Unisart 500586972 CN95 - 25 mm Unisart 500677182 FR1 -10 mm Mdi R2990/998/1 Vivid Plasma Separation Pall 79EXPPA0200S0X Cytosep 1660 Ahlstrom-Munksjo MHPM1660 Triton x-100 Sigma Aldrich T9284 96 F well High bind plate Costar 9018 96 F well Low bind plate Medline 9000 11SP Deionised water ELGASystem Mologic 30 mL Polystyrene universal container STARLAB E1412-3011 1.5 mL Microcentrifuge tubes STARLAB S1615-5500 10 ml K2E(EDTA) Vacutainer Becton Dickinson 367525 Histopaque Sigma Aldrich 10771 LP3 tubes/Rohren tubes Sarstedt, Inc 55.484 Rohren push cap lids Sarstedt, Inc 65.809 200 mM Tris Mologic N/A 1-Methyl-1-piperidinomethane Sigma M8640 sulfonate 3-(4,5-Dimethyl-2-thiazolyl)-2,5- Sigma M2128 diphenyl-2H-tetrazolium bromide β-Nicotinamide adenine dinucleotide Sigma N8129 Sodium-L-Lactate Sigma/Aldrich 71718 L-Lactate dehydrogenase Roche 10127230001 Cube reader Optricon, Berlin 875-1622-0016

Conjugation of CD16b Antibody to Gold Nanoparticles

Buffer Screening Test

[0279] The buffer type, molarity and pH can affect the antibody binding to the gold particles because the binding is largely due to the charge and hydrophobic interactions. To test a range of buffers, 20 mM buffers were made up at the pH ranging from 6.7 to 9.3

[0280] The antibody was loaded at 15 μg/mL for all buffers investigated. A volume of 1.5 μL antibody was added to the bottom of a clean 96 μlate well, 5 μL of conjugation buffer was then added and finally 100 μL of gold OD5. These are incubated for 10 minutes at room temperature whilst stationary. The plate was then read to measure absorbance using a plate reader at 550 and 600 nm; the ratio of 550/600 was then calculated to give an aggregation ratio. If the aggregation ratio was ≥3.5 then the buffer was deemed suitable for conjugation.

CD16b Antibody Loading Test

[0281] Once a suitable buffer had been chosen from above the optimal loading of the antibody was determined. The loading concentrations 0 μg/mL to 30 μg/mL in 5 μg/mL increments were investigated in the buffer of choice. The respective volume of antibody (0, 0.5, 1.0 μL etc. . . . ) were added to the bottom of a clean 96 well plate, 5 μL of conjugation buffer was added followed by 100 μL of OD5 gold. Again, these were incubated for 10 minutes at room temperature. Once incubated, 10 μL of 1M NaCl was placed into each well. If the gold was not sufficiently coated, then the concentrated salt causes the gold conjugate to become unstable and aggregate, indicated by a colour change from bright pink to purple/grey. To determine the degree of aggregation, the plate was read on a plate reader and, again, an absorbance of 550 and 600 nm was measured. The ratio of 550/600 was taken and if the conjugate again met an aggregation ratio of 3.5 then it was deemed suitable.

Conjugation of CD16b Antibody for the Assay

[0282] The conjugation of the antibody at 15 μg/mL was achieved by the following method: [0283] Place 15 μL of 1 mg/mL antibody at the bottom of a clean 1.5 mL centrifuge tube [0284] Then add 50 mL of conjugation buffer to the antibody [0285] Add 1000 μL of OD 5 40 nm gold to the tube, lightly vortex, and then leave stationary for 10 minutes [0286] After 10 minutes, add 10 μL of 200 mg/mL BSA (aq.) and leave stationary for a further 30 minutes [0287] Place the tube into a suitable centrifuge and spin at 4000 g for 10 minutes [0288] Remove the supernatant and replace with the same volume of gold drying buffer
The conjugation of the antibody at 20 μg/mL was achieved by the following method: [0289] Place 20 μL of 1 mg/mL antibody at the bottom of a clean 1.5 mL centrifuge tube [0290] Then follow steps 2-6.
The conjugation of the antibody at 30 μg/mL was achieved by the following method: [0291] Place 30 μL of 1 mg/mL antibody at the bottom of a clean 1.5 mL centrifuge tube [0292] Then follow steps 2-6.

Nitrocellulose Plotting

[0293] Three different NCs were plotted (CN95, CN140 and CN180) using the CD16b antibody of interest, these have differing pore sizes and hence run at different speeds. The antibody was prepared in PBS+1% Sucrose to a final concentration of between 0.5 mg/mL-1 mg/mL. The antibody was plotted in 10 cm bands and then dried at 37° C. for a minimum of 30 minutes. Strip Construction The antibody plotted onto NC bands were mounted on adhesive backing card at the base of the card. The absorbent pad/sink pad was mounted at the top of the backing card with a 7 mm overlap on the NC. These bands were then cut into 3 mm wide strips using the Biodot Guillotine Cutter.
The assay was run using the two following procedures:

‘Wet’ Assay:

[0294] 1. Place 4 ml Gold Conjugate (ODS) into the well of a low bind microtitre plate and add 20 ml of sample or CD16b analyte in buffer (PBS).
2. Place the test strip into the well and allow to run until the well is empty.
3. Add 30 ml of chase buffer (PBST) and allow to run until the well is empty.
4. Place strip into a suitable holder and read the line values using an Optricon cube reader.

Isolated WBC Assay:

[0295] 1. Place 4 μl Triton X-100 (0.482%) into the well of a low bind microtitre plate and add 28 μl of cell suspension and preincubate for 5 mins. (Final dilution of TX-100 is 0.125%)

(Lysed Cells)

[0296] 2. Transfer 20 μl of lysed cell mixture from to a fresh well containing 4 μL Gold conjugate (ODS).
3. Proceed as from Step 2 using ‘Wet’ Assay protocol.

Blood Samples

[0297] Blood samples were taken from 13 anonymous donors at Mologic Ltd, in line with the Human Tissue Act license number 12647. These were used as required for the experiments, stored for no longer than two weeks and disposed of in line with Mologic's Code of Practice.

TABLE-US-00002 Mologic Blood Donor Number Volume/tube type 1095 2x 10 mL/EDTA 1079 2x 10 mL/EDTA 1099 1x 10 mL/EDTA 1x 1101 2x 10 mL/EDTA 1103 1x 10 mL/EDTA 1x 1104 1x 10 mL/EDTA 1x 1105 1x 10 mL/Heparin 1107 2x 10 mL/EDTA 1109 2x 10 mL/EDTA 1113 2x 10 mL/EDTA 1114 2x 10 mL/EDTA 1117 2x 10 mL/EDTA 1118 2x 10 mL/EDTA

Example 1 Initial Conjugation and Testing of Anti-CD16b Antibodies

Buffer Screening Test

[0298] The first experiments performed involved the conjugation of selected antibodies to gold nanoparticles with a buffer screen (at a set 15 μg/mL loading). 8 buffers were chosen to give the widest range of pH and buffer type. The antibodies that were tested were AF1597, Mab 1597 and NBP-2. When performing the buffer screen, the absorbance for the gold particles was read at 550 and 600 nm, a ratio of 550/600 nm was taken; if the ratio was found to be ≥3.5 then it met the criteria required to be suitable for conjugation. The results for this can be found in Table 1.

TABLE-US-00003 TABLE 1 Table to show the aggregation ratios for varying conjugation buffers at an antibody loading of 15/Ag/mL (15 μg/mL). Aggregation Ratio 550/600 nm MES BES TES TAPS TAPS Borate Borate Borate 6.7 6.9 7.1 7.5 8.5 8.5 9.0 9.3 AF1597 3.2874 4.1212 3.9935 3.9126 4.2276 3.9040 4.0498 3.9773 Mab1597 4.1444 1.4042 2.1059 1.9489 4.2687 2.1553 2.2187 2.5224 NBP-2 4.5191 4.2500 4.0586 2.3903 4.3822 4.3011 4.1679 4.0334
As can be seen from Table 1, 7 of the 8 buffers tested gave an acceptable aggregation ratio for the AF1597 and NBP-2, plus 2 out of 8 were suitable for the Mab1597. The buffer that was chosen to take forward for conjugation was TAPS pH 8.5 as all three antibodies gave an aggregation ratio of >4.2. Once the gold conjugation conditions were established, batches of gold were made and all three were resuspended in gold drying buffer and tested on strips.

Initial Orientation Testing

[0299] The antibodies were then deposited onto nitrocellulose (NC, CN140) by placing a 1 μL drop of 1 mg/mL antibody in a solution of PBS+1% sucrose onto the NC. This allowed for an initial orientation experiment to be performed against the newly conjugated gold nanoparticles. These strips and gold particles are tested with a blank buffer (0 ng/mL PBS) and varying concentrations of recombinant CD16b analyte (in PBS). It should be noted that all 9 combinations of NC and gold were tested but only those combinations that gave a visible response are shown in Table 2.

TABLE-US-00004 TABLE 2 Raw data obtained for hand deposited antibodies (in PBS + 1% sucrose) on CN140 strips with varying gold conjugations at different concentrations of CD16b analyte. AB (CN140) Gold (AB) Concentration (ng/mL) T AF1597 AF1597 0 1.5 10 6.3 100 32 1000 72 Mab1597 AF1597 1000 8.6 NBP-2 NBP-2 0 29 1000 59 Mab1597 0 41 1000 57 AF1597 1000 3
As can be seen from Table 2, when the AF1597 (AF) was on the NC and on the gold it gave the best curve response with a sensitivity of 100 ng/mL being seen.
When the Mab1597 (Mab) and NBP-2 were deposited on the NC and the AF1597 gold was tested, there was no response for a 1000 ng/mL sample. The NBP-2 on the NC and also on the gold showed a high non-specific interaction (spot seen for 0 ng/mL) and no real increase in signal for a 1000 ng/mL sample. The same response was seen when the NBP-2 was deposited on NC and the Mab1597 (Mab) was conjugated to the gold, with a high non-specific interaction seen and no increase in signal to a 100 ng/mL sample. There was some form of aggregation of the gold, which could be seen as a visible red line at the bottom of the NC. To confirm the orientation of the assay and to determine if the AF1597 self-pair was correct, small bands of antibody were deposited using a standard Isoflow machine to plot the antibody onto the NC (10 cm band). This gave a line response which could be more accurately read on the cube reader as opposed to a spot. Additionally, another monoclonal antibody (MM01) was also tested. The results for the Isoflow deposited antibodies can be found in Table 3.

TABLE-US-00005 TABLE 3 Raw data obtained for Isoflow deposited antibodies (in PBS + 1% sucrose) on CN140 strips with varying gold conjugations at two different concentrations of CD16b analyte. NC Gold 0 ng/mL 1000 ng/mL AF1597 AF1597 3.1 91 Mab1597 AF1597 2 6 NBP-2 AF1597 3.6 17 AF1597 Mab1597 2.6 31 Mab1597 Mab1597 45 71 NBP-2 Mab1597 110 156 AF1597 NBP-2 1.5 146 Mab1597 NBP-2 106 72 NBP-2 NBP-2 71 145 AF1597 MM01 1.6 4.8 Mab1597 MM01 47 29
As can be seen from Table 3, when the AF1597 was conjugated to the gold, the best specific signal (high CD16b test response with a low blank background) was seen when the AF1597 was deposited on the NC. It was therefore decided to continue with the development of the AF1597 pair system (using this antibody on both the test line and gold) as it showed the most reproducible results. To confirm the initial result, a buffer curve was run to determine assay sensitivity and range.

Development of AF1597 Self Paired System

Buffer Curve

[0300] To find the initial assay sensitivity and range, CD16b was spiked into PBS at 10, 100, 1,000 and 10,000 ng/mL and the hand deposited strips, Isoflow deposited antibody on the NC with old gold and Isoflow deposited antibody on the NC with new gold conjugates were investigated. The results for the buffer curve can be found Table 4.

TABLE-US-00006 TABLE 4 Raw data obtained for AF1597 antibody deposited by different methods (in PBS + 1% sucrose) with two different batches of gold and varying concentrations of analyte Isoflow/Gold Isoflow/Gold Concentration Hand Plot (old batch) (new batch) 0 1.5 4.3 3.1 10 6.3 4.3 25 100 32 46 59 1000 72 72 91
As can be seen from Table 4, the non-specifics for the AF1597 pair system are low for all three golds tested and all show a specific test line signal over the range tested. For the hand plotted antibody system, the 100 and 1,000 ng/mL gave specific signal; these signals were comparable when tested on the Isoflow plotted antibody system.
Fatty Acid Free BSA Vs. Normal BSA
An optimisation was attempted to see if a change of gold blocking agent could improve assay performance (gain in specific signal). For this, two forms of BSA were tested to block the gold conjugates, these were fatty acid free BSA and normal BSA. The results for the comparison can be found in Table 5.

TABLE-US-00007 TABLE 5 Raw data obtained for AF1597 antibody deposited on CN95 (in PBS + 1% sucrose) with two different blocking agents (fatty acid free and normal BSA) on conjugated gold with varying concentrations of analyte. AB on CN95 Gold (AB) Concentration (in PBS) T AF1597 AF1597 FaF BSA 0 ng/mL 3 10 ng/mL 26 50 ng/mL 73 100 ng/mL 98 200 ng/mL 107 AF1597 BSA 0 ng/mL 1.5 10 ng/mL 27 50 ng/mL 73 100 ng/mL 129 200 ng/mL 108
As can be seen from Table 5, the results for the two blocking agents were very comparable, with both giving a sensitivity of 10 ng/mL and increasing specific signal up to 100 ng/mL.

Increased Antibody Loading on Gold

[0301] The method for conjugating 15, 20 and 30 μg/mL to gold is described above. To determine the effect of a higher loading of the gold conjugates, the same concentrations of analyte were run again. The results for the higher loaded gold conjugates can be found in Table 6.

TABLE-US-00008 TABLE 6 Raw data obtained for AF1597 antibody deposited on CN95 (in PBS + 1% sucrose) with two different blocking agents (fatty acid free and normal BSA) on conjugated gold with varying concentrations of analyte. Antibody loading on gold NC Concentration 15 μg/mL 20 μg/mL 30 μg/mL AF1597 0 ng/mL 1.3 1.7 1.3 on CN95 10 ng/mL 5.4 6.2 15 50 ng/mL 47 54 41 100 ng/mL 96 86 84 200 ng/mL 94 110 100 1000 ng/mL 98 122 137

[0302] As can be seen from Table 6, the 20 μg/mL gold conjugate showed an increase in signal for all concentrations tested when compared to the 15 μg/mL coat; the lowest increase in specific signal was seen between 200 and 1,000 ng/mL. This suggested the higher gold loading gave an improvement in the assay performance by giving a larger range to the assay. This was more apparent when the 30 μg/mL coat was tested, with a specific signal gain at both the low end (10 ng/mL) and an enhanced specific signal gain between 200 and 1,000 ng/m L. This suggested that the best gold conjugate loading concentration at this stage was 30 μg/mL.

Increasing Test Line Antibody Concentration

[0303] As seen above, an increase in antibody loading on the gold conjugate gave a better assay response. To investigate if this could be improved further, the antibody deposited onto the test line was also increased. The test line antibody concentrations investigated were the current 1.0 mg/mL along with 1.5 and 2.0 mg/mL, using the 30 μg/mL gold conjugate; the results are displayed in Table 7.

TABLE-US-00009 TABLE 7 Raw data obtained for AF1597 antibody deposited on CN95 (in PBS + 1% sucrose) at varying concentrations with increasing concentrations of CD16b analyte. Test Line Concentration 13/1 - 14/1 - 15/1 - 15/1 - 15/1 - NC (AB) Concentration 1 mg/mL 1 mg/mL 1 mg/mL 1.5 mg/mL 2.0 mg/mL CN95 0 ng/mL 1.3 3.2 2.1 1.6 2 (AF1597) 10 ng/mL 15 19 26 18 19 50 ng/mL 41 78 93 53 77 100 ng/mL 84 90 107 98 88 200 ng/mL 100 134 148 118 121 500 ng/mL — — 153 114 133 1000 ng/mL 137 135 144 107 135
As can be seen from Table 7, the 1 mg/mL data showed the best response and was very comparable to the previous results obtained. The results suggested that there was enough antibody in the system at 1 mg/m L. As there was no benefit at this stage of increasing the antibody concentration, it was decided to keep the current system with 1.0 mg/mL being deposited on the test line.

Change of Nitrocellulose

[0304] Another change that can improve assay sensitivity and range of a lateral flow assay is the NC membrane used. The majority of the development work had been performed on CN95, so two other NCs were investigated to see if the assay sensitivity and range could be improved. The CN140 and CN180 NCs are slower running membranes with smaller pores and can give increased sensitivity and range by allowing more binding event complexes to be formed on the test line.

TABLE-US-00010 TABLE 8 Raw data obtained for AF1597 antibody deposited on CN95, CN140 and CN180 (in PBS + 1% sucrose) with AF1597 conjugated to gold with varying concentrations of analyte. Nitrocellulose Type Antibody Concentration CN95 (13/1) CN95 CN140 CN180 AF1597 0 ng/mL 1.3 3.2 3.2 2.2 (1 mg/mL) 10 ng/mL 15 19 13 21 50 ng/mL 41 78 74 70 100 ng/mL 84 90 89 92 200 ng/mL 100 134 100 95 1000 ng/mL 137 135 104 99
As can be seen from Table 8, the best results were obtained when CN95 was used as the NC and these results were very comparable to the previous data obtained with the same NC. Whilst giving good specific signal for all concentrations, the CN140 and CN180 gave lower specific signal for both 200 and 1,000 ng/mL. Between 10 and 200 ng/mL, the three NCs gave very comparable specific signal, this suggested that there was no benefit to changing the NC at this stage.
The system taken forward for testing in plasma and blood samples was CN95 with 1 mg/mL AF1597 antibody deposited in PBS+1% sucrose and AF1597 loaded at 30 μg/mL on gold.

Example 2— Detection of CD16b in Spiked Samples

Spiked Plasma Curve

[0305] It was decided initially to see if the CD16b could be spiked and recovered in a simpler matrix than blood. The matrix chosen for this was plasma, with whole blood being drawn and collected in EDTA and lithium heparin tubes. Once collected, the blood was spun down at 1,228x g for 10 minutes, the blood separates into three distinct layers at this point; plasma, buffy coat and erythrocytes. The plasma was collected from the top layer and placed into polypropylene tubes. The analyte (CD16b) was then spiked in the plasma and run on the strips to see if it could be recovered successfully; the results for this experiment can be found in Table 9 and FIG. 1.

TABLE-US-00011 TABLE 9 Raw data obtained for AF1597 antibody deposited on CN95 with varying concentrations of CD16b analyte spiked into either EDTA or lithium heparin collected plasma. Concentration NC (AB) Gold (A/B) Anticoagulant CD16b in Plasma T CN95 AF1597 EDTA 0 ng/mL 37 (AF1597 1 (30 μg/mL) 10 ng/mL 39 50 ng/mL 50 100 ng/mL 60 200 ng/mL 87 1000 ng/mL 103 CN95 AF1597 Lithium 0 ng/mL 13 (AF1597 1 (30 μg/mL) 100 ng/mL 34 1000 ng/mL 55
As can be seen from Table 9 and FIG. 1, there was specific signal gained for 50-1,000 ng/mL CD16b in EDTA plasma (plus a signal was also obtained for 100 and 1,000 ng/mL CD16b in lithium heparin collected plasma). The EDTA plasma showed greater specific signal differential for the CD16b concentrations measured, suggesting it was the better anticoagulant to collect the venous blood.
If fingerstick whole blood is used there may not be a need for an anticoagulant collection method in the final form. It was also noticed that there was some endogenous the soluble form of CD16b in the plasma of the blood sample taken. This is not surprising as neutrophils are known to shed CD16b as part of their turnover in blood. The neutrophils can be found in the buffy coat layer of the blood sample that has been spun down. To investigate the measurement of cellular CD16b, buffy coat isolation is required. This can then be evaluated in the assay, utilising detergent lysis/solubilisation to release any cellular CD16b present.

Example 3—CD16b Detection in Buffy Coat and Cell Preparations Buffy Coat and Histopaque Investigations

[0306] For a reproducible buffy coat collection methodology, a histopaque gradient separation column method was used. This involved carefully layering 2 mL of blood over 1 mL of histopaque and leaving it at room temperature for 145 mins. This then gently separates the erythrocytes from the plasma and white blood cells to leave a plasma/cells mix that can be removed without disturbance of the erythrocytes. See FIG. 2.

[0307] Using the histopaque method, it was possible to take the plasma/cells mix and separate by centrifugation to give plasma and a pellet of white blood cells. As flow cytometry was being used to count the number of cells present, a sample of the cells was investigated along with the plasma/cell mix from EDTA and lithium heparin blood, with and without lysis.

[0308] The lysis of the buffy coat was performed by adding 0.125% Triton x100 to the running buffer (PBS). Another lysis method was also investigated which is the addition of water, which causes lysis of cells by osmosis. The results from this can be found in Table 10.

TABLE-US-00012 TABLE 10 Raw data obtained for AF1597 antibody deposited on CN95 with cells and histopaque plasma/cells mix (BC) collected from lithium heparin and EDTA tubes with and without lysis buffer (TX100 and H.sub.2O). Sample Neat +0.125% +Water Cell Prep (GNvC) 7.5 12 3.5 Histopaque BC EDTA 81 62 60 Histopaque BC Lith. Hep. 58 31 19

[0309] As can be seen from Table 10, when the cells prepared for flow cytometry (so not freshly isolated) were tested in the assay there was a much lower signal compared to plasma. When the cells were lysed/solubilised there was little to no specific signal gain with either Triton or water. The cells provided in this preparation were suspended in a buffer that was preferred for flow cytometry; so it was unclear if this buffer was having a detrimental effect on the assay at this stage. When the buffy coat was removed from the histopaque treated blood, and lysed, there was a drop in specific signal from the neat with both lysis methods. This suggested that the cells need to be collected from the histopaque method and resuspended in the current running buffer (PBS) to see if the lysis was having the desired effect.

[0310] To isolate the cells from the histopaque buffy coat mix, two aliquots were centrifuged at 400×g for 10 minutes to sufficiently pellet the cells. The supernatant was then removed (retained for testing) while the cells were resuspended in the same volume of PBS, one was kept (and labelled “cells”), the other was centrifuged again to completely wash the cells of any the soluble form of CD16b component (labelled “washed cells”). These were investigated with three lysis methods, TX100 (0.125%), H.sub.2O and a cell extraction buffer provided by ABCAM in their ELISA pack. The results for this testing can be found in Table 11 and FIG. 3.

TABLE-US-00013 TABLE 11 Raw data obtained for AF1597 antibody deposited on CN95 with histopaque plasma/cells mix (BC), cells and washed cells collected from EDTA tubes with and without lysis buffer (TX100, H.sub.2O and CEB). Sample Neat +0.125% TX +Water CEB(1%) Histopaque BC 51 54 Histopaque BC supernatant 72 77 Cells 14 27 12 3.1 12 33 Washed Cells 3.7 64 2.5 15 3.6 33 5.5 56
As can be seen from FIG. 3, when the buffy coat from the histopaque was measured, there was a small increase in signal obtained with the supernatant from the cell preparation step. This suggested that there was a small release of CD16b from the cells through the process and these values were not affected from the addition of lysis buffer (TX100). When the cells were isolated and measured in PBS, there was a significant drop in signal compared to the plasma. This suggested that the soluble component of the CD16b was in the plasma and had been measured throughout. When the cells were lysed with TX100, there was an increase in specific signal which was not seen when water and CEB were used as lysis buffers. To see if this effect was real, the cells were again lysed with TX100. This showed an increase in signal again suggesting the effect was real. To conclusively know if lysis was indeed solubilising cellular CD16b and the number of cells present, the washed cells were also tested with all three lysis buffers. The washed cells showed a significant increase in signal from neat to those lysed by TX100. There was a small increase in signal for the CEB but it was not as high as the TX100. The water, again, showed no increase in signal, this suggested that water was not suitable for lysis/solubilisation. The washed cells were repeated twice with and without lysis, and this again showed that a significant increase in signal could be obtained. As the methodology had been established to now measure cellular CD16b, it was decided to look at five further blood donors to confirm the result and also get the cells counted by flow cytometry.

Example 4—CD16b Detection in Cell Preparations

[0311] To confirm the results of Example 3 that the signal obtained from the lysed cells correlated to the number of cells present, five further blood donors were investigated. The cells were prepared and washed as described above. The cells were prepared at different dilutions to gain a better idea of how the cell number changes affect the assay. The dilutions prepared for the first blood sample were 4×, 2×, neat and ½ cells. Blood donors 1113, 1114, 1117 and 1118 were prepared at 2×, neat and ½ cells. To prepare a stock of 2×cells, 800 μL plasma/cell mix was centrifuged, the cells were resuspended in 400 μL PBS. Once the solutions of the cells were prepared, they were split in two and half went to flow cytometry for neutrophil counting and the other half were read in the assay. The results for the five blood donors with the corresponding neutrophil count can be found in Table 12 and FIGS. 4-6.

TABLE-US-00014 TABLE 12 Raw data obtained for AF1597 antibody deposited on CN95 with washed cells collected from EDTA tubes with and without lysis buffer (TX100) and corresponding cell count from flow cytometry. Blood Donor Washed Cell Neat +0.125% TX Cell Count (x1000) 1109 x4 1.7 45 — x2 3 22 1333.5 x1 2.7 11 788 x½ 1.5 7.9 325 1113 x2 4.9 33 6820 x1 4.3 16 1250 x½ 2.6 5.2 670 1114 x2 15 85 6240 x1 9.2 66 4270 x½ 6.6 36 1710 1117 x2 9.5 66 3120 x1 7.5 32 790 x½ 3 16 360 1118 x2 9.5 53 4730 x1 6.3 30 1180 x½ 2.1 15 455
As can be seen from FIGS. 4-6, there is a trend for higher number of neutrophils to generate a higher assay signal, and then this count drops along with assay signal suggesting there is a clear relationship between the two. There is one anomalous point in blood sample 1113 where the x2 sample gave a very high cell count but the assay signal is not as high as the 2×sample for 1114. It was discovered in the testing that the white blood cells in the x2 sample had sedimented prior to flow cytometry so this could be the reason the count is higher. The highest assay signal and corresponding neutrophil count is found in FIG. 5 for blood donor 1114. The cell count from flow cytometry shows a high neutrophil count and the assay signal recorded with these was also correspondingly high. This suggests that the higher the neutrophil count the higher the assay signal will be which is to be expected if more cells are available to be lysed. To gain a better understanding of the relationship between the neutrophil count and the assay signal, correlation graphs were plotted. As blood donor 1113 contained an anomalous cell count for the 2×cells, it was removed from the correlation graph. The results for the cube units against the cell count can be found in FIG. 7.
As can be seen from FIG. 7, there is a good correlation between the test line measurement (cube units) obtained for the lysed cells and the neutrophil count obtained by flow cytometry. As can also be seen from FIG. 7, the correlation value (R2) was 0.861. This suggests a good correlation between the cube units obtained and the cell count.
As a correlation had been found between the cellular CD16b (lysed cells) and the neutrophil count, it was decided to see if there was any link between the plasma levels for the different donors and their cellular levels (lysed cells) and cell count. As the plasma/cell mix would contain the same number of cells as a whole blood samples, the comparison was only made with the values obtained for neat cells. The test line measurement (cube unit) results for the plasma against lysed cells and cell count can be found in Table 13.

TABLE-US-00015 TABLE 13 Raw data obtained for centrifuged plasma samples from five blood donors against cube units for lysed washed cells and the corresponding cell counts obtained from flow cytometry. Donor Plasma Cell Lysis (Neat) Cell Count 1109 47 11 788 1113 184 16 1250 1114 22 66 4270 1117 163 32 790 1118 103 30 1180

Example 5—Blood Transport Study

[0312] To determine how long a blood sample could be stored, processed and used in the assay, an initial stability experiment was performed. The time points for placing two different blood donors onto the Histopaque were 0, 2, 3.5 and 24 hours. The isolated cells were then measured at 2×, neat and ½ concentration using PBS+0.06% triton as lysis buffer. The results for this blood stability experiment can be found in Table 14.

TABLE-US-00016 TABLE 14 Raw data (cube units for CD16b) obtained for different concentrations of washed cells for two donors at 0, 2, 3.5 and 24 hr time points in wet assay system. Lysed Washed Cells (+0.06% TX100) Blood Donor Cell Dilution 0 h +2 h +3.5 h +24 h 1149 x2 25 28 27 17 x1 15 18 16 19 x½ 13 12 11 15 1151 x2 40 31 31 37 x1 36 24 37 23 x1 /2 38 17 16 25
The results show a good reproducibility of results for 0, 2 and 3.5 hour time points. At 24 hours, the results began to plateau suggesting that the blood needed to be processed or sampled prior to this time. From this data it can be determined that the blood can be stored at ambient temperature for 3.5 hours without impact.

Example 6—Further Anti-CD16b Antibody Testing

RP02 Conjugation and Optimisation

[0313] A new antibody (RP02) was identified for testing as it was cheaper than the AF1597 antibody. To determine RP02's suitability, a gold optimisation test was performed with the antibody (as was described in Example 1). The results for the aggregation ratio testing (which informs which buffer is most suitable for conjugation) can be found in FIG. 9. As can be seen from FIG. 9, there was only one buffer that met the criteria for suitable conjugation of RP02 antibody and that buffer was borate pH 9.3. After conjugation, the gold is usually resuspended in a gold drying buffer and tested. In this case, there was no drying buffer that was suitable for the antibody as they caused aggregation after being resuspended. This suggested that RP02 could not be used on the gold and as a result it was deposited onto the test line and tested against AF1597 gold.

RP02 Wet System Testing

[0314] To test the suitability of the RP02 for use on the nitrocellulose, RP02 was deposited onto CN140 nitrocellulose in 1% sucrose and tested against AF1597 AuC (at 30 μg/mL). The initial results showed that the use of RP02 on the test line gave an increase in non-specific binding (NSB) whilst giving a good specific signal increase. In an attempt to lower the NSB, the running buffer was changed from PBS to PBST. The system was also tested using two different CD16b antigens (sourced from Sinobio and R&D systems) which were used to produce the two test antibodies, RP02 and AF1597.
A good standard curve was obtained for both types of CD16b. There was a specific signal gain with 10 ng/ml for both types of analyte. The top end of the assay (1000 ng/ml) also gave good comparability for both CD16b antigens, although differences were observed in mid CD16b range (SO-200 ng/mL), with the R&D systems antigen (AF1597 immunogen) having a lower response when compared to the Sinobio systems antigen (RP02 immunogen). As the AF1597 self-pair system showed good response with washed cells, it was decided to see how the RP02/AF1597 system would compare.
RP02 Testing with Washed Cells
To test the RP02/AF1597 test strips using CN95 NC with lysed washed cells, two blood donors were obtained and the white blood cells processed as described in Example 3. The RP02/AF1597 system was then compared to the AF1597 self-pair to ensure good comparability between the two systems. The various processed components of the blood were also measured to include, plasma (from spun blood), plasma (buffy coat after Histopaque treatment) and different concentrations of the washed cells.

[0315] The cube units obtained for the washed cells at the different concentrations gave very comparable results for both systems. Differences were observed when measuring the different plasmas for the two donors. The AF self-pair system gave very comparable results for the spun and buffy coat plasmas when both donors were measured. The RP02 system, however, showed differences between the types of plasma, with the spun plasma giving a much lower value when compared to the buffy coat plasma.

Initial RP02/AF1597 Dry Down System

[0316] To further the development of the RP02 (CN95 NC)/AF1597 (AuC) test system, the reagents were dried down. Initially, the volume of the gold conjugate (2.4, 4 and 6 μL) to be dried down was investigated to determine the best specific signal gain. The CD16b standards (Sinobio) were initially spiked into PBST and were also spiked into PBS for comparison.
The results show that when CD16b was spiked into PBST, the specific signal was lower than that obtained when spiked into PBS, for all volumes of deposited gold conjugate. When CD16b was spiked into PBS and tested, a good specific signal increase was observed at 200 ng/ml and then 1 μg/ml at the 4 and 6 μL gold conjugate volumes. In contrast with 2.4 μL gold, the curve plateaued with 1 μg/ml giving no increase in signal above 200 ng/ml. At the top end the 4 μl gold gave the best signal in PBS, the signal being reduced at 6 μl. To explore this further, two additional volumes were assessed to see if a better signal could be found.
The 200 ng/ml standard gave very comparable results for all volume of gold conjugate. The 500 ng/ml standard was found to be highest on the 3 μL deposit which was then lower but comparable for both the 4 and 5 μL deposits. The 1000 ng/ml standard was very comparable for the 3 and 4 μL deposits which then decreased at 5 μL of deposit. The best condition was seen with 4 μL of gold deposit as there was good specific signal difference for all concentrations of CD16b tested, comparable to a high concentration of analyte with wet gold conjugate at the same volume. As the 4 μL showed the best performance it was taken forward for further testing with two different conjugate pads (8951 and 8980). Here the AF1597 AuC conjugate was sprayed onto the two pads using an Isoflow plotter.
The results show a good curve was obtained for both conjugates pads in the dry assay system. The 8980 conjugate pad performed better than the 8951 giving a better specific signal across the standard curve range. This suggested that the best conjugate pad for testing in subsequent sections would be the 8980 conjugate pad.

Example 7—Biotin Testing

Initial Biotin System Testing

[0317] To see if the sensitivity of the system could be enhanced, a polystreptavidin-biotin system was evaluated; this can also help reduce costs as antibodies are typically more expensive. In this system, polystreptavidin was plotted on the test line of CN140 NC and the antibodies RP02 or AF1597 conjugated to biotin and AF1597-gold were combined and dotted onto the conjugate pad. In the presence of the CD16b antigen the biotinylated and gold antibodies form a sandwich with CD16b and the resulting complex will then bind to the polystreptavidin test line. This is shown schematically in FIG. 10. A slower CN140 NC was used in this set up to allow more time for the sandwich to form before reaching the test line.
The antibodies RP02 and AF1597 were biotinylated using a Biotinylation Lightning Link Kit (@ 2 mg/ml) and then diluted 1 in 100 to a final concentration of 16.7 μg/ml. In the initial testing, 4 μL of the biotin conjugate was mixed with 4 μL of the gold conjugate and 20 μL of CD16b analyte in PBS.
The results show that a good specific signal was obtained at 10-200 ng/ml CD16b for both the AF1597 and RP02 biotin systems, with both demonstrating a hook effect at 1000 ng/ml. It can also be seen that the Sinobio CD16b analyte, whilst giving the full assay range, showed lower specific signal for the lower end of the curve. As a hooking effect was observed for high concentrations, it suggested that there was a lack of biotin conjugated material and that a higher volume would be required. To test this, 8 μL of the biotin conjugate was used with the same volume of gold.
The results show that when the volume of biotin conjugate was increased from 4 to 8 μL, a better standard curve was obtained. A difference in specific signal was seen between 200-1000 ng/ml suggesting the hook effect observed using 4 μL of conjugate had been overcome. As a good standard curve had been obtained for the biotin system, a comparison to the previous system where antibody is deposited on the nitrocellulose was performed. Here, 4 μL of 1/50 diluted biotin conjugates were used to mimic the 8 μL of 1/100 dilutions used in the previous experiments.
The results show that there is very good comparability between both the biotin and the non-biotin system with both giving a good standard curve. That being the case, the biotin system was dried down to assess its performance and suitability compared to the gold conjugate system.

Biotin Dry Down Testing

[0318] As a dry system is required for the final device, the biotin system was dried down to assess its performance and suitability as a system to be taken forward. As the dry down requires two components (gold and biotin conjugate), it was decided to investigate if the two conjugates could be dried down mixed together or if they needed to be separated on the pad.
The results show that when the biotin and gold conjugates were mixed together and dried down, high non-specific binding was seen along with only small increases in specific signal and poor standard curve resolution. However, when the two were dried down separately on the conjugate pad lower non-specifics were observed for both systems suggesting the mixing of the materials had led to the increase in non-specific signal. The AF1597 biotin conjugate (with AF1597 AuC) showed a very shallow standard curve with low specific signal increase between 10-200 ng/ml. The RP02 biotin conjugate (with AF1597 AuC) showed a better standard curve response with good signal difference seen between 10-200 ng/ml which was comparable to the initial dry down curve for the RP02 antibody on the test line format. Thus, a good proof of concept was achieved for the biotin system.

Example 8— Whole Blood Testing

Lysis Time Course

[0319] As the design of the device requires the lysis of cells isolated from whole blood, it was important to try and determine the time this process would take. It was found that a lysis time of 5 minutes gave a good signal response. This would require a pre incubation of the cells with lysis buffer prior to being run on the lateral flow strip. To determine at which time the cells gave the most consistent signal, a time course was set up sampling at intervals of 0, 1, 3 and 5 minutes. The results for this testing can be found in FIG. 11.
As can be seen from FIG. 11, when the cells are lysed for 5 minutes a good curve is obtained for both blood donors. When the cells are lysed for 3 minutes, a similar curve is obtained for both donors. The biggest difference is noticed for the x2 cells after 3 minutes lysis, where a lower signal was obtained. This could be an anomalous result as this is the lowest signal recorded for all three concentrations for this blood donor. As the results showed that 3 and 5 minutes were the best it was decided to keep 5 minutes as a lysis time for further experiments.

Example 9—Trapping and Lysis of White Blood Cells within the CD16b Lateral Flow Device

[0320] The trapping and lysis of washed white blood cells was demonstrated within the CD16b lateral flow assay utilising a filter in the form of a sample pad membrane under the sample application window to trap the cells. Washed white blood cells from EDTA blood were used. It was determined that a range of filters may be used to trap neutrophils, for example PES10 (2 orientations), MLRF-NANO-1 (LF1) and MLRF-NANO-2 (LF2) or no cell membrane (filter). It was determined that a range of surfactants may be used for the cell lysis step, for example 0.03% TX-100, 0.03125% SDS or 0.0125% Sarkosyl.

Example 10—Dry Assay Standard Curve

[0321] In the dry assay format of the lateral flow test, the test strip was prepared using a CD16b antibody-gold conjugate sprayed and dried onto two different conjugate pads (8951 & 8980; Ahlstrom Munksjo). Dry strips were constructed using conjugate pads at the base, overlapping with a CN95 nitrocellulose membrane (Sartorius) plotted with an anti CD16b antibody (11046-RP02-SIB; Stratech) as a test line, then a sink pad at the top. CD16b standards (Stratech) were spiked into PBS at a range of concentrations from 10-1000 ng/mL. 50 μL of each standard was run up the dry strips followed by 30 μL of PBS and then the test read @ 10 minutes on a cube reader. The results for this testing can be found in Table 15.

TABLE-US-00017 TABLE 15 Raw data obtained for varying concentrations of CD16b in PBS with dried sprayed gold conjugate on two conjugate pads (8951 and 8980) with a 10 minute read time. Standard 10 min read CD16b ng/mL 8951(CP) 8980(CP) 0 5.6 9.6 10 9.8 25 50 32 41 100 55 71 200 65 83 500 95 134 1000 152 157

Example 11—Lactate Assay

[0322] The format of the Lactate assay is based on the enzymatic activity of lactate dehydrogenase. This was achieved by immobilisation of the lactate dehydrogenase enzyme on a nitrocellulose membrane and the inclusion of a dye in the form of a yellow tetrazole (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)) which can be converted to a purple precipitate called Formazan ((E, Z)-5-(4, 5-dimethylthiazol-2-yl)-1, 3-diphenylformazan) in the presence of lactate through the reduction of NAD+ to NADH during its conversion to pyruvate. (NB: other reagents can be used here to form different formazans). The overall proposed format can be found in FIG. 12.

Lactate Assay Standard Curve

[0323] A dry lateral flow assay was constructed using a dye mix (containing MTT) deposited and dried onto a conjugate pad (8951; Ahlstrom), overlapping with a CN95 nitrocellulose membrane with an LDH test line plotted at 1 mg/mL. Sodium Lactate was spiked into PBS at a range of concentrations from 0.1-10 mM and 80 μL of each standard was run up the dry strips (in triplicate) and read at 10 minutes on a cube reader. The results for this testing can be found in Table 16 and FIG. 13.

TABLE-US-00018 TABLE 16 Raw data obtained for varying concentrations of Sodium Lactate, dye mix dried on conjugate pad & triplicate LDH test line signal measured in Cube units. Test Line Signal (Cube Units) Lactate (mM) Rep1 Rep2 Rep3 Mean Stdev % CV 0 18 19 26 21.0 4.36 20.8 0.1 55 61 56 57.3 3.21 5.6 0.5 120 125 144 129.7 12.66 9.8 1 188 134 149 157.0 27.87 17.8 2.5 218 171 210 199.7 25.15 12.6 5 267 266 254 262.3 7.23 2.8 10 301 299 294 298.0 3.61 1.2

Combined Detection of CD16b and Lactate

[0324] The strip design for whole blood requires a blood separator to retain the cells and stop them being lysed prior to running the CD16b portion of the assay. This blood separator will allow the plasma to be run through the strip which will give the lactate assay result. The initial volume of whole blood to be tested was 20 μL with an additional 50 μL of PBS to potentially fully wash all the plasma from the blood (and therefore from the sample pad). It was hoped that the higher volume of blood would give a higher volume of plasma, and thus, would give a better lactate response.

[0325] The results show that when 20 μL of blood is used on FRI separator pad, there is little to no haemolysis onto the conjugate pad which suggests that there is little to no lysis of the blood when running the lactate part of the assay. It can also be seen that after 5 minutes there is a clear response for both the 0 mM (very feint) and 10 mM spiked lactate which is stronger and more intense for the higher concentration. After 10 mins, the response for the 10 mM sample had increased in intensity with a darker and wider line seen. The 0 mM, however, showed a small increase in response but was very similar to that at 5 mins. As the volume of whole blood could be lower for the final device (finger prick volume can be low), it was decided to investigate if a lower volume of whole blood with a higher volume of PBS would still give a response whilst flushing the plasma through the strip better. The results for this testing can be found in FIG. 14.

[0326] As can be seen from FIG. 14, when 10 μL of whole blood was used with 60 μL of PBS to wash through the plasma there was again little to no haemolysis onto the conjugate pad. This again suggested that even with a lower volume of blood and higher volume of PBS the cells are unaffected. It can also be seen that there is also a clear difference in intensity at 5 minutes between the 0 mM and 10 mM spiked lactate which becomes more apparent at 10 minutes. It can also be seen that there is little to no movement of the formazan product from the test line. These results suggest that it is possible to measure the lactate content of a blood sample whilst also retaining the cells that can then be transferred to the second strip for measurement of CD16b.

Example 12— Comparative Data for Correlation of the Levels of Soluble CD16b Vs. Membrane-Anchored and Intracellular CD16b with the Number of Neutrophils in a Sample

[0327] Blood samples were obtained from 11 individuals. Levels of soluble CD16b were examined for correlation with the number of neutrophils. Separately, levels of membrane-anchored and intracellular CD16b (after removal of soluble CD16b and subsequent cell lysis) were examined for correlation with the number of neutrophils. The results show that the cell-associated (intracellular and membrane-anchored) CD16b positively correlates with neutrophil levels (FIG. 15), while soluble CD16b negatively correlates with neutrophil concentration (FIG. 16). The data shows better correlation between the membrane-anchored and intracellular CD16b and neutrophil counts than the soluble CD16b and neutrophil counts (r-squared correlation coefficient of 0.53 (intracellular and membrane-anchored) vs. 0.41 (soluble)). After correction for outliers (3 samples), the R-squared correlation coefficient was 0.8014 for intracellular and membrane-anchored CD16b and 0.3478 for soluble CD16b samples.
The correlation of membrane-anchored and intracellular CD16b with the number of neutrophils is substantially better than the correlation of soluble CD16b with the number of neutrophils. Thus, detection of the levels of membrane-anchored and intracellular CD16b provides a more accurate method for determining the number of neutrophils in the sample.

Example 13—Typical CD16b Standard Curve in a Wet Assay Format

[0328] In the wet assay format of the lateral flow test, strips were constructed using a CN95 nitrocellulose membrane (Sartorius) at the base, plotted with an anti CD16b antibody (AF1597) as a test line, then a sink pad at the top. CD16b standards (R&D Systems) were spiked into PBS at a range of concentrations from 5-200 ng/mL. 20 μL of each standard was mixed with 2.4 uL of AF Gold conjugate mixed and run up the wet strips followed by 30 μL of PBS and then the test read @ 10 minutes on a cube reader. The results for this testing can be found in Table 17 and FIG. 1.

TABLE-US-00019 TABLE 17 Raw data obtained for varying concentrations of CD16b in PBS in a wet assay format with a 10 minute read time. CD16b Standard (ng/mL) Cube Units 0 (PBS) 5.0 5 21 20 78 50 123 100 174 200 225