RAPID TEST FOR CELLULAR FIBRONECTIN
20200284799 ยท 2020-09-10
Assignee
Inventors
Cpc classification
G01N2333/78
PHYSICS
International classification
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a rapid assay for detection of human cellular fibronectin (c-Fn) where ELISA-based assays have previously been developed for detecting and measuring cellular fibronectin in biological fluids, but these methods are too time-consuming for practical clinical diagnostic use. The assay of the present invention enables prediction of bleeding events on a rapid timescale. Described as well are high affinity human monoclonal antibodies, particularly those directed against isotopic determinants of cellular fibronectin (c-Fn), as well as direct equivalents and derivatives of these antibodies. These antibodies bind to their respective target with an affinity at least 100 fold greater than they do to plasma fibronectin, and enable assays to be created that detect c-Fn in less than 30 minutes in a variety of detection formats, and in some detection formats less than 15 minutes. These antibodies are useful for diagnostics, particularly prediction of bleeding events, prophylaxis and treatment of disease.
Claims
1.-22. (canceled)
23. A rapid assay for the prediction of bleeding in a human test subject that determines in 60 minutes or less the level of human cellular fibronectin (c-Fn) in a test sample taken from the human test subject; wherein the test sample is selected from the group consisting of whole blood, serum, and a plasma sample, wherein said rapid assay comprises (a) obtaining a test sample from the human test subject, (b) mixing the test sample with an antibody and a reference epitope, wherein the antibody is P4F6, P3A3, P1H1, 19B12-3H5, IST-9 or FN-3E2 and wherein the antibody binds to c-Fn with an affinity at least 100 fold greater than it binds to plasma fibronectin and the antibody binds to amino acid sequence 1631-1721 of the c-Fn molecule (the EDA region), wherein the reference epitope is a synthetic polypeptide consisting of an amino acid sequence selected from the group consisting of TABLE-US-00001 (SEQIDNO:1) (i) DGEEDTAELQGLRPGSEC, (SEQIDNO:2) (ii) ESPQGQVSRYRVTYSSPEDC, and (SEQIDNO:3) (iii) HDDMESQPLIGTQSC wherein either (1) said antibody or an antigen-binding portion thereof are bound to magnetic particles and said reference epitope is labeled or (2) said reference epitope is bound to magnetic particles and said antibody or an antigen-binding portion thereof are labeled, and wherein said assay is a competitive assay where the c-Fn antigen of the test sample competes with the reference epitope for the binding sites on said antibody, (c) forming a complex between the antibody and either said EDA region of the c-Fn of the test sample or the reference epitope of (i), (ii) or (iii), and (d) measuring the amount of labeled epitope bound to the magnetic particles or labeled antibody or labeled antigen-binding portion thereof bound to the magnetic particles, and wherein the obtaining, mixing, forming and measuring steps occur in 60 minutes or less.
24. The rapid assay according to claim 23, wherein said isolated human monoclonal antibody is IST-9.
25. The rapid assay according to claim 23, wherein said isolated human monoclonal antibody is P3A3, P1H1 or P4F4.
26. The rapid assay according to claim 23, wherein said isolated human monoclonal antibody is P4F4.
27. The rapid assay according to claim 26, wherein said isolated human monoclonal antibody is P4F4 with Goat Anti-Mouse IgG ALP as detective enzyme.
28. The rapid assay according to claim 23, wherein the amount of bound labeled antibody or bound labeled reference epitope is inversely proportional to the amount of human cellular fibronectin levels in the test sample and the amount of human cellular fibronectin levels in the test sample is predictive of bleeding in a human test subject.
29. The rapid assay according to claim 23, which does not require the antibody to be fixed to a solid matrix to immunoreact with the EDA region of the c-Fn molecules or the reference epitope in said test sample.
30. The rapid assay according to claim 23 wherein the amount of the labeled antibody bound to either said EDA region or the labeled reference epitope of (i), (ii) or (iii) in the test sample is determined in 20 minutes or less.
31. The rapid assay according to claim 23 wherein the test subject has a neurological disease, cardiovascular disease and/or is a candidate for anti-coagulant therapy and the rapid assay is predictive of future bleeding following treatment of the test subject for these diseases or anti-coagulant therapy.
32. The rapid assay according to claim 31, which can determine the level of human cellular fibronectin in 20 minutes or less.
33. The rapid assay according to claim 23, wherein said assay is a fluidic assay using paramagnetic beads free flowing in liquid media.
34. The rapid assay according to claim 23, wherein c-Fn is determined in said test sample in the range of 0-20 mg/mL.
35. The rapid assay according to claim 23, wherein the antibody is labeled.
36. The rapid assay according to claim 23, wherein the reference epitope is labeled.
37. A rapid assay according to claim 23, wherein said antibodies immunoreact with said synthetic polypeptide and denatured human cellular fibronectin, but do not substantially immunoreact with human plasma fibronectin in either the denatured or native states.
38. A rapid assay according to claim 37, wherein said antibodies also immunoreact with native, non-denatured human cellular fibronectin.
39. A rapid assay according to claim 23, wherein a FastPack IP system is used.
40. A rapid assay for the prediction of bleeding in a human test subject that determines in 60 minutes or less the level of human cellular fibronectin (c-Fn) in a test sample taken from the human test subject; wherein the test sample is selected from the group consisting of whole blood, serum, or a plasma sample, wherein said rapid assay comprises (a) obtaining a test sample from a human test subject, (b) mixing the test sample with an isolated human monoclonal antibody, or an antigen-binding portion thereof, or both the antibody and its antigen-binding portion, wherein the antibody is P4F6, P3A3 or P1H11, and wherein the antibody and/or or the antigen-binding portion thereof binds to c-Fn with an affinity at least 100 fold greater than it binds to plasma fibronectin and wherein the antibody or the antigen-binding portion thereof binds to an amino acid sequence, selected from the group consisting of TABLE-US-00002 (SEQIDNO:1) a) DGEEDTAELQGLRPGSEC, (SEQIDNO:2) b) ESPQGQVSRYRVTYSSPEDC, (SEQIDNO:3) c) HDDMESQPLIGTQSC, and (SEQIDNO:4) d) NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYS SPEDGIFIELFPAPDGEEDTAELQGLRPGSEYTVSV VALHDDMESQPLIGTQSTA, (c) forming complexes between the antibody and c-Fn in the test sample, mixing the complexes with coated paramagnetic particles that bind the antibody, and (d) detecting the complexes in the test sample, wherein the obtaining, mixing, forming and detecting steps occur in 60 minutes or less.
41. A rapid assay according to claim 40 which does not require the antibody be fixed to a solid matrix to immunoreact with the c-Fn in said test sample.
42. The method of claim 40, wherein the complexes of antibody and c-Fn in the test sample are detected in a sandwich assay using a labeled antibody, wherein the amount of a labeled antibody bound to the complexes is detected.
43. A rapid assay according to claim 42, wherein the amount of labeled antibody bound to the complexes in the test sample is indicative of c-Fn levels in the test sample, and the c-Fn level is predictive of bleeding in the human test subject.
44. The rapid assay according to claim 31 wherein the neurological disease is Parkinson's disease.
45. The rapid assay according to claim 31 wherein the neurological disease is Alzheimer's disease.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The drawings are described as follows:
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DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0035] As used herein, regardless of whether fragments or portions are specified, the term antibody as used herein includes such fragments or portions as well as single chain forms unless otherwise indicated. As long as the protein retains the ability to specifically or preferentially bind the EDA region of c-Fn molecule and includes a sequence or sequences disclosed herein, it is included within the term antibody. An antibody may be derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone. Preferably an antibody of the invention exists in a homogeneous or substantially homogeneous population. An antibody can be intact, comprising complete or full length constant regions, including the Fc region, or a portion or fragment of such an antibody provided that any shortened form comprises the antigen-binding portion and retains antigen-binding capability. Such shortened forms include, e.g., a Fab fragment, Fab fragment or F(ab) 2 fragment that includes the CDRs or the variable regions of the anti-c-Fn antibodies disclosed. Furthermore, such shortened antibody forms can be a single chain Fv fragment that may be produced by joining the DNA encoding the LCVR and HCVR with a linker sequence. (See, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, N.Y., pp 269-315, 1994). Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art.
[0036] The term >Antigen refers to antigenic determinants, whether natural or synthesized, for which the analyte antibodies are specifically reactive when used according to the present invention. Synthesized antigens include those which are constructed according to conventional chemical syntheses as well as those constructed according to recombinant DNA techniques. Antigen materials may also be labeled with colloidal particles, fluorescent markers or chemi-luminescent particles according to the invention and used in sandwich type assays for the detection of antibody analytes or in competition assays for the detection of antigen analytes.
[0037] The term label as used herein refers to any molecule or particle bound or conjugated to a specific binding member, or general marker protein which can produce a signal. A label can also include a substrate capable of producing a visually detectable signal when reacted with an enzyme conjugated to the general marker protein. In one embodiment, the label is preferably a direct label which is capable of spontaneously producing a detectible signal without the addition of ancillary reagents and will be easily detected by visual means without the aid of instruments. For example, colloidal gold particles can be used as the label. Other suitable labels may include other types of colloidal metal particles, minute colored particles, such as dye sols, and colored latex particles. Many such substances will be well known to those skilled in the art.
[0038] The term human engineered antibody refers to an antibody having frameworks, hinge regions, and constant regions of human origin that are identical with or substantially identical (substantially human) with frameworks and constant regions derived from human genomic sequences. Fully human frameworks, hinge regions, and constant regions are those human germline sequences as well as sequences with naturally-occurring somatic mutations. A human engineered antibody may comprise framework, hinge, or constant regions derived from a fully human framework, hinge, or constant region containing one or more amino acid substitutions, deletions, or additions therein. Often, a human engineered antibody is preferably substantially non-immunogenic in humans.
[0039] The term prediction is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive, following administration or treatment with by a surgical procedure. The predictive methods of the present invention are valuable tools in predicting if a patient is likely to suffering bleeding side effects to a treatment regimen or surgical intervention.
[0040] As used herein, the term Rapid Assay refers to be an assay that can be performed in less than 60 minutes and preferably less than 20 minutes.
[0041] The term treatment as used herein is defined as the management of a patient through medical or surgical means. The treatment improves or alleviates at least one symptom of a medical condition or disease and is not required to provide a cure. The term treatment outcome as used herein is the physical effect upon the patient of the treatment.
[0042] The term sample as used herein indicates a patient sample containing bodily fluid from a human patient, most preferably from whole blood, blood plasma, or blood serum.
2. Detailed Description
2.1 Antibody (Ab) Development and Evaluation
[0043] We used several methods of antibody development. The first two methods we tried yielded antibodies that could recognize their antigen to which it was screened against, but had weak affinity to or was unable to recognize the whole c-Fn molecule. Three additional methods were tried. The five different antibody development methods are listed below along with the antigens used in generating the antibodies.
Methods Used Included:
[0044] Panning of ABD Serotec HUCAL library using Peptide A1 [0045] Standard immunization of mice against whole c-Fn molecule, negatively screened against binding to whole p-Fn molecule. [0046] Panning of ABD Serotec HUCAL library using Full ED-A region [0047] Standard immunization of mice against 3 peptide mixture (B1, B2, B3) [0048] Standard immunization of mice against peptide A2 and Full ED-A domain
[0049] A positive Ab would show strong positive binding to the c-Fn whole molecule and zero binding to p-Fn. Ultimately 4 clones met this criteria and were selected for purification and continued testing, along with a commercially available IST-9 Ab.
[0050] Anti-c-FN Ab's passing screening included: [0051] P4F6 [0052] P3A3 [0053] P1H11 [0054] 19B12-3H5 [0055] commercially available IST-9
[0056] After extensive affinity testing, three capture antibodies a) anti-peptide P3A3, b) anti-peptide P4F6, and c) commercially available anti-cFN clone IST-9 were evaluated as capture antibodies (coated under various conditions onto microplates) with a Rabbit anti-Fibronectin detection system. Currently there is one commercially available c-Fn ELISA kit, manufactured by BIOHIT, however the results of that assay had many limitations in linear range, accuracy, controls, and the sample medium. Development of an accurate ELISA kit was thus a high priority for correlation to the eventual rapid immunoassay.
[0057] Overall the test results demonstrated a P4F6 capture ab with rabbit anti-Fibronectin while a secondary ab was identified as the best combination 1) specific for c-Fn with no cross-reactivity with fibronectin 2) low background with zero standard, and 3) adequate spike and recovery values.
[0058] Initial testing has demonstrated that the described antibody pairs provided acceptable results in our relevant ranges 0, 0.10-20.0 mg/mL; 1) acceptable discrimination between zero and low-end standards (sensitivity), 2) recognition of c-Fn with no cross-reactivity with fibronectin and 3) P4F6 is able to recognize c-Fn in both serum and plasma.
[0059] After considerable testing we optimized condition and buffers and were able to obtain more than acceptable spike and recovery in various blood, serum, plasma matrices, consistent % CV with both standards and samples. As a result of this process, we standardized the controls and are able to make c-Fn ELISA kits that are superior to the current commercially available kit from Biohit in both linearity and accuracy.
[0060] Testing compatibility with POC platform specific properties and components.
[0061] There is immense difficulty in obtaining a quality antibody to c-Fn is due to two key reasons. The ED-A region, which is present in c-Fn and not present in p-Fn is not a very prevalent region for antibody binding. Additionally due to this small prevalent region for binding, it is impossible to get ab pairs specific just for c-Fn with high affinity. Thus one of the ab's in an ELISA system would be non-specific. Additionally, we discovered that c-Fn degrades rapidly and or loses ED-A antigenicity quickly and with freeze thaw cycles.
[0062] The inability to get 2 separate c-Fn specific monoclonal abs to bind as a pair posed a significant problem due to the abundance of p-FN in blood. The 100 fold greater abundance of p-Fn would remove the secondary ab and interfere with detection in this system. This is not a problem in the ELISA because of the wash step that removes all unbound p-Fn prior to the non-specific ab being applied. Thus because of these reasons, we chose Qualigen, Inc.'s FASTPACK IP System as a platform upon which to demonstrate the instant invention.
[0063] Both competitive and sandwich type chemiluminescence assays may be run on the FASTPACK IP System, which has six separate chambers in a flat package attached to an injection port in which the blood/sera/blood plasma sample is inserted in Chamber 1. In a sandwich assay, a sample with an unknown concentration of the analyte is mixed with excess amounts of capture antibody and labeled antibody. The mixture is incubated for a specified time to allow both antibodies to bind to the analyte in a sandwich format (in Chamber 2). The mixture is then brought into contact with coated paramagnetic particles, which bind to the capture antibody (and thus the analyte). The analyzer uses a small magnet to hold the paramagnetic particles with their attached sandwiched analyte while they are washed repeatedly (from Chamber 4). The wash removes any unbound antibody (all done in Chamber 3) to the waste chamber (Chamber 5). Finally, a substrate solution (from Chamber 6) is added, which reacts with the labeled antibody and emits light, which is directly proportional to the concentration of the analyte in the sample (all in Chamber 3).
[0064] Because the FASTPACK IP System is a fluidic test, there were no physical mediums (ie. sample pad, conjugate pad, and lateral flow strip) to test with our antibody other than the plastic pouch, and chemiluminescence reagents.
[0065] To overcome the problem of c-Fn molecule stability, we lyophilized our c-Fn controls and standards. We then tested them in extensive stability studies versus in solution and concluded that the lyophilized controls were stable. For eventual distribution and use of our ELISA and rapid assay, these would be reconstituted fresh.
3. Examples
[0066] The present invention will be better understood with reference to the specific embodiments set forth in the following examples.
3.1 Example I
[0067] In this example, we demonstrate a competitive assay format for the rapid test of c-Fn. Two basic competitive assay formats were employed during development. In one format (
Materials and Methods
Raw Materials
[0068] Materials are given in
Biotin and Conjugate Methods
[0069] Table 1 summarizes the biotinylations and alkaline phosphatase conjugations performed. Biotinylations of cFn antigen were performed using PEG4 Biotin at antigen:biotin molar ratios of 1:5, 1:10, 1:20 and 1:50. Biotinylations of P4F6 antibody were performed using PEG4 Biotin at antibody:biotin ratios of 1:5 and 1:20. Biotinylations of P1H1, and P3A3 were performed using PEG4 Biotin at antibody:biotin ratios of 1:20. Alkaline phosphatase conjugations were performed to the enzyme alkaline phosphatase (ALP) according to Qualigen protocol for enzyme-Ab conjugation utilizing SMCC and SPDP heterobifunctional reagents.
FASTPACK Reagents and Assay Protocol
[0070] Reagent packs used in the FASTPACK system were filled on Filler II. During development, multiple combinations of biotinylated and ALP conjugated reagents were tested. A summary of the components follows: [0071] Antibody solution (100 L/pack) containing ALP conjugate (antigen, peptide or antibody) in buffer solution. [0072] Paramagnetic particles (150 L/pack): Speedbead PMP (Seradyne) 0.5 mg/mL coupled to streptavidin, containing either biotinylated antigen, biotinylated peptide or biotinylated antibody in buffer solution. [0073] Wash solution (2.1 mL/pack): Tris buffer with detergents. [0074] Substrate (140 L/pack): Immuglow Plus.
[0075] Table 2 summarizes the basic FASTPACK assay protocol. As one can see, the total time is approximately 18 minutes to completion.
Development and Optimization Studies
[0076] Screening Studies
[0077] Antigen, Peptide and Antibody Screening
[0078] Biotinylated antigen, peptide and antibody were paired with corresponding antibody, antigen and peptide alkaline phosphatase (ALP) conjugates to determine pairing combinations that generated optimal assay performance. Table 3 below details the biotin and conjugate pairings tested during development. Pairs were tested for maximum assay response and % relative displacement of spiked samples containing known concentrations of cFn. Results from these analyses are detailed in Appendix 1 attached to this report. From these screening evaluations, two pairs were ultimately chosen for continued evaluation: [0079] 1) EDA-Fc Biotin/P4F6 Antibody Conjugate [0080] 2) cFn Biotin/P4F6 Antibody Conjugate
a) Buffer Study
[0081] Various pH buffers, detergents, proteins and other additives were tested in the cFn FASTPACK assay in order to optimize stability, response and % relative displacement of cFn antigen. The Coupling buffers were used in the coupling of the biotin solution to the paramagnetic particles (PMP) and the Conjugate buffers were used to dilute the ALP conjugates.
[0082] The Coupling and Conjugate buffer compositions are detailed below: Coupling buffers:
#1) 0.1M TBS pH 8+0.1% Prionex+0.02% Tween 20
#2) 25 mM PBS pH 6+1% BSA+0.1% Prionex+0.02% Tween 20
#3) 25 mM PBS pH 6+0.1% Prionex+0.02% Tween 20
#4) BBC pH 8 (Tris+3% BSA)+0.02% Tween 20
[0083] #5) BBC pH 8 (Tris+3% BSA)+0.25 mg/mL -Cyclodextrin+0.02% Tween 20
#6) BBC pH 8 (Tris+3% BSA)+5% Trehalose+0.02% Tween 20
[0084] Conjugate buffers;
#1) PBS pH 6+1% BSA
#2) PBS pH 6+0.1% Prionex
[0085] Each combination of Coupling buffer and Conjugate buffer was tested on Nov. 22, 2011 and on Nov. 28, 2011, to demonstrate stability over a period of 6 days. Table 4 below contains data from the study including RLU of Buffer Blank and c-Fn fibronectin at 10 g/mL.
[0086] The results from the Coupling/Conjugate buffer study indicate instability with the addition of BSA in buffer; causing a dramatic decrease of response over a period of 6 days using a Coupling buffer containing 3% BSA. The % displacement of the c-Fn is not significant using any of the buffer combinations, therefore the optimal buffer combination was chosen based upon maximum response and stability. The following details the buffer compositions determined to generate optimal response and stability:
Coupling buffer=25 mM PBS pH 6+0.1% Prionex+0.02% Tween 20
Conjugate buffer=PBS pH 6+0.1% Prionex
[0087] In an attempt to optimize cellular fibronectin (cFn) antigen displacement in the assay, several sample pretreatment studies were performed that included the testing of various pH buffers, protein, detergents, reduction agents and other additives.
i)
Sample Buffer Additive Experiment
[0088] Known concentrations of cellular fibronectin (cFn) sample were treated with various buffer additives and tested in the assay, comparing the response generated to buffer blank containing no cFn. A 2 stock solution of additive was prepared in PBS pH 6 buffer Immediately prior to testing, the sample (blank or cFn) was diluted 1:2 with the appropriate 2 stock buffer. Table 5 details the buffer additives and responses generated.
SDS Sample Pre-Treatment
[0089] This study details the effects of using SDS (Sodium Dodecyl Sulfate) as a sample pre-treatment. SDS was chosen for its ability to change protein structure; thereby creating opportunity for improved cFn binding. SDS 2 stock buffers were prepared in PBS pH 6, 0.1% Prionex, 0.02% Tween-20 buffer. Samples were diluted 1:2 in each SDS buffer immediately prior to testing. Table 6 shows data from initial SDS titer screen. Table 7 shows data from narrow SDS titer screen.
[0090] SDS used as a sample pre-treatment appears to improve cFn displacement as compared to samples with no pre-treatment. Comparing response and displacement ratios, SDS at 0.05% (SDS 0.1% stock) appears to be the optimal concentration for use as a sample pre-treatment.
pH Buffers
[0091] Two studies are detailed for comparison of pH in assay and sample (control) buffers. One study compared pH 6 and pH 8 buffers used in PMP coupling, conjugate and sample buffers (Table 8). Another study compared the use of sample buffers at pH 4, 5 and 6 containing SDS additive (Table 9).
[0092] Based upon the data generated, the assay appears to be pH and/or buffer dependent. A very low assay response was generated using TBS pH 8, whereas buffers tested at pH 6 and below generated acceptable assay response. The data form Table 9 shows an increasing assay response with a decreasing buffer pH. The displacement ratio is optimal using Phosphate pH 6 as assay and control (sample) buffer.
Comparison of ALP Labeled Vs Unlabeled P4F6 Antibody
[0093] In order to optimize assay response and reduce effects of conjugate instability, ALP labeled P4F6 Antibody (conjugate) was compared to unlabeled P4F6 antibody using Goat
[0094] Anti-Mouse IgG (Fab specific) (GAM) ALP as detection enzyme. Stability of the ALP conjugated P4F6 antibody has not yet been determined, however day to day variability has been noted. Table 10 details data from this comparison.
[0095] The use of the unlabeled P4F6 antibody with Goat Anti-Mouse ALP as detection enzyme greatly improves assay response and the displacement ratio is marginally improved. Further studies were performed (results not listed) using the P4F6 unlabeled antibody with GAM ALP detection enzyme.
Monoclonal Anti-Cellular Fibronectin Clone FN-3E2
[0096] A monoclonal antibody to cellular fibronectin was purchased from Sigma and tested in the assay to compare to current anti-cFn clone P4F6 antibody. Both antibodies were detected using Goat Anti-Mouse IgG (Fab) ALP. Assay response and cFn displacement was compared. Table 11 details results of this study.
[0097] Although an acceptable assay response was generated using the purchased monoclonal anti-cFn antibody, no cFn displacement was apparent. Therefore no improvement is apparent as compared to the current P4F6 antibody.
Assay Incubation Study
[0098] Various assay incubation times were tested in order to optimize assay response and cFn displacement. The FASTPACK assay system includes 2 incubations; the first is a sample/antibody incubation and the second incubation occurs during the mixing of sample/antibody and Biotin coupled streptavidin PMP immunocomplex. Table 12 shows assay responses and displacement ratios for the combinations of incubations tested.
[0099] Optimal response and displacement appears to be generated from the use of 1.5 minute sample/antibody incubation with 5 mixes (approx. 4 min) PMP/antibody complex incubation.
Optimized Assay
[0100] The following is a summary of the optimized cFn Competitive Format FASTPACK Assay:
Antibody solution (100 L/pack) contains: P4F6 anti-cFn monoclonal antibody (0.5 g/mL) with Goat anti-Mouse IgG (Fab specific) ALP (1:2000 dilution) in PBS pH 6 with 0.1% prionex and 0.02% Tween 20.
Paramagnetic particles (150 L/pack): cFn Biotin (2 g/mL) coupled to Speedbead streptavidin PMP (Seradyne) at 0.5 mg/mL in PBS pH 6 with 0.1% prionex and 0.02% Tween 20.
Substrate (140 L/pack): Immuglow Plus.
[0101] Wash Solution (2.1 mL/pack): Tris buffer with detergents.
[0102] An assay protocol with a total time of approximately 7 minutes was employed. This protocol consists of a 1.5 minute incubation of sample (100 L pre-treated in buffer containing SDS) with antibody solution, followed by mixing of the immunocomplex with c-Fn biotin coupled streptavidin PMP (5 mixes/approx. 4 min). The final steps include a 3 wash and substrate read. The sample pre-treatment is performed immediately prior to testing. Samples and blanks are diluted 1:2 in SDS 0.1% buffer, and immediately loaded into packs.
[0103] Titers of c-Fn were tested in duplicate using assay conditions detailed above. The mean, standard deviation, % CV and displacement ratios were calculated. Table 13 details results of the study. See
4. Conclusion
[0104] Using materials supplied to Qualigen by Prediction BioSciences, as well as purchased reagents, an immunoassay for cFn in competitive assay format was optimized on the FASTPACK System, with a total incubation time of approximately 7 minutes.
[0105] Although the EDA-Fc peptide generated acceptable response in the assay, the cFn native whole molecule was chosen in the final assay configuration due to the improvement in the ability of cFn in sample to displace labeled whole molecule cFn versus labeled EDA-Fc peptide.
[0106] A sample pre-treatment step using sodium dodecyl sulfate (SDS) must be employed in order to generate cFn displacement. In the competitive assay format, the assay response is inversely related to the amount of c-Fn in the sample. The decrease in response generated with a sample of c-Fn as compared to a sample containing no cFn is considered displacement.
Example II
[0107] In this example, we demonstrate a sandwich assay for the c-Fn rapid test using paramagnetic bead free flowing in liquid media. Experiments performed were: preparation of biotin-monoclonal antibody, polyclonal antibody coupled to the enzyme alkaline phosphatase, assay optimization and Master Standard Curve generation. In addition, the performance characteristics of the assay were evaluated for linearity, sensitivity and imprecision. Although the P4F6 antibody proved to be the best in the ELISA system, that doesn't always translate to a fluidic rapid immunoassay system, thus 3 c-Fn monoclonal antibodies were tested. Fibronectin, from human foreskin fibroblast, was purchased from Sigma Aldrich P/N F2518 L/N 031M4065V.
[0108] Anti-cFn monoclonal antibody was biotinylated using PEG4-Biotin (5:1 ratio of biotin:Ab); anti-cFn rabbit antibody was conjugated to the enzyme alkaline phosphatase (RIgG-ALP) according to Qualigen's internal protocol for enzyme-Ab conjugation utilizing SMCC and SPDP heterobiofunctional reagents.
[0109] Reagent packs filled on Filler III with the following components:
Antibody solution (100 L/pack) contains: IgG-Biotin (2.5 g/mL) and RIgG-ALP (1.5 g/mL) in Qualigen antibody solution containing PBS with stabilizers and 0.05% Tween 20.
Paramagnetic particles (150 L/pack): Speedbead PMP (Seradyne) at 0.5 mg/mL in Tris buffer containing 3% BSA and 0.02% Tween 20.
Substrate (140 L/pack): Immuglow Plus.
[0110] Wash Solution (2.1 mL/pack): Tris buffer with detergents.
[0111] An assay protocol with a total time of approximately 11 minutes was employed for the evaluation of cFn on the FASTPACK System. This protocol consists of a 5 minute incubation of sample (25 L) with antibody solution, then mixing the immunocomplex with the PMP-streptavidin (5 mixes for a total time of 2 minutes), followed by 3 washes. Finally, the substrate is added to the reactant and read for 1 minute. See outline protocol in Table 2. Standards: Standards were prepared by diluting cFn antigen in a BSA-based matrix (Qualigen Multi Analyte Stabilizing Solution). Standards were prepared at the following concentrations: 0, 0.25, 0.5, 1.0, 2.5, 5.0, 6.0, 7.5 and 10 g/mL. The standards were stored at -20C until use.
[0112] Controls: Three levels of cFn controls were prepared in Qualigen Multi Analyte Stabilizing Solution for use in the imprecision study.
[0113] Linearity Samples were prepared by spiking cFn into delipidated human serum (Goldenwest cat #420-1L LN 08E1206), then interdiluted with unspiked serum.
[0114] Sensitivity: cFn concentrations of 0.0, 0.25, 0.5 and 1.0 g/mL were prepared. Each level was tested in 20 replicates on 3 FASTPACK Analyzers for a total of 60 determinations.
[0115] All the antibodies provided by Predication BioSciences were biotinylated and enzyme conjugated. The screening experiments showed that all of the monoclonals paired with the rabbit antibody. None of the mono mono pairs gave sufficient signal for use in the evaluation; therefore this viability study is based on the use of the monoclonal P4F6 biotin with RIgG-ALP.
[0116] A total assay time of 11 minutes was employed in this study. The first incubation step consists of mixing the antibodies (mIgG-biotin and RIgG-ALP) with sample.
[0117] A standard curve was generated by testing each cFn standard in five replicates, on six different FASTPACK Analyzers. A total of 30 replicates per standard level were generated. The RLU values were then normalized using a standard level at a concentration of 5 g/mL and the trimmed mean was calculated for each standard level.
[0118] We demonstrated the viability of a chemiluminescent immunoassay for c-Fn on the FASTPACK System, with a total incubation time of approximately 11 minutes. The assay dynamic range tested was 0.0 to 10.0 g/mL, with a detection limit of 0.31 g/mL.
[0119] Assay imprecision was tested by analyzing controls prepared in-house on multiple FASTPACK Analyzers. The within-analyzer and total imprecision were 4.7% and 14.9%, respectively. Assay linearity was tested by spiking a high standard into a patient serum sample, which was then diluted.
[0120] It will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense. It will further be understood that it is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.
Derivation of, and Conclusions from, the Invention in Brief
[0121] The instant invention allows for the rapid testing, in less than 15 minutes, of the blood for the dimeric glycoprotein cellular fibronectin. In one aspect of the present invention, there are provided antibodies to bind to the EDA region of cellular fibronectin, said antibodies having high affinity for said EDA region of cellular fibronectin and at least two orders of magnitude less affinity for plasma fibronectin. The invention further encompasses diagnostic assays and kits comprising antibodies generated against the EDA region of cellular fibronectin.
[0122] In another aspect of the present invention, antibodies to c-Fn are used in a competitive assay format with paramagnetic beads to allow for a quantitative measurement of c-Fn in a blood sample.
[0123] A preferred embodiment of this aspect is a competitive assay format using the FASTPACK IP system by Qualigen, Inc.
[0124] In another aspect of the present invention, antibodies to c-Fn are used in a sandwich assay format with paramagnetic beads to allow for a quantitative measurement of c-Fn in a blood sample.
[0125] A preferred embodiment of this aspect is a sandwich assay format using the FASTPACK IP system by Qualigen, Inc.
[0126] In a specific embodiment of the present invention, the rapid assay allows the prediction of bleeding at the point of care in a specific patient.
[0127] In a preferred aspect of this embodiment, the rapid assay allows for the prediction of the likelihood of internal neurological or cardiovascular bleeding in a human subject, or likelihood of a neurological or cardiovascular bleeding following administration to the subject of a therapeutic agent to a human subject.
[0128] In another preferred aspect of this embodiment, the rapid assay allows prediction of hemorrhagic bleeding after administration of a human subject with a thrombolytic therapeutic.
[0129] In another preferred aspect of this embodiment, the rapid assay allows prediction of major bleeding after administration of a human subject with an anti-coagulation therapeutic agent.
[0130] In another preferred aspect of this embodiment, the rapid assay allows prediction of major bleeding after administration of a human subject with a Factor Xa inhibitor.
[0131] In yet another preferred aspect of this embodiment, the rapid assay allows prediction of major bleeding after administration of a human subject with a therapeutic agent against Alzheimer's or Parkinson's disease.
[0132] In yet another preferred aspect of this embodiment, the rapid assay allows prediction of major bleeding before administering a surgical procedure.
5. Recapitulation of the Invention
[0133] The present invention has been seen to relate to a rapid assay for detection of human cellular fibronectin where ELISA-based assays have been developed for detecting and measuring cellular fibronectin in biological fluids, but these methods are too time-consuming for practical clinical diagnostic use. This assay enables prediction of bleeding events on a rapid timescale. Described as well are high affinity human monoclonal antibodies, particularly those directed against isotopic determinants of cellular fibronectin (c-Fn), as well as direct equivalents and derivatives of these antibodies. These antibodies bind to their respective target with an affinity at least 100 fold greater than they do to plasma fibronectin, and enable assays to be created that detect c-Fn in less than 30 minutes in a variety of detection formats, and in some detection formats less than 15 minutes. These antibodies are useful for diagnostics, particularly prediction of bleeding events, prophylaxis and treatment of disease.
[0134] Together, the disclosed rapid assay and methods of use enjoy substantial benefits over the prior art, including simplicity of use by an operator, rapid in situ determinations of cellular fibronectin, and single-use methodology that minimizes the risk of contamination of both operator and patient. The disclosed invention is adaptable to the point-of-care clinical diagnostic field, including use in accident sites, emergency rooms, surgery, nursing homes, intensive care units, and non-medical environments.
[0135] For the first time, the instant invention allows a quantitative, accurate measurement of cellular fibronectin from a human fluid sample that is simple to perform, takes less than 20 minutes, and can be performed by a tabletop instrument. This will allow point-of-care testing for the prediction of bleeding in administering a treatment or surgical procedure where time is often of the essence, as major bleeding can predicted for the first time on a molecular level. It will allow physicians to make >go or >no-go decisions about treatments and procedures that affect the cardiovascular and neurovascular systems of a human patient without facing the uncertainty of sometimes fatal bleeding side effects.