MICROTITER - SURFACE - FLOCCULATION ASSAY FOR ANTIGEN OR ANTIBODY SCREENING

Abstract: An objective microtiter-surface-flocculation assay for antigen or antibody screening. The assay employs slanting solid surface coated with an antibody to a protein in a 'V' shaped microtiter well. The assay is readable by instrument means and is automatable fully or partially. A kit containing various components of the assay is also disclosed.

Description: MICROTITER - SURFACE - FLOCCULATION ASSAY FOR ANTIGEN OR ANTIBODY SCREENING. BACKGROUND OF THE INVENTION Technical Field The present invention is related to the detection of antigens or antibodies in a sample. More particularly, the present invention is related to a icrotiter - surface-flocculation (MSF) assay for screening the presence of specific antigens or antibodies in a sample of the body fluid. State of the Art The most common current test used for screening blood for antibodies, e.g. to syphilis antigen, is by flocculation of charcoal particles coated with cardiolipins from beef heart. The test is done on plastic coated cards and results are determined subjectively, i.e. by visual examination. Clearly, such tests are prone to judgmental errors and are slow due to the necessity of manual manipulation. Almost all current tests using agglutination/flocculation particles coated with antigen rely on differences in settling patterns of particles in the presence or absence of antibodies. These tests depend for their accuracy and sensitivity upon the training and experience of the person reading the test. Significant variations occur due to the subjectivity of different individuals. The MSF assay of the present invention eliminates subjectivity being machine readable without sacrificing the sensitivity. Some of the aspects in which the present inventions differs from the currently known assays may be summarized as follows: Unique features of the present invention: a) Coating of surface of microtiter plate with antibodies to imπiunoglubulins resulting in specific differences in the sliding properties of antigen coated particles has not heretofore been used for agglutination/flocculation assays. b) Coating of solid surfaces with proteins including immunoglobulins is standard laboratory procedure, but it has not been used to capture specific antibodies that result in changes in flocculation/agglutination pattern specific of antigen-coated particles. c) The use of microtiter plates to automate the test is not unique, but reading flocculation or agglutination reaction in a coated plate is novel. d) The use of antibodies to human immunoglobulins to enhance agglutination has been reported, but only in liquid phase and without objective, instrumental reading. The present method is the first to provide a solid phase assay readable by instrument means and being automatable. SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide an objective MSF assay for screening the presence of specific antigens or antibodies in a serum, plasma or a body-fluid sample. If is a further object of the present invention to provide at least a partially or fully automated MSF assay capable of mechanical reading. It is yet another object of the present invention to provide a microtiter method for detecting antigen or antibodies in blood which comprises capturing particles coated with specific antigen or antibodies on a slanting surface and determining the amount of flocculation resulting from specific antigen-antibody reaction. Other objects and advantages of the present invention will become apparent as the detailed description thereof proceeds. BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Figure 1 shows various symbols used in figures 2 and 3 hereof. Figure 2 shows schematic representaion of various steps for the detection of specific antibodies using MSF assay. Figure 3 is a schematic representation of various steps for the detection of specific antigens using MSF assay. Figure 4 is a schematic representation of an embodiment of an automated system for MSF assay. DETAILED DESCRIPTION OF THE INVENTION These and other objects of the present invention are achieved by a microtiter-surface-flocculation assay which comprises the steps of (a) coating a slanting solid surface in a microtiter well with antibodies to a protein; (b) adding test sample to the well and incubating the sample for sufficient time at a suitable temperature for binding reaction between the test sample and coated solid surface to be substantially complete; (c) removing unbound sample from step (b) ; (d) adding to the well particles coated with antibodies or antigens specific for antigens or antibodies, respectively, the presence of which in the sample is to be detected; (e) separating captured antigen-antibody ligand or complex after reaction in step (d) is substantially complete; and (f) reading agglutination reaction by instrument means. The test sample is not limited to blood or blood products, e.g., serum or plasma, but may be any sample of the body fluid from humans or animals if such body fluid contains or is suspected to contain antigens or antibodies of interest. The term "substantially complete" as used herein means that the reaction is as complete as can be expected to occur under the conditions within a reasonable time period. The term "objective" as used herein means that the test .result is determined, read- or evaluated not by subjective judgment of a person but by instrument means. Such instruments include a spectrophotometer adopted to read "off-the center" of the microtiter well, a printout or display device to record the reading and the like. The principle of the MSF assay described herein is that the surface of 'V 1 wells of microtiter plates is coated with antibodies to a protein, including poly or monoclonal antibodies, preferably human immunoglobulins (IgG, IgM and/or IGA) and the like. These antibodies capture immunoglobulins from the test sample and when antigen coated charcoal or other suitable paticles are added, these in turn are captured by the specific antibody as shown in Figure 2. Presence of charcoal on the surface of 'V 1 well interfers with light transmission proportional to the presence of specific antibody in test serum when read in spectrophotometer set to read "off the center" as shown in Figure 4. Particles such as red blood cells, latex, charcoal magnetic or plastic spheres, fixed stained bacteria and the like can be coated with specific antigen(s) by chemical or physical methods well known in the art. These coated particles when mixed with the body fluid sample, e.g., serum or plasma containing specific antibodies to antigen(s) coated on the particles, cause flocculation or agglutination by forming antigen-antibody linkage or complex with various particles. The difference between negative and positive serum reactions is determined by the pattern and degree of flocculation when particles have settled. The slanting solid surface augments sliding of the flocculated complex to the bottom of the microtiter well and is a unique feature of the present invention. The present assay can be performed in any suitable plate, utilizing currently FDA licensed reagents and be evaluated automatically or se i-automatically by a machine to provide results in the form of a digital display, printout and the like. The test described herein meets all these criteria. The present assay is particularly suitable in a blood bank type setting where screeing of the samples is required. The following examples illustrate the preferred embodiments of the present MSF assay. Example l-"Test for Syphilis" 100 μl of test serum/plasma are added to a well of coated plate ('V') bottom microtiter plate coated with antibodies to human IgG and IgM heavy chain specific. A set of 3 negatives and 2 positive samples are similarly applied to serve as controls. The plate is covered and incubated at about 37° C for about 60 minutes to allow binding of immunoglobulins in test/control specimens by antibodies coated on the wells. Unbound proteins are removed from the wells by aspiration and washing with phosphate buffered saline (PBS). 100 μl of 1:5 dilution of charcoal particles coated with syphilis antigen is added to each well except the first well (blank) to which 100 μl of PBS is added. Plates are shaken on rotor (100 rpm) for 10 minutes and then centrifuged at approxi atley 1500g for about 1 minute. Reading is taken by employing microplate reader (MR-580, Dynatech) specially modified to read light transmission 'off the center 1 of the well of microtiter plate as shown in Figure 4. Plates are read using 450 nm as reference and 610 nm as transmission beam in MR-580. Test Results: In tests conducted on a panel of positives, 20 samples gave higher light absorbence than negatives. The test values were normalized by determining the net light absorbence i.e., sample-negative control mean or (S-N) value. A sample was considered reactive when S-N was equal or greater than 0.05. The cutoff value may be further adjusted with a larger number of samples. Example 2: Test for Antibodies to Cytomegalovirus (CMV) in Human Blood Wells of 'V bottom plates are coated with anti-human IgG/IgM. After incubating serum or plasma in the well for appropriate time and temperature, unbound serum/plasma proteins are removed by washing with buffer. Latex particles coated with CMV antigen are then added to 'V wells. After proper incubation, the plate is centrifuged and read for light transmission through the wells. Specimens containing anti-CMV activity block more light than negative specimens. Significant difference between negative and positive samples is obtained. Example 3: Test for Hepatitis B Surface Antigen (HBsAg) : As shown in Figure 3, 'V bottom plates are coated with antibodies to HBsAg. HBsAg in test specimen is captured by anti-HBs on 'V plate and when particles (latex, charcoal or red cells) coated with anti-HBs are added, these bind to HBsAG already captured by anti-HBs coated on the wells of the plate. After centrifugation plates are read for light absorbance as described supra. Similar tests may be utilized for antibodies to Brucellosis. Of course, the present MSF assay can be employed for antigen as well as antibodies in any system where particle agglutination occurs. Clearly, the MSF assay of the present invention is inherently convenient, efficient and superior to currently employed manual subjective assays. An MSF kit and an MSF apparatus are two other embodiments of the present invention. The components of the kit and/or the appartus comprise microtiter plate having a plurality of 'V shaped wells; solid surface coated with an antibody to a protein, preferably to IgG/IgM/IgA; container(s) containing specific antigen or antibody coated particles; container containing a suitable buffer or washing medium, e.g., PBS; a microtiter reader assembly, preferably with a printout or display device; instructions for earring out the assay and other accessories, e.g., micropipette, and the like commonly included in such kits or devices. An at least partially automated device for microtiter surface flocculation assay for detecting the presence of a specific antibody in a sample is now described. The device comprises a 'V shaped container transparent to light in the visible spectrum for receiving said sample; a solid surface coated with an antibody to a protein, said surface being slantingly disposed in said container; means for introducing in said container a predetermined quantity of particles coated with an antigen specific to the antibody presence of which is to be detected; means for separating from the sample in said container antigen-antibody complex formed as a result of reaction between said antigen coated particles and the antibody in said sample; means for detecting the presence of said complex in said container and means for recording the result thereof. The recording means may be any suitable assembly, preferably a printout or a display device. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims: IN THE CLAIMS: 1. A microtiter-surface-flocculation (MSF) assay for detecting the presence of an antibody, comprising the steps of: a) coating a slanting solid surface in a microtiter well with poly- or mono-clonal antibodies to a protein; b) adding test sample to the well and incubating the sample for sufficient time at a suitable temperature for binding reaction between the test sample and coated solid surface to be substantially complete; c) removing unbound sample from step (b) ; d) adding to the well particles coated with antigens specific for antibodies the presence of which in the sample is to be detected; e) separating captured antigen-antibody ligand or complex after reaction in step (d) is substantially complete; and f) reading agglutination reaction by instrument means. 2. The assay of claim 1 wherein said microtiter well is a 'V shaped well. 3. The assay of claim 2 wherein said protein is selected from the group consisting of immunoglobulin IgG, IgM and IgA. 4. The assay of claim 3 wherein said test sample is a body fluid. 5. The assay of claim 4 wherein said body fluid is a blood sample. 6. The assay of claim 5 wherein said blood sample is plasma or serum. 7. The assay of claim 6 wherein said particle is selected from the group consisting of charcoal, latex and red blood cells, magnetic spheres, plastic spheres and fixed stained bacteria. 8. A method of screening blood samples in a blood bank setting for the presence of specific antibodies, comprising testing said samples by the assay of claim 1. 9. A kit for detecting the presence of antigen or antibodies in a sample comprising: a microtiter plate with 'V shaped wells; a solid surface coated with antibodies against a protein, said surface being slantingly disposed in said well; a container containing particles coated with a specific antigen or antibody; and instruction for using the kit. 10. An at least partially automated device for microtiter surface flocculation assay for detecting the presence of a specific antigen or antibody in a sample, comprising: a 'V 1 shaped container transparent to light in the visible spectrum for receiving said sample; a solid surface coated with an antibody to a protein, said surface being slantingly disposed in said container; means for introducing in said container a predetermined quantity of particles coated with an antigen specific to the antibody the presence of which is to be detected; means for separating from the sample in said container antigen-antibody complex formed as a result of reaction between said antigen or antibody coated particles and antigen or antibody in said sample; means for detecting the presence of said complex in said container and means for recording the result thereof. 11. The device of claim 10 having a plurality of said container. 12. The device of claim 11 wherein said detecting means is a spectrophotometer. 13. The device of claim 12 wherein said recording means is a printout or display assembly. 14. A microtiter-surface-flocculation (MSF) assay for detecting the presence of an antigen, comprising the steps of: a) coating a slanting solid suface in a microtiter well with poly- or mono-clonal antibodies to a protein; b) adding test sample to the well and incubating the sample for sufficient time at a suitable temperature for binding reaction between the test sample and coated solid surface to be substantially complete; c) removing unbound sample from step (b) ; d) adding to the well particles coated with antibodies specific for antigens the presence of which in the sample is to be detected; e) separating captured antigen-antibody ligand or complex after reaction in step (d) is substantially complete; and f) reading agglutination reaction by instrument means. 15. The assay of claim 14 wherein said microtiter well is a 'V shaped well. 16. The assay of claim 15 wherein said protein is selected from the group consisting of immunoglobulin IgG, IgM and IgA. 17.. The assay of claim 16 wherein said test sample is a body fluid. 18. The assay of claim 17 wherein said body fluid is a blood sample. 19. The assay of claim 18 wherein said blood sample is plasma or serum. 20. The assay of claim 19 wherein said particle is selected from the group consisting of charcoal, latex, red blood cells, magnetic spheres, plastic spheres and fixed stained bacteria. 21. A method of screening blood samples in a blood bank setting for the presence of specific antigens comprising testing said samples by the assay of claim 14.

Inventors: NATH NRAPENDRA, NATH, NRAPENDRA
Applicants: AMERICAN NAT RED CROSS, AMERICAN NATIONAL RED CROSS
Application year: 1986
Main classification: G01N33
IPC codes: G01N33