Research in Veterinary Science 1991, 51, 292-298
Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay R. G. DHAWEDKAR*, N. C. JAIN, Department of Clinical Pathology, M. E. MOUNT, Department of Pharmacology and Toxicology, A. T. BOWLING, Serology Laboratory, J. L. VEGAD*, Department of Clinical Pathology, School of Veterinary Medicine, University of California, Davis, California 95616, USA
An enzyme-linked immunosorbent assay (ELISA) w a s standardised and applied for the detection of antiplatelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardlsed technique consisted of using Immulon type 3 plate, 1 per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 /~1 test serum, horseradish peroxidase conjugated a n t i b o d y and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophii antigens could be stored at 4 ° and - 70°C for four to five weeks without significant loss of antigenicity. The ELISAwas very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for nentrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplate!et serum. Standardised ELISA detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologons equine blood typing sera. IMMUNE-mediated thrombocytopenia and neutropenia occur in human beings (Sears et al 1986, Bussel and Abboud 1987, Schwartz 1988, Madyastha and Glassman 1989, Menitove et al 1989, Busse11990) and animals (Linklater et al 1973, Leidl et al 1980, Jain 1986, Swenson et al 1988, McVey and Shuman 1989). Several serological techniques are available to detect *Present address: College of Veterinary Science and Animal Husbandry, Jabalpur-482001, India
putative antibodies in serum or adsorbed to the platelets ar/d neutrophils (Schwartz 1988, yon dem Borne 1988, Mueller-Eckhardt and Kiefel 1989). Application of some highly specific and sensitive techniques such as the immunofluorescence test, the enzyme-linked immunosorbent assay (ELISA) procedure, and flow cytometry has made the detection of antiplatelet and antineutrophil antibodies (APA and ANA) a common diagnostic approach for immunemediated thrombocytopenia and neutropenia in human beings (Nel and Stevens 1980, Doughty et al 1981, Gudino and Miller 1981, Hegde et al 1981, Horai et al 1981, Taaning 1985, Sears et al 1986, Rosenfeld et al 1987, Sintnicolaas et al 1987, Menitove et al 1989, Veys et al 1989). However, diagnosis of these disorders in common domestic animals is lagging behind because of the inadequate availabilitty of appropriate sensitive serological assays. The present report describes the application and standardisation of an ELISA technique for the detection of APA and ANA using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. Materials and methods
Platelet isolation The method described by Schmidt and Rasmussen (1979) for isolating platelets from human blood was used with some modifications. Briefly, jugular blood from seven healthy horses was collected into a 35 ml polypropylene disposable syringe containing 7 ml sterile acid-citrate dextrose-A solution. Blood was kept on ice and processed within 30 minutes. Blood was equally distributed into eight polycarbonated tubes (15 ml) and centrifuged at 200 g for five minutes. Platelet-rich-plasma (PRP) with an uppermost erythrocyte layer was removed and pooled into four tubes and centrifuged at 3000 g for 20 minutes. The platelet pellet was suspended in 5 ml citrate buffer
ELISA detection of equine antibodies (0"05 M, pH 6-2) containing 2.5 per cent dextrose and washed three times by centrifugation at 3000 g for 10 minutes. Then the pellet was suspended in 5 ml of isotonic verenol buffer-EDTA (VBE) and centrifuged at 200 g for five minutes. The supernatant fluid containing platelets was carefully removed and the pellet was reprocessed after suspension in VBE to obtain additional platelets. The number of platelets tzl- 1was determined using an electronic counter (Baker Platelet Analyzer-810; Baker I n s t r u m e n t ) a n d adjusted to 3×108 ml -~. Platelets were used immediately or within 48 hours of storage at 4°C after the addition of sodium azide at a final concentration of 0.02 per cent. All steps of platelet isolation were carried out at 22°C.
Neutrophil &olation Jugular blood from eight healthy horses was collected using EDTAas the anticoagulant and neutrophils were isolated using a discontinuous Percoll density gradient technique described earlier (Jain et al 1990).
Antibody production Antineutrophil antibody (Jain et al 1990) and antiplatelet antibody (Jain et al 1991a) were raised in rabbits as described previously. Both antineutrophil and antiplatelet sera were absorbed three times with washed equine red blood cells for 30 minutes each at 22°C and twice with equine heart tissue powder (H4882; Sigma) overnight at 4°C to remove nonspecific antibodies. The absorbed antisera were stored in 2 ml samples at - 70°C. Normal rabbit serum was similarly absorbed and stored frozen until used.
Enzyme-linked immunosorbent assay (ELISa)for antiplatelet and antineutrophil antibodies in rabbit antisera The procedure was essentially that described by Tamerius and Tani (1983) for detecting antiplatelet antibodies in humans. All solutions were prepared as described and specific steps were as follows. Poly-L-lysine (P 0879; Sigma) (PLL, 70 /zg ml-~) coating solution (50 ~1) was added to each well of flatbottom microtitre plates (Dynatech) and allowed to react at 22°C for 30 minutes. The wells were washed three times with VBE and 50/~1 of unfixed platelet (5 × 107 m l - 1) or neutrophil (5 × 106 m l - 1) suspension was added to each well. The plates were centrifuged at 350 g for three minutes and kept for 30 minutes at 22°C. The unbound antigen was removed by washing the wells three times with VBE. The plates were examined under a microscope (100 ×) to determine
uniform attachment of the antigen to wells. Then 50 ~1 of VBE was added to each well, and the plates were covered with sealing tape and kept at 4°C overnight. The next day, VBE was drained and 200/zl of blocking solution (1 per cent gelatin in VBE) was added to each well. After incubation at 37°C for 60 minutes, the wells were washed mechanically (mechanical washer; Dynatech) six times with ELISA washing solution (EWS) (0"01 M phosphate buffer, 0.15 M sodium chloride, and 0- 05 per cent Tween 20, pH 7.4). Twofold dilutions of specific antiserum were made in 90 #1 samples in each well as needed. Suitable controls were included in each test. The plates were incubated at 37°C for 60 minutes and were washed again six times with EWS. Then 50/zl of peroxidaseconjugated anti-rabbit IgG (Sigma) (diluted 1:5000 in VBE) was added to each well and the plates were reincubated for 30 minutes. After rinsing the wells six times with EWS, freshly prepared 100 /~1 of ELISA substrate solution [40 mg O-phenylene diamine dihydrochloride (Sigma), 40/zl 30 per cent hydrogen peroxide in 100 ml substrate buffer (0"02 M citric acid, 0.05 phosphate buffer, pH 5)] was added to each well and the plates were kept at 22°C for 10 to 15 minutes in the dark to develop optimal colour. Finally, 100/zl of ELrSA stopping solution (ice-cold 4 N sulphuric acid) was added to each well. The optical density at 490 nm was determined within an hour using an automatic microtitre ELISA reader (Biotek). Antibody titres were calculated after correction of optical density readings for background obtained with the antiserum alone.
ELISA for antiplatelet and antineutrophil antibodies in equine sera The above described ELISA procedure was used to test the antiplatelet and antineutrophil antibodies in 23 normal equine sera and 100 equine blood typing sera. The latter were from a collection of isoimmunisation of horses with whole blood or obtained from unimmunised horses (natural source). Briefly, antigen-coated plates were incubated with test sera (1:10 to 1:80) and suitable controls. Then 50 /zl of peroxidase-conjugated anti-horse IgG (Sigma) (1:2000) was added to each well and the plates were incubated at 37°C for 30 minutes. After the removal of excess conjugate by washing the plates, freshly prepared 100 #1 quantities of substrate solution (5-aminosalicylic acid (Sigma), 1 mg m1-1) in prewarmed 0"2 M sodium phosphate buffer, pH 6-8, with 0"01 per cent fresh hydrogen peroxide) was added to each well and the plates were kept at 22°C for 10 to 15 minutes in the dark to develop optimal colour. Finally, 100 /zl of 3 M sodiu hydroxide
R. G. Dhawedkar, N. C. .lain, M. E. Mount, A. T. Bowling, J. L. Vegad
(stopping solution) was added to each well and optical density was determined at 450 nm. Test sera with optical density readings greater than the mean plus two standard deviations of 23 normal equine sera were considered positive (Gudino and Miller 1981). Some of the blood typing sera were reacted with equine platelets to detect antiplatelet antibodies also by the platelet suspension immunofluorescence test (Jain et al 1991a).
Platelet and neutrophil fixation An equal volume of 1 per cent paraformaldehyde was added to the platelet or neutrophil suspension and allowed to react for five minutes at 22°C. Fixed platelets and neutrophils were washed three times with VBE by centrifugation at 3000 g and 400 g, respectively, for 10 minutes and resuspended in VBE to a final concentration of 3 × 108 and 107 cells m1-1.
Platelet and neutrophil extracts Extracts were prepared by freezing and thawing platelet (3 × 108 ml-1) and neutrophil (1 × 107 ml-1) suspensions three times in dry ice-actone mixture followed by sonication (10 to 15 seconds) and centrifugation at 20,000 g for 15 minutes at 4°C (Hegde et al 1981). The supernatant fluid was designated as soluble antigen. Platelet extracts were also prepared from platelets reacted with antiplatelet serum (1:2, 1:8, 1:200, and 1:800). Two ml of platelets (3 × 108 m l - 1) were centrifuged at 3000 g for 10 minutes and suspended in equal volume of diluted antiserum. After incubation at 37°C for 30 minutes, the platelets were washed three times with VBE and reconstituted to the original volume for extraction. The supernatant fluid was used to detect eluted antiplatelet antibody.
Storage o f antigen-coated microtitre plates Microtitre plates coated with antigen (platelets or neutrophils) were stored at 4°C and - 7 0 ° C . For storage at 4°C, 200 ~1 of VBE containing 0.02 per cent sodium azide was added to each well and the plates were covered with sealing tape. Similarly, 200 t~l of freezing solution (10 per cent dimethyl sulphoxide and 10 per cent heat-inactivated fetal calf serum in VBE) was added to each well for storage at - 70°C. After specific period of storage, the plates were thawed at 37°C and washed six times with EWS for use in the ELISA procedure.
Removal of endogenous peroxidase from neutrophil antigen Before the addition of blocking solution to the plate
containing neutrophil antigen, 100/~1 of 1 per cent hydrogen peroxide in 95 per cent methanol was added to each well. The plate was incubated for 20 minutes at 22°C, rinsed and washed three times with VBE.
Cross absorption of antiplatelet and antineutrophil sera One ml of platelet suspension (3 × 108 m l - 1) was pelleted by centrifugation at 3000 g for 10 minutes. The pellet was suspended in 1 ml antineutrophil serum and incubated at 22°C for 30 minutes. The platelet suspension was recentrifuged and the supernatant (absorbed serum) was used in the test. A similar procedure was also carried out with neutrophil suspension (107 ml-1) and antiplatelet serum.
Experiments Various experiments were performed with antiplatelet sera to standardise the ELISA test procedure. Microtitre plates coated with PLL and alkaline buffer (pH 9.6) were compared with uncoated plates to determine effective attachment of antigen to plastic surfaces. Paraformaldehyde-fixed platelets, unfixed platelets, and platelet extracts were used as antigen. Different dilutions of platelet suspensions were used to determine the optimal number of platelets giving satisfactory antibody titres. Effectiveness of 1 per cent gelatine in VBE and 2.5 per cent bovine calf serum in VBE as blocking solutions, to reduce nonspecific IgG binding by microtitre plates, was evaluated. Different types of microtitre plates (Dynatech) were compared to determine the type of plate giving highest titres with the least non-specific reaction. Microtitre plates coated with platelets were stored at 4°C and - 7 0 ° C and periodically used to determine antibody titres. Similarly, standardisation of ELISA procedure was also carried out with antineutrophil serum and neutrophils as antigen.
Results Procedural aspects of the ELISA test Immulon types 1 to 4 microtitre plates were compared using platelets as antigen and identical dilutions of antiplatelet sera. All plates were equally sensitive in detecting antiplatelet antibody, but the type 3 plate was selected for further experiments because it had the lowest non-specific binding with the conjugate. Gelatine (1 per cent) and bovine calf serum (2.5 per cent) were equally effective in reducing the non-specific binding of the ELISAconjugate to the surface of microtitre plates unoccupied by the antigen.
ELISA detection o f equine antibodies TABLE 1: Relative ELISA readings (mean optical densities) for antiplatelet and antineutrophil sera using paraformaldehydefixed and unfixed platelet and neutrophil antigens Reciprocal of antiserum dilution
1600 3200 6400 12,800 25,600
1 .277 1 '085 0'858 0-551 O' 349
Unfixed 1 '449 1 '390 1 ' 172 0'953 O" 572
0"717 0"673 0"285 0.184 O" 120
1 "093 0'981 O' 596 0'373 O" 248
Values for fixed and unfixed antigens of respective cell types differed significantly (P .
Platelet and neutrophil antigens adhered equally well to microtitre plates coated with PLL solution or alkaline buffer (pH 9" 6). Unfixed platelet and neutrophil antigens gave higher (P