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Platelet immuno-assay (PIA) as a simple and sensitive technique for the detection of antigens
Journal of Immunological Methods, 17 (1977) 47--55
47
© Elsevier/North-Holland Biomedical Press
P L A T E L E T IMMUNO-ASSAY (PIA) AS A SIMPLE AND SENSITIVE TECHNIQUE F O R THE DETECTION OF ANTIGENS
JONATHAN SCHULTZ, STEVEN 0. DISCIULLO and J. GARY ABUELO 1
Division of Medical Research, Rhode Island Hospital, Providence, R.I. 02902 and Division of Biological and Medical Sciences, Brown University, Providence, R.I. 02912, U.S.A. (Received 6 December 1976, accepted 21 March 1977)
ABSTRACT The platelet immuno-assay (PIA) is a simple and inexpensive technique to detect antigens with ng/ml sensitivity using micro-titer equipment and commercially available antisera. It can be easily adapted for virtually any antigen to which rabbit antiserum containing specific IgG-antibodies can be obtained. The assay is inhibited by IgG and C l q in amounts />125 pg/ml and 12.5 pg/ml respectively. Its application is thus limited to those fluids where IgG and C l q are present in less than these concentrations.
INTRODUCTION
In 1968 Penttinen and Myllyla introduced the sedimentation pattern test to detect platelet aggregation caused by IgG-immune complexes. While these authors were interested in the measurement of circulating immune complexes and of specific post infection anti-viral antibodies (Myllyla et al., 1969, 1971; Paluoso et al., 1970; Penttinen et al., 1970), Myllyla (1973) suggested that the platelet aggregation test could be a sensitive and general m e t h o d for the detection of antigens. He reported that as little as 1.6 ng/ml 5-nitro-3-iodo-4-hydroxyphenylacetic acid-bovine serum albumin (NIP-BSA) antigen added to specific anti-NIP serum formed sufficient immune complexes to be detected by platelet aggregation (Myllyla, 1973). In our study, we were able to use this technique to assay several other antigens, exploiting the fact that specific rabbit IgG-antibody in complex with small amounts of antigens induced detectable aggregation of human blood platelets (Myllyla, 1973). We measured as little as 0.25 ng/ml IgG, 1 ng/ml BSA, and 100 ng/ml ~ galactosidase in pure antigen solution, and 3.5 ng/ml IgA in a semipurified IgA-rich solution. The test is set up in three hours with simple, inexpensive microtiter equipment and commercial antisera and results are read after a 16 to 18 h incubation period. 1 Reprint requests should be directed to: Dr. J. Gary Abuelo, Rhode Island Hospital, Providence, R.I. 02902.
48 MATERIALS AND METHODS
Reagents All antisera used in this study were prepared in rabbits. Antisera specific for human C l q , IgG, and IgA and for BSA were purchased from Miles Laboratories. Anti-/~ galactosidase serum and fl galactosidase were generous gifts from Dr. Boris Rotman and Richard Roth of Brown University, Providence, R.I. Human IgG was obtained by concentrating the 7S peak of Cohn Fraction II (Sigma Chemicals) from a Biogel A5M (Biorad) gel filtration column. BSA was purchased from Sigma Chemicals. Partially pure human IgA was obtained from the American Red Cross National Fractionation Center, Bethesda, MD. Pure human C l q was the generous gift of Eric Einstein and Dr. Walter Thayer, Rhode Island Hospital. Heat aggregated IgG was prepared by a modification of the method of Hannestad (1969). Cohn Fraction II solution (3 mg/ml in phosphate buffered saline as above) was heated to 63°C for 12 min followed by immediate cooling in an ice bath. The sample was applied to a Sephadex G-200 gel filtration column (Pharmacia Fine Chemicals). The protein peak coming off the column in the void volume was collected and concentrated. Anti-human IgG serum was depleted of its specific anti-IgG activity by adsorbing the antiserum with polymerized Cohn Fraction II prepared by the glutaraldehyde cross-linking m e t h o d of Avrameas and Ternynch (1969). Adsorption was considered to be complete when there was no anti-IgG activity detectable b y Ouchterlony gel diffusion or immunoelectrophoresis. Pure anti-human IgG antibody was obtained by eluting the b o u n d antibody from the polymerized Cohn Fraction II according to Avrameas and Ternynch (1969). All reagents were frozen in aliquots at --20°C and thawed shortly before use. Micropipets, diluters, and microtiter plates were purchased from Flow Laboratories. Quantitation of serum IgG was carried out by single radial immunodiffusion (Quantiplate, Kallestad Laboratories, Inc. Chaska, Minn.).
Platelet harvest Platelets were obtained by drawing 70 ml of blood from human volunteers into plastic centrifuge tubes containing 15 ml. ACD anticoagulant (30.4 g sodium citrate, 13.6 g citric acid, 20 g dextrose/in 1 liter H20). Platelets were isolated by a modification of the method of Aster and Jandl (1964). The citrated blood was divided into 4 aliquots and centrifuged at 325 g for 14 min at 15°C. The platelet rich plasma was collected in plastic tubes and centrifuged at 4 0 0 g for 5 min, 15°C. The platelet-rich supernatant was decanted and centrifuged at 2000 g for 10 min at 15 ° C. The supernatant was discarded and the platelet pellet was gently resuspended in 10 ml 0.9% NaC1.
49 After 3 washes in (0.01 M NaH2PO4, 18 mg% glucose, to platelets during the problem.
saline, the platelets were resuspended in PBS buffer 0.15 M NaC1, adjusted to pH 7.6 with NaOH) with a concentration of 200,000 platelets/ml. Clumping of wash procedure occurred only rarely and was n o t a
Initial platelet aggregation test An initial platelet aggregation test was performed by a modification of the m e t h o d of Penttinen and Myllyla (1969). The appropriate dilution of antiserum to achieve maximum sensitivity, yet minimum non-specific platelet aggregation, is determined b y adding dilutions of antiserum in PBS buffer with 18 rag% glucose to a row of wells at 0.05 ml/well. The first well in each horizontal row receives twice the concentration of antiserum as the remaining wells. Antigen (0.05 ml/well) is added to the first column of wells at a known concentration varying between 400 and 1000 ng/ml. The first well in the row contains a dilution of antiserum equal to that of the other wells in the row. Using microdiluters with a 0.05 ml capacity, the antigen is serially diluted in the diluted antiserum such that a checkerboard titration is produced (see fig. la). A row containing serial dilutions of antigen alone, a column containing serial dilutions of antiserum alone, and a well containing only buffer provide negative controls. A row of wells containing serum dilutions of heat-aggregated IgG may be used to provide a positive control, since heat-aggregated IgG causes platelet aggregation in this system in concentrations as low as 100 ng/ml (Paluoso and Leikola, 1975). The plates are incubated for 1 h at room temperature after the addition of antigen to the antiserum. Then 0.1 ml of platelets, 200,000/ml are added to each well. The plates are placed at 4°C for 16--18 h. They are then returned to room temperature for interpretation. The results are read with indirect lighting against a dark background. Frequently at this point all the wells have a uniform layer of platelets at the b o t t o m . Within a few minutes to a half hour the platelets in the negative controls settle into a b u t t o n in the center of the well (a negative pattern). The microplates must then be read promptly. A positive result is either ring formation or a uniform layer of platelets at the b o t t o m of the wells. Even in these wells with a positive pattern platelet settling continues, so that within 30 rain to several hours the platelets will form a b u t t o n pattern as in the negative control. Certain antisera were found to cause non-specific platelet aggregation (give a positive platelet pattern) at low dilutions even in the absence of antigen. One must choose a dilution of antiserum high enough so that antiserum alone does n o t aggregate platelets. On the other hand higher dilutions of antisera become increasingly less sensitive in the detection of antigen. Examination o f the initial platelet aggregation test allows the choice of a dilution of antiserum which is quite sensitive in the detection of antigen y e t
50 DILUTION
OF I g G ( 5 O o n g / m l ) ,~
o0
¢',,t I000
uJ o3
2000 Z '< 3 0 0 0 i..i.. 0 4000 Z 0
6000
_J O m
o,I
'~
DILUTION
OF ANTIGEN
CO t~
~
~
~
0
t :1000 NHS
GAL
500 rig/rot
IgO
Fig. 1. a) Dilutions o f rabbit anti-human IgG antiserum are incubated with serial dilutions o f 500 ng/ml 7S IgG and platelets to d e t e r m i n e the appropriate dilution o f antiserum to use for IgG detection. A dilution of 1 : 3000 proves appropriate since lower dilutions cause t o o great a degree of non-specific aggregation as seen in the last c o l u m n which contains no IgG. Dilutions greater than 1 : 3000 give l o w e r titers of platelet aggregation in response to the IgG, and are thus less sensitive, b) Using the same platelet d o n o r and batch o f antiserum as in (a) a 1 : 3000 antiserum dilution is used in all the wells of the 'a' rows to semi-quantify the a m o u n t of IgG in the samples. The wells in the 'b' rows contain a 1 : 3 0 0 0 dilution o f normal rabbit serum with n o anti-IgG activity as a negative control. A sample of a 1 : 1000 normal h u m a n serum (NHS) gives a positive titer for IgG to the m a x i m u m titer tested (2048). A sample of pure ~ galactosidase 2 5 0 / l g / m l (fl G A L ) gives a negative result for IgG. 500 ng/ml IgG is positive to a titer of 128 which corresponds to a test sensitivity of 3.8 ng/ml.
causes no non-specific platelet aggregation by itself. The appropriate dilution of antiserum must be determined for each donor and antiserum, but once determined may be employed repeatedly with the same platelet donor.
51
Platelet immuno-assay After choosing the o p t i m u m dilution of antiserum with the initial platelet aggregation test, the concentration of antigen in a series of samples can be measured by running serial dilutions of the sample with this antiserum dilution in the presence of platelets from the same donor. The amount of antigen in the u n k n o w n is semi-quantitated by comparing its platelet aggregation titer (last dilution showing platelet aggregation) to that of a known concentration of the same antigen. Diluted antiserum alone and a row of serially diluted antigen in which diluted normal rabbit serum is substituted for specific antiserum are included as negative controls. Serial dilutions of the antigen in specific antiserum or heat aggregated IgG serve as positive controls. RESULTS Fig. l a and l b illustrate the two phases of the platelet aggregation test in determining the a m o u n t of antigen in a series of samples. In fig. la, the o p t i m u m dilutiori of rabbit anti-human IgG serum for the particular platelet donor is determined as described in the M e t h o d s section. In fig. l b , the o p t i m u m antiserum dilution of 1 : 3000 is used throughout to quantify the concentration of human IgG in u n k n o w n samples (see legend and methods). In this manner, as little as 0.25 ng/ml IgG, 1 ng]ml BSA, and 100 ng/ml galactosidase could be detected in pure antigen soluti(~n. As little as 3.5 ng/ml human IgA could be detected in a semi-pure IgA-rich solution. To assess the sensitivity and reproducibility of the assay, an initial platelet aggregation test was performed on three separate days on each of three platelet donors using a known quantity of IgG. The o p t i m u m antiserum dilution observed was constant from day to day for each donor. However, as seen in table 1, this dilution was different from donor to donor. The sensitivities of the three donors varied widely, b u t even when least sensitive 16 ng IgG/ml was detected. The ability of the assay to reproducibly measure IgG in a sample of human serum was examined b y performing the PIA on three separate days in each of the three platelet donors mentioned above. The quantity of IgG was calculated by comparing the titer of the serum sample to the titer of the IgG standard. The platelet test measured amounts varying from 3.2 to 102 mg/ml in a serum sample containing 17 mg/ml IgG b y the radial immunodiffusion technique (table 1). Rabbit antisera to C l q , IgG, IgA and BSA were found to cause a high degree of non-specific platelet aggregation in the absence of antigen. In contrast, rabbit anti-fi-galactosidase serum caused no non-specific platelet aggregation (table 2). In an effort to understand the basis for non-specific platelet aggregation, rabbit antiserum to human IgG was completely adsorbed of anti-IgG activity. This adsorption completely removed nonspecific platelet aggregation in the absence of antigen. A large amount of
52 TABLE 1 Sensitivity and r e p r o d u c i b i l i t y of PIA in t h e m e a s u r e m e n t of h u m a n IgG. Range of optimum d i l u t i o n s of antiserum
Sensitivity n g / m l IgG
S e m i - q u a n t i t a t i o n o f IgG ( n g / m l ) in h u m a n s e r u m sample * * *
Donor A
2000--4000
4.1 (range 0 . 2 5 - - 8 ) *
53 (range 3 . 2 - - 1 0 2 ) **
Donor B
2000
1.1 (range 0 . 2 5 - - 2 ) *
51 (range 2 5 . 6 - - 1 0 2 ) *
Donor C
1000--2000
6.8 (range 0 . 4 - - 1 6 ) *
51 (range 2 5 . 6 - - 1 0 2 ) *
4.0 ~ 5.2 (S.D.)
51.5 ± 43 (S.D.)
Total
* 3 s e p a r a t e assays o n d i f f e r e n t days. ** 2 s e p a r a t e assays o n d i f f e r e n t days. *** C o n t a i n i n g 17 m g / m l IgG as d e t e r m i n e d by radial i m m u n o d i f f u s i o n .
non-specific platelet aggregating activity was present, however, in purified anti-human IgG antibody. Preliminary studies were carried out on inhibitors of platelet aggregation known to be present in serum. Inhibition of the platelet aggregation test was caused by adding 7S human IgG and pure human C l q to the wells prior to addition of antigen. The detection of human IgA in a sample containing 15 pg/ml IgA was completely inhibited by 125 pg/ml human IgG. 12.5-100 pg/ml of human C l q decreased platelet aggregation titers of a sample of BSA by 4 to 32-fold.
TABLE 2 Specific and n o n s p e c i f i c p l a t e l e t aggregating activities of a n t i s e r a in a typical p t a t e l e t donor. Antiserum
N o n s p e c i f i c activity : highest dilution causing s p o n t a n e o u s p l a t e l e t aggregation
Specific activity : highest dilution causing p l a t e l e t aggregation in presence of antigen
A n t i IgG
1000
4000
A n t i IgA
100
400
4
50
3200
64
< 10
3200
> 320
Anti BSA Anti B GAL
R a t i o o f specific to nonspecific activity
4
53 DISCUSSION
There are a great variety of immunologic methods reported that can detect antigens, particularly immunoglobulin, in the ng/ml range (Salmon et al., 1969; Mann et al., 1969; Arbesman and Ito, 1971; Avrameas and Guilbert, 1971; Engvall and Perlmann, 1971; VanWeeman and Schuurs, 1971; Leute et al., 1972; Ugguis, 1976), however, all these methods involve sophisticated immuno-chemical techniques and equipment. The platelet immuno-assay in microtiter equipment can detect antigens in the ng/ml range simply and inexpensively using commercially obtainable antisera. The test can be set up in 3 h and can be easily adapted for virtually any antigen to which rabbit antiserum containing specific IgG antibodies can be obtained. For every new platelet d o n o r and antiserum preparation, an initial platelet test must be done to determine the appropriate dilution of antiserum to be used (see Methods). It is necessary to find a dilution of antiserum low enough to create a sufficient number of immune complexes to aggregate platelets, y e t one high enough to avoid non-specific platelet aggregation. Fortunately, once a satisfactory dilution of antiserum is found for the particular donor and batch of antiserum, it varies little from day to day. The results shown in table 1 demonstrate the great sensitivity of the PIA (0.25--16 ng/ml IgG was detected). This assay is based on a comparison of the titer of an u n k n o w n quantity of antigen to the titer of a known quantity of antigen. As might be anticipated the results of the assay vary considerably on repeated determinations for the same sample. This would restrict the usefulness of the PIA to those situations where one wishes to k n o w whether an antigen is present or not or where a semiquantitative method is satisfactory. The non-specific aggregation of platelets by certain antisera (e.g. anti-Clq, --IgG, --BSA, --IgA) is probably due to the reaction of these antisera with the specific antigen on the surface of platelets. The human blood platelet is known to adsorb many serum proteins such as IgG and complement components onto its surface (Salmon et al., 1969; Penttinen et al., 1970; Wautier et al., 1976). To demonstrate that the non-specific platelet aggregation was caused b y the specific antibody fraction of the antiserum, we found a high degree of non-specific aggregation caused by pure anti-human IgG a n t i b o d y alone. Non-specific aggregation did not occur with anti-human IgG serum that had its anti-IgG activity removed. Anti-fl galactosidase antiserum also failed to cause non-specific platelet aggregation, since it is expected that there is little or no fl galactosidase on the surface of platelets. The slight degree of non-specific platelet aggregation caused by anti-BSA serum is probably due to cross-reactivity between anti-BSA and human serum albumin. Inhibition of immune complex-induced platelet aggregation has been reported with native IgG and C1 (Penttinen et al., 1970; Pfueller and Li]scher, 1972). Myllyla (1973) found the detection of NIP-BSA antigen in the presence of specific antiserum was inhibited b y 25 pg/ml native IgG.
54
Pfueller and Luscher (1972) report that the release reaction of platelets induced by immune complexes could be inhibited by 30 pg/ml of human IgG with maximal effect by 500 pg/ml and by 1 pg/ml of human C1 with maximal effect by 75 pg/ml. They suggest that native IgG competes with immune complexes for a platelet Fc receptor and that C1 competes with platelets for the Fc portion of the IgG in immune complexes. In our study, the detection of BSA in pure antigen solution with specific antiserum was inhibited by 12.5 #g/ml Clq. Similarly, the detection of human IgA was inhibited by 125 pg/ml, 7S human IgG. It must be pointed out that the presence of large amounts of certain proteins (e.g. IgG, Clq), in biological fluids like serum might interfere with the detection of antigens by this technique. This precludes the use of PIA in the assay of specific antigens present in small concentrations in serum. However, other applications are forseeable. The great sensitivity of this technique will permit the rapid and simple assay of specific antigens in IgG poor fractions of biological fluids separated by a variety of methods such as gel filtration or preparative electrophoresis. Similarly the PIA could be used for detection of antigens in bacterial or cell culture supernatants or in body fluids such as spinal fluids where large quantities of interfering proteins are not present. ACKNOWLEDGMENTS
The authors gratefully acknowledge the helpful comments and assistance of Dr. Gilbert R. DiLeone, R.I. Hospital, and Dr. Paul M. Knopf, Brown University, during the preparation of the manuscript and thank Mr. Milton Lipsky for the preparation of the figure, and also wish to thank Mrs. Kathleen Lund for her secretarial assistance. REFERENCES Adelson, E., J.J. Rheingold and W.H. Crosby, 1964, Blood 17,767. Arbesman, C.E. and K. Ito, 1971, J. Allergy 47, 85. Aster, R.H. and J.H. Jandl, 1964, Clin. Invest. 43,843. Avrameas, S. and T.Ternynch, 1969, Immunochemistry 6, 53. Avrameas, S. and B. Guilbert, 1971, C.R. Acad. Sci. Ser. D, 272, 2705. Engvall, E. and P. Perlmann, 1971, Immunochemistry 8,871. Hannestad, K., 1969, J. Clin. Immunol. 4, 555. Leute, R., E. Ullman, A. Goldstein and L. Herzenberg, 1972, Nature New Biol. 236, 93. Mann, D., H. Granger and J.L. Fahey, 1969, J. Immunol. 102, 618. Myllyla, G., A. Vaheri, T. Vesikari and K. Penttinen, 1969, Clin. Exp. Immunol. 4 , 3 2 3 Myllyla, G., A. Vaheri and K. Penttinen, 1971, Clin. Exp. Immunol. 8,399. Myllyla, G., 1973, Scand. J. Haematol. Suppl. 19. Nagaki, K., K. Fujikawa and S. Inai, 1965, Biken J. 8,129. Paluoso, T., K. Penttinen and G. Myllyla, 1970, Archiv. Virusforsch. 31, 11. Paluoso, T. and J. Leikola, 1975, Clin. Exp. Immunol. 20,371. Penttinen, K. and G. Myllyla, 1969, Ann. Med. Exp. Fenn. 46, 188. Penttinen, K., L. Kaavainen and G. Myllyla, 1970, Arch. Ges. Virusforsch. 29,189.
55 Pfueller, S.L. and E.F. Liischer, 1972, Immunochemistry 9, 1151. Salmon, S.E., G. Mackey and H.H. Fudenberg, 1969, J. Immunol. 103,129. Ugguis, E.E., 1976, J. Immunol. Methods 10, 85. VanWeeman, B. and A. Schuurs, 1971, FEBS Letters 15,232. Wautier, J.L., G.M. Tobelem, A.P. Peltier and J.P. Caen, 1976, Immunol. 30,459.
47
© Elsevier/North-Holland Biomedical Press
P L A T E L E T IMMUNO-ASSAY (PIA) AS A SIMPLE AND SENSITIVE TECHNIQUE F O R THE DETECTION OF ANTIGENS
JONATHAN SCHULTZ, STEVEN 0. DISCIULLO and J. GARY ABUELO 1
Division of Medical Research, Rhode Island Hospital, Providence, R.I. 02902 and Division of Biological and Medical Sciences, Brown University, Providence, R.I. 02912, U.S.A. (Received 6 December 1976, accepted 21 March 1977)
ABSTRACT The platelet immuno-assay (PIA) is a simple and inexpensive technique to detect antigens with ng/ml sensitivity using micro-titer equipment and commercially available antisera. It can be easily adapted for virtually any antigen to which rabbit antiserum containing specific IgG-antibodies can be obtained. The assay is inhibited by IgG and C l q in amounts />125 pg/ml and 12.5 pg/ml respectively. Its application is thus limited to those fluids where IgG and C l q are present in less than these concentrations.
INTRODUCTION
In 1968 Penttinen and Myllyla introduced the sedimentation pattern test to detect platelet aggregation caused by IgG-immune complexes. While these authors were interested in the measurement of circulating immune complexes and of specific post infection anti-viral antibodies (Myllyla et al., 1969, 1971; Paluoso et al., 1970; Penttinen et al., 1970), Myllyla (1973) suggested that the platelet aggregation test could be a sensitive and general m e t h o d for the detection of antigens. He reported that as little as 1.6 ng/ml 5-nitro-3-iodo-4-hydroxyphenylacetic acid-bovine serum albumin (NIP-BSA) antigen added to specific anti-NIP serum formed sufficient immune complexes to be detected by platelet aggregation (Myllyla, 1973). In our study, we were able to use this technique to assay several other antigens, exploiting the fact that specific rabbit IgG-antibody in complex with small amounts of antigens induced detectable aggregation of human blood platelets (Myllyla, 1973). We measured as little as 0.25 ng/ml IgG, 1 ng/ml BSA, and 100 ng/ml ~ galactosidase in pure antigen solution, and 3.5 ng/ml IgA in a semipurified IgA-rich solution. The test is set up in three hours with simple, inexpensive microtiter equipment and commercial antisera and results are read after a 16 to 18 h incubation period. 1 Reprint requests should be directed to: Dr. J. Gary Abuelo, Rhode Island Hospital, Providence, R.I. 02902.
48 MATERIALS AND METHODS
Reagents All antisera used in this study were prepared in rabbits. Antisera specific for human C l q , IgG, and IgA and for BSA were purchased from Miles Laboratories. Anti-/~ galactosidase serum and fl galactosidase were generous gifts from Dr. Boris Rotman and Richard Roth of Brown University, Providence, R.I. Human IgG was obtained by concentrating the 7S peak of Cohn Fraction II (Sigma Chemicals) from a Biogel A5M (Biorad) gel filtration column. BSA was purchased from Sigma Chemicals. Partially pure human IgA was obtained from the American Red Cross National Fractionation Center, Bethesda, MD. Pure human C l q was the generous gift of Eric Einstein and Dr. Walter Thayer, Rhode Island Hospital. Heat aggregated IgG was prepared by a modification of the method of Hannestad (1969). Cohn Fraction II solution (3 mg/ml in phosphate buffered saline as above) was heated to 63°C for 12 min followed by immediate cooling in an ice bath. The sample was applied to a Sephadex G-200 gel filtration column (Pharmacia Fine Chemicals). The protein peak coming off the column in the void volume was collected and concentrated. Anti-human IgG serum was depleted of its specific anti-IgG activity by adsorbing the antiserum with polymerized Cohn Fraction II prepared by the glutaraldehyde cross-linking m e t h o d of Avrameas and Ternynch (1969). Adsorption was considered to be complete when there was no anti-IgG activity detectable b y Ouchterlony gel diffusion or immunoelectrophoresis. Pure anti-human IgG antibody was obtained by eluting the b o u n d antibody from the polymerized Cohn Fraction II according to Avrameas and Ternynch (1969). All reagents were frozen in aliquots at --20°C and thawed shortly before use. Micropipets, diluters, and microtiter plates were purchased from Flow Laboratories. Quantitation of serum IgG was carried out by single radial immunodiffusion (Quantiplate, Kallestad Laboratories, Inc. Chaska, Minn.).
Platelet harvest Platelets were obtained by drawing 70 ml of blood from human volunteers into plastic centrifuge tubes containing 15 ml. ACD anticoagulant (30.4 g sodium citrate, 13.6 g citric acid, 20 g dextrose/in 1 liter H20). Platelets were isolated by a modification of the method of Aster and Jandl (1964). The citrated blood was divided into 4 aliquots and centrifuged at 325 g for 14 min at 15°C. The platelet rich plasma was collected in plastic tubes and centrifuged at 4 0 0 g for 5 min, 15°C. The platelet-rich supernatant was decanted and centrifuged at 2000 g for 10 min at 15 ° C. The supernatant was discarded and the platelet pellet was gently resuspended in 10 ml 0.9% NaC1.
49 After 3 washes in (0.01 M NaH2PO4, 18 mg% glucose, to platelets during the problem.
saline, the platelets were resuspended in PBS buffer 0.15 M NaC1, adjusted to pH 7.6 with NaOH) with a concentration of 200,000 platelets/ml. Clumping of wash procedure occurred only rarely and was n o t a
Initial platelet aggregation test An initial platelet aggregation test was performed by a modification of the m e t h o d of Penttinen and Myllyla (1969). The appropriate dilution of antiserum to achieve maximum sensitivity, yet minimum non-specific platelet aggregation, is determined b y adding dilutions of antiserum in PBS buffer with 18 rag% glucose to a row of wells at 0.05 ml/well. The first well in each horizontal row receives twice the concentration of antiserum as the remaining wells. Antigen (0.05 ml/well) is added to the first column of wells at a known concentration varying between 400 and 1000 ng/ml. The first well in the row contains a dilution of antiserum equal to that of the other wells in the row. Using microdiluters with a 0.05 ml capacity, the antigen is serially diluted in the diluted antiserum such that a checkerboard titration is produced (see fig. la). A row containing serial dilutions of antigen alone, a column containing serial dilutions of antiserum alone, and a well containing only buffer provide negative controls. A row of wells containing serum dilutions of heat-aggregated IgG may be used to provide a positive control, since heat-aggregated IgG causes platelet aggregation in this system in concentrations as low as 100 ng/ml (Paluoso and Leikola, 1975). The plates are incubated for 1 h at room temperature after the addition of antigen to the antiserum. Then 0.1 ml of platelets, 200,000/ml are added to each well. The plates are placed at 4°C for 16--18 h. They are then returned to room temperature for interpretation. The results are read with indirect lighting against a dark background. Frequently at this point all the wells have a uniform layer of platelets at the b o t t o m . Within a few minutes to a half hour the platelets in the negative controls settle into a b u t t o n in the center of the well (a negative pattern). The microplates must then be read promptly. A positive result is either ring formation or a uniform layer of platelets at the b o t t o m of the wells. Even in these wells with a positive pattern platelet settling continues, so that within 30 rain to several hours the platelets will form a b u t t o n pattern as in the negative control. Certain antisera were found to cause non-specific platelet aggregation (give a positive platelet pattern) at low dilutions even in the absence of antigen. One must choose a dilution of antiserum high enough so that antiserum alone does n o t aggregate platelets. On the other hand higher dilutions of antisera become increasingly less sensitive in the detection of antigen. Examination o f the initial platelet aggregation test allows the choice of a dilution of antiserum which is quite sensitive in the detection of antigen y e t
50 DILUTION
OF I g G ( 5 O o n g / m l ) ,~
o0
¢',,t I000
uJ o3
2000 Z '< 3 0 0 0 i..i.. 0 4000 Z 0
6000
_J O m
o,I
'~
DILUTION
OF ANTIGEN
CO t~
~
~
~
0
t :1000 NHS
GAL
500 rig/rot
IgO
Fig. 1. a) Dilutions o f rabbit anti-human IgG antiserum are incubated with serial dilutions o f 500 ng/ml 7S IgG and platelets to d e t e r m i n e the appropriate dilution o f antiserum to use for IgG detection. A dilution of 1 : 3000 proves appropriate since lower dilutions cause t o o great a degree of non-specific aggregation as seen in the last c o l u m n which contains no IgG. Dilutions greater than 1 : 3000 give l o w e r titers of platelet aggregation in response to the IgG, and are thus less sensitive, b) Using the same platelet d o n o r and batch o f antiserum as in (a) a 1 : 3000 antiserum dilution is used in all the wells of the 'a' rows to semi-quantify the a m o u n t of IgG in the samples. The wells in the 'b' rows contain a 1 : 3 0 0 0 dilution o f normal rabbit serum with n o anti-IgG activity as a negative control. A sample of a 1 : 1000 normal h u m a n serum (NHS) gives a positive titer for IgG to the m a x i m u m titer tested (2048). A sample of pure ~ galactosidase 2 5 0 / l g / m l (fl G A L ) gives a negative result for IgG. 500 ng/ml IgG is positive to a titer of 128 which corresponds to a test sensitivity of 3.8 ng/ml.
causes no non-specific platelet aggregation by itself. The appropriate dilution of antiserum must be determined for each donor and antiserum, but once determined may be employed repeatedly with the same platelet donor.
51
Platelet immuno-assay After choosing the o p t i m u m dilution of antiserum with the initial platelet aggregation test, the concentration of antigen in a series of samples can be measured by running serial dilutions of the sample with this antiserum dilution in the presence of platelets from the same donor. The amount of antigen in the u n k n o w n is semi-quantitated by comparing its platelet aggregation titer (last dilution showing platelet aggregation) to that of a known concentration of the same antigen. Diluted antiserum alone and a row of serially diluted antigen in which diluted normal rabbit serum is substituted for specific antiserum are included as negative controls. Serial dilutions of the antigen in specific antiserum or heat aggregated IgG serve as positive controls. RESULTS Fig. l a and l b illustrate the two phases of the platelet aggregation test in determining the a m o u n t of antigen in a series of samples. In fig. la, the o p t i m u m dilutiori of rabbit anti-human IgG serum for the particular platelet donor is determined as described in the M e t h o d s section. In fig. l b , the o p t i m u m antiserum dilution of 1 : 3000 is used throughout to quantify the concentration of human IgG in u n k n o w n samples (see legend and methods). In this manner, as little as 0.25 ng/ml IgG, 1 ng]ml BSA, and 100 ng/ml galactosidase could be detected in pure antigen soluti(~n. As little as 3.5 ng/ml human IgA could be detected in a semi-pure IgA-rich solution. To assess the sensitivity and reproducibility of the assay, an initial platelet aggregation test was performed on three separate days on each of three platelet donors using a known quantity of IgG. The o p t i m u m antiserum dilution observed was constant from day to day for each donor. However, as seen in table 1, this dilution was different from donor to donor. The sensitivities of the three donors varied widely, b u t even when least sensitive 16 ng IgG/ml was detected. The ability of the assay to reproducibly measure IgG in a sample of human serum was examined b y performing the PIA on three separate days in each of the three platelet donors mentioned above. The quantity of IgG was calculated by comparing the titer of the serum sample to the titer of the IgG standard. The platelet test measured amounts varying from 3.2 to 102 mg/ml in a serum sample containing 17 mg/ml IgG b y the radial immunodiffusion technique (table 1). Rabbit antisera to C l q , IgG, IgA and BSA were found to cause a high degree of non-specific platelet aggregation in the absence of antigen. In contrast, rabbit anti-fi-galactosidase serum caused no non-specific platelet aggregation (table 2). In an effort to understand the basis for non-specific platelet aggregation, rabbit antiserum to human IgG was completely adsorbed of anti-IgG activity. This adsorption completely removed nonspecific platelet aggregation in the absence of antigen. A large amount of
52 TABLE 1 Sensitivity and r e p r o d u c i b i l i t y of PIA in t h e m e a s u r e m e n t of h u m a n IgG. Range of optimum d i l u t i o n s of antiserum
Sensitivity n g / m l IgG
S e m i - q u a n t i t a t i o n o f IgG ( n g / m l ) in h u m a n s e r u m sample * * *
Donor A
2000--4000
4.1 (range 0 . 2 5 - - 8 ) *
53 (range 3 . 2 - - 1 0 2 ) **
Donor B
2000
1.1 (range 0 . 2 5 - - 2 ) *
51 (range 2 5 . 6 - - 1 0 2 ) *
Donor C
1000--2000
6.8 (range 0 . 4 - - 1 6 ) *
51 (range 2 5 . 6 - - 1 0 2 ) *
4.0 ~ 5.2 (S.D.)
51.5 ± 43 (S.D.)
Total
* 3 s e p a r a t e assays o n d i f f e r e n t days. ** 2 s e p a r a t e assays o n d i f f e r e n t days. *** C o n t a i n i n g 17 m g / m l IgG as d e t e r m i n e d by radial i m m u n o d i f f u s i o n .
non-specific platelet aggregating activity was present, however, in purified anti-human IgG antibody. Preliminary studies were carried out on inhibitors of platelet aggregation known to be present in serum. Inhibition of the platelet aggregation test was caused by adding 7S human IgG and pure human C l q to the wells prior to addition of antigen. The detection of human IgA in a sample containing 15 pg/ml IgA was completely inhibited by 125 pg/ml human IgG. 12.5-100 pg/ml of human C l q decreased platelet aggregation titers of a sample of BSA by 4 to 32-fold.
TABLE 2 Specific and n o n s p e c i f i c p l a t e l e t aggregating activities of a n t i s e r a in a typical p t a t e l e t donor. Antiserum
N o n s p e c i f i c activity : highest dilution causing s p o n t a n e o u s p l a t e l e t aggregation
Specific activity : highest dilution causing p l a t e l e t aggregation in presence of antigen
A n t i IgG
1000
4000
A n t i IgA
100
400
4
50
3200
64
< 10
3200
> 320
Anti BSA Anti B GAL
R a t i o o f specific to nonspecific activity
4
53 DISCUSSION
There are a great variety of immunologic methods reported that can detect antigens, particularly immunoglobulin, in the ng/ml range (Salmon et al., 1969; Mann et al., 1969; Arbesman and Ito, 1971; Avrameas and Guilbert, 1971; Engvall and Perlmann, 1971; VanWeeman and Schuurs, 1971; Leute et al., 1972; Ugguis, 1976), however, all these methods involve sophisticated immuno-chemical techniques and equipment. The platelet immuno-assay in microtiter equipment can detect antigens in the ng/ml range simply and inexpensively using commercially obtainable antisera. The test can be set up in 3 h and can be easily adapted for virtually any antigen to which rabbit antiserum containing specific IgG antibodies can be obtained. For every new platelet d o n o r and antiserum preparation, an initial platelet test must be done to determine the appropriate dilution of antiserum to be used (see Methods). It is necessary to find a dilution of antiserum low enough to create a sufficient number of immune complexes to aggregate platelets, y e t one high enough to avoid non-specific platelet aggregation. Fortunately, once a satisfactory dilution of antiserum is found for the particular donor and batch of antiserum, it varies little from day to day. The results shown in table 1 demonstrate the great sensitivity of the PIA (0.25--16 ng/ml IgG was detected). This assay is based on a comparison of the titer of an u n k n o w n quantity of antigen to the titer of a known quantity of antigen. As might be anticipated the results of the assay vary considerably on repeated determinations for the same sample. This would restrict the usefulness of the PIA to those situations where one wishes to k n o w whether an antigen is present or not or where a semiquantitative method is satisfactory. The non-specific aggregation of platelets by certain antisera (e.g. anti-Clq, --IgG, --BSA, --IgA) is probably due to the reaction of these antisera with the specific antigen on the surface of platelets. The human blood platelet is known to adsorb many serum proteins such as IgG and complement components onto its surface (Salmon et al., 1969; Penttinen et al., 1970; Wautier et al., 1976). To demonstrate that the non-specific platelet aggregation was caused b y the specific antibody fraction of the antiserum, we found a high degree of non-specific aggregation caused by pure anti-human IgG a n t i b o d y alone. Non-specific aggregation did not occur with anti-human IgG serum that had its anti-IgG activity removed. Anti-fl galactosidase antiserum also failed to cause non-specific platelet aggregation, since it is expected that there is little or no fl galactosidase on the surface of platelets. The slight degree of non-specific platelet aggregation caused by anti-BSA serum is probably due to cross-reactivity between anti-BSA and human serum albumin. Inhibition of immune complex-induced platelet aggregation has been reported with native IgG and C1 (Penttinen et al., 1970; Pfueller and Li]scher, 1972). Myllyla (1973) found the detection of NIP-BSA antigen in the presence of specific antiserum was inhibited b y 25 pg/ml native IgG.
54
Pfueller and Luscher (1972) report that the release reaction of platelets induced by immune complexes could be inhibited by 30 pg/ml of human IgG with maximal effect by 500 pg/ml and by 1 pg/ml of human C1 with maximal effect by 75 pg/ml. They suggest that native IgG competes with immune complexes for a platelet Fc receptor and that C1 competes with platelets for the Fc portion of the IgG in immune complexes. In our study, the detection of BSA in pure antigen solution with specific antiserum was inhibited by 12.5 #g/ml Clq. Similarly, the detection of human IgA was inhibited by 125 pg/ml, 7S human IgG. It must be pointed out that the presence of large amounts of certain proteins (e.g. IgG, Clq), in biological fluids like serum might interfere with the detection of antigens by this technique. This precludes the use of PIA in the assay of specific antigens present in small concentrations in serum. However, other applications are forseeable. The great sensitivity of this technique will permit the rapid and simple assay of specific antigens in IgG poor fractions of biological fluids separated by a variety of methods such as gel filtration or preparative electrophoresis. Similarly the PIA could be used for detection of antigens in bacterial or cell culture supernatants or in body fluids such as spinal fluids where large quantities of interfering proteins are not present. ACKNOWLEDGMENTS
The authors gratefully acknowledge the helpful comments and assistance of Dr. Gilbert R. DiLeone, R.I. Hospital, and Dr. Paul M. Knopf, Brown University, during the preparation of the manuscript and thank Mr. Milton Lipsky for the preparation of the figure, and also wish to thank Mrs. Kathleen Lund for her secretarial assistance. REFERENCES Adelson, E., J.J. Rheingold and W.H. Crosby, 1964, Blood 17,767. Arbesman, C.E. and K. Ito, 1971, J. Allergy 47, 85. Aster, R.H. and J.H. Jandl, 1964, Clin. Invest. 43,843. Avrameas, S. and T.Ternynch, 1969, Immunochemistry 6, 53. Avrameas, S. and B. Guilbert, 1971, C.R. Acad. Sci. Ser. D, 272, 2705. Engvall, E. and P. Perlmann, 1971, Immunochemistry 8,871. Hannestad, K., 1969, J. Clin. Immunol. 4, 555. Leute, R., E. Ullman, A. Goldstein and L. Herzenberg, 1972, Nature New Biol. 236, 93. Mann, D., H. Granger and J.L. Fahey, 1969, J. Immunol. 102, 618. Myllyla, G., A. Vaheri, T. Vesikari and K. Penttinen, 1969, Clin. Exp. Immunol. 4 , 3 2 3 Myllyla, G., A. Vaheri and K. Penttinen, 1971, Clin. Exp. Immunol. 8,399. Myllyla, G., 1973, Scand. J. Haematol. Suppl. 19. Nagaki, K., K. Fujikawa and S. Inai, 1965, Biken J. 8,129. Paluoso, T., K. Penttinen and G. Myllyla, 1970, Archiv. Virusforsch. 31, 11. Paluoso, T. and J. Leikola, 1975, Clin. Exp. Immunol. 20,371. Penttinen, K. and G. Myllyla, 1969, Ann. Med. Exp. Fenn. 46, 188. Penttinen, K., L. Kaavainen and G. Myllyla, 1970, Arch. Ges. Virusforsch. 29,189.
55 Pfueller, S.L. and E.F. Liischer, 1972, Immunochemistry 9, 1151. Salmon, S.E., G. Mackey and H.H. Fudenberg, 1969, J. Immunol. 103,129. Ugguis, E.E., 1976, J. Immunol. Methods 10, 85. VanWeeman, B. and A. Schuurs, 1971, FEBS Letters 15,232. Wautier, J.L., G.M. Tobelem, A.P. Peltier and J.P. Caen, 1976, Immunol. 30,459.