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Enzyme-linked immunosorbent microassay for quantification of specific antibodies to collagen type I, II, III
Journal o f Immunological Methods, 29 (1979) 71--77 Q Elsevier/North-Holland Biomedical Press
71
ENZYME-LINKED IMMUNOSORBENT MICROASSAY FOR QUANTIFICATION OF SPECIFIC ANTIBODIES TO COLLAGEN TYPE I, II, III
BIRGIT GOSSLAU and HANS-JURGEN B A R R ACH
Institu t fiir Toxikologie und Embryopharmakologie, Freie Universitiit Berlin, D-1000 Berlin 33, G.F.R. (Received 26 October 1978, accepted 28 February 1979) An enzyme-linked immunosorbent assay (ELISA) is described for determination of antibodies to collagen type I, II, III in serum or other body fluids. Polystyrene microplates are coated with either collagen I, II or III, and samples of body fluids being tested are incubated in the plates. Antibodies which attach specifically to the collagen are detected with anti-IgG antibody conjugated with peroxidase. Bound peroxidase is quantitatively estimated by the colour reaction produced with the substrate 5-aminosalicylic acid.
INTRODUCTION
Detection of anticollagen antibodies has been performed by various methods. Passive hemagglutination tests have been extensively used (Steffen et al., 1968; Timpl et al., 1970; Beil et al., 1972; Michaeli and Fudenberg, 1974; Andriopoulos et al., 1975). Radioimmunoassay has been demonstrated to be more sensitive for quantitation of antibodies to collagen (Lindsley et al., 1971; Adelmann et al., 1973; Rohde et al., 1976; Menzel, 1977). We expected the micro-sandwich ELISA to be a useful alternative, since it does not require isotopes. Engvall and Ruoslahti (1977) have used ELISA for binding tests. Collagen and other proteins were adsorbed to a plastic surface and a fibronectin solution was added. The bound fibronectin was detected using fibronectin antibodies. We use collagen coated microplates to identify antibodies to collagen type I, II and III in body fluids by an ELISA system adapted from the m e t h o d developed by Engvall and Perlmann (1972). The test is used routinely to determine the titer of antibodies to collagen type I, II and III in hyperimmune sera derived from rabbits and sheep and to examine the sera of patients with polyarthritis or collagen diseases (Gosslau and Barrach, in preparation). MATERIALS AND METHODS
Preparation of antigen Collagen type I and IIi were prepared by acetic acid extraction of the skin of mice of postnatal day 3--6 (Miller and Matukas, 1974; Von der Mark et
72 al., 1976). Separation of the two collagen types was performed by repeated sequential.salt precipitation at pH 7.5 (50 mM Tris buffer) with 1.2 M, 1.7 M (collagen type III) and 2.5 M NaC1 (collagen type I). Collagen type II was isolated from Swarm rat chondrosarcoma according to Smith et al. (1975). It was extracted with 200 mM NaC1, precipitated twice by 20% NaC1 at pH 7.6 and further purified by diethylaminoethyl cellulose chromatography.
Coating o f microplates with antigen 5 mg collagen type I (or type III) were dissolved in 100~ml coating buffer I containing 0.02% NAN3. 5 mg collagen type II were dissolved in 100 ml coating buffer II containing 0.02% NAN3. Coating buffer I (pH 7.5) contained 7 mM CH3COOH, 7 mM KzHPO4, 0.17 M NaC1. Coating buffer II (pH 8.3) contained 0.1 M NaHCO3, 0.5 M NaC1. The test was performed in polystyrene Microtiter Plates M29A (Greiner, Nfirtingen, G.F.R.). The trays were coated with 100 pl antigen solution per well for at least 1 day at 4 ° C. Routinely the plates were allowed to stand for 6 days. They were then washed 3 times. For each wash the plates were filled with 100 pl cold PBS pH 7.8 per well. After 5 min the PBS was removed and the plates were emptied and shaken dry. Then the plates were stored over P205 (Sicapent, Merck, Darmstadt, G.F.R.) in vacuum until use. They were kept in this way up to 4 months. PBS = 0.12 M NaC1, 0.01 M Na2HPO4, 0.004 M KH2PO4, pH 7.8.
Enzyme-linked antiglobulin conjugates The conjugates were horseradish peroxidase labeled goat antirabbit IgG (Miles Laboratories, Frankfurt, G.F.R.) and horseradish peroxidase labeled rabbit antisheep IgG (Nordic Immunological Laboratories, Dietzenbach, G.F.R).
Substrate solution 80 mg 5-aminosalicylic acid (Ferak, Berlin, G.F.R.) were dissolved in 100 ml distilled water at 70°C. The solution was cooled to room temperature. Before use the pH was adjusted to 6.0 by adding 1 N NaOH. 9 ml of this stock solution were mixed with 1 ml of 0.05% H202 (Voller et al., 1976).
ELISA The assay is a variation of the ELISA m e t h o d described by Engvall and Perlmann (1972) and modified by Ruitenberg et al. (1976). For antibodies against collagen type I, II or III the following procedure was used: serial dilutions of sera were made using PBS, pH 7.8. Of each serum dilution 100 ~l, containing 1% bovine serum albumin (BSA) fraction V (Sigma Chemie GmbH, Munich, G.F.R.), were transferred to the collagen coated wells. The trays were incubated for 45 min at room temperature on a Microshaker (Flow Laboratories GmbH, Bonn, G.F.R.). After washing 3 times with PBS,
73 pH 7.8, 100 pl of PBS-diluted conjugate containing 1% BSA were added. The microplates were again incubated on the Microshaker for 45 min at r o o m t e m p e r a t u r e and then washed 3 times with PBS pH 7.8. The a m o u n t of b o u n d peroxidase was det er m i ned by adding 100 pl of the substrate solution. After 1 h the reaction was stopped by 100 pl 1 N NaOH. 100 pl from each well was transferred into microcuvettes containing 900 pl distilled water. Th e absorbance of each sample was measured at 450 nm with distilled water as the blank. In all tests the following controls were included: ( 1 ) p o o l e d negative serum control, (2) pooled positive serum control, (3) conjugate control {i.e. instead o f serum, PBS pH 7.8 containing 1% BSA was added), (4) substrate c on tr o l (i.e. instead of serum and conjugate PBS pH 7.8 containing 1% BSA was added). Sera ex p ected to contain anticollagen antibodies were tested in serial dilutions. The results were expressed as titers, defined as serum dilutions which give higher absorbance than the pooled negative serum in the first dilution. Visual reading is also possible.
Passive hemagglutination test Passive hemagglutination test was p e r f o r m e d according to the m e t h o d of Beil et al. (1972) using 10 mg collagen per 1 ml h u m a n e r y t h r o c y t e s . RESULTS AND DISCUSSION Several experiments were p e r f o r m e d in order to determine optimal conditions for the assay. In all tests the same collagen t y p e I preparation and the same pooled positive and pooled negative rabbit sera were used. Only the variable to be investigated differed.
Determination o f optimal antigen concentration A 1 : 50 dilution of the pooled positive and negative sera was tested against various antigen concentrations. As shown in Fig. 1, 50 /~g/ml dilution of antigen provided consistent results. Effect o f conjugate dilution on the sensitivity o f the assay Conjugate dilutions of 1 : 500, 1 : 1500 and 1 : 4500 were tested against two-fold serial dilution (1 : 16 to 1 : 2048) of the pooled positive serum and c o m p a r e d to 1 : 16 diluted, pooled negative serum. The best discrimination b etween positive and negative serum was obtained with 1 : 1500 diluted conjugate (Fig. 2). Effect o f adding B S A during serum and conjugate incubation stages on the sensitivity o f the assay To decrease non-specific color d e v e l o p m e n t in conjugate and negative
74 E03 c
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20 50 100 soc bg,m~ CONCE~T~AnON OF C O L L A S E ~ < ¥ P E
Fig. 1. D e t e r m i n a t i o n of optimal antigen concentration for coating. T h e plotted absorbance values express the differences b e t w e e n the absorbance values of pooled positive a n d negative rabbit sera diluted 1 : 50. T h e conjugate dilution w a s I : 1500. $025-,0~
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Fig. 2. Effect o f conjugate dilution. The p l o t t e d absorbance values were o b t a i n e d by subtracting the absorbance o f the reference serum pool (1 : 16) from the absorbances o f the positive serum dilutions. The antigen c o n c e n t r a t i o n for coating was 2 0 / l g / m l .
75 serum controls, addition of BSA to serum and conjugate during incubation was necessary. Fig. 3 shows t hat the presence of BSA during incubation improves discrimination between positive and negative sera and lowers the absorbance o f t he conjugate control. It probabl y prevents non-specific adsorption o f serum protein to the plate. Omission of the BSA during incubation with the conjugate resulted in increased absorbance in the conjugate and negative serum controls (Fig. 4), probably due to non-specific attachm e n t o f conjugate to the microplates. No distinct difference was seen between the relative absorbances of positive and negative sera in the BSA c o n c e n t r a t i o n range (0.5--5%) tested. 1% BSA was a d o p t e d as a convenient working c o n c e n t r a t i o n in b o t h cases. Addition of Tween 20 to the washing buffer as suggested by Voller et al. (1976) p r o d u c e d no improvement.
Reproducibility We d eter mine d the titer of the pooled positive serum in a 4-fold serial dilution (1 : 8 to 1 : 512) and com pa r ed it with the absorbance of pooled negative serum diluted 1 : 8. Assays were carried out on 4 different days using plates coated in different batches. The resulting titer (1 : 128) was always the same (Table 1).
[] POSrIIVESERUM [] NEUAIIVESERUM • CONJUGAIECONTROL{[o)
I
i !
015
010
I
i
O05
1
3 -5 -7 -9
-11 -13 -15f0 2 1
0 % BSA
3
5
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9
11 -13 -15C0 2-1
05 % BSA
-3
5 -7
-9 -11
I °£ BSh
13 -15C0 ~ 1
3 -5
-7
-9 -11 -13 -15[0
SERUMDILUIIOR 5 % 8Sh
Fig. 3. The i n f l u e n c e o£ a d d i n g B S A at the serum i n c u b a t i o n stage on the s e n s i t i v i t y o f t h e assay. Absorbanee values were read using the substrate c o n t r o l as the b l a n k . 1% B S A
was p r e s e n t during c o n j u g a t e i n c u b a t i o n . T h e antigen c o n c e n t r a t i o n for c o a t i n g was 20 p g / m l . T h e c o n j u g a t e d i l u t i o n was 1 : 500.
76 ~ 25 ~
m]
r
[] POSIIIVE SERUM D NEGAIIVE SERUM • CONJUGME CON]ROL (Co)
~030 < 0 Z5
020
015
i i
OlO
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7
8
9
10 11 12 13(0 2-5 -7 -8 -9 -10 -11 -12 -13C0 2- 5 -7 -8 -9 -10 -11 -12 -13(0 ~-5 -7 -8 -9 -10 -11 -12 -13C0 SERUM DKUHON
o °/o BSA
0 5 °/o RSA
~ % BSA
S °/o BSA
Fig. 4. T h e i n f l u e n c e o f a d d i n g B S A at t h e c o n j u g a t e i n c u b a t i o n stage o n t h e s e n s i t i v i t y o f t h e a s s a y . A b s o r b a n c e v a l u e s w e r e r e a d u s i n g t h e s u b s t r a t e c o n t r o l as t h e b l a n k . 1% B S A w a s p r e s e n t d u r i n g s e r u m i n c u b a t i o n . A n t i g e n c o n c e n t r a t i o n for c o a t i n g w a s 50 p g / ml. C o n j u g a t e d i l u t i o n w a s 1 : 500.
Comparison of E L I S A with passive hemagglutination test We found ELISA more sensitive than the passive hemagglutination test. Titers in both tests did not always correlate well. This may be explained as suggested by Menzel (1977) since the passive hemagglutination test detects
TABLE 1 REPRODUCIBILITY OF ELISA F o u r d i f f e r e n t d e t e r m i n a t i o n s o n t h e s a m e s a m p l e s yield t h e s a m e t i t e r (1 : 128). A b s o r b a n c e was m e a s u r e d at 4 5 0 n m . Test no.
1 2 3 4
Serum
positive negative positive negative positive negative positive negative
Serum dilution ] :8
1 : 32
1:128
1 : 512
0.323 0.065 0.307 0.057 0.278 0.055 0.297 0.085
0.328
0.225
0.064
0.342
0.216
0.057
0.264
0.122
0.053
0.280
0.190
0.080
77 IgM a n t i b o d i e s m o r e r e a d i l y , w h e r e a s E L I S A d e t e c t s I g G a n t i b o d i e s . In this s t u d y we h a v e s h o w n t h a t E L I S A is a suitable m e t h o d f o r t h e r o u tine d e t e c t i o n o f a n t i b o d i e s t o collagen I, II a n d I I I in sera. T h e p e r f o r m a n c e o f t h e t e s t d o e s n o t r e q u i r e a n y special e q u i p m e n t a n d can be carried o u t within a few hours. Handling could be further facilitated by mechanization. ACKNOWLEDGEMENTS T h e a u t h o r s wish t o t h a n k Dr. P. Peters, Dr. F. van K n a p e n a n d Dr. A. H a g e n a a r s at t h e N a t i o n a l I n s t i t u t e o f Public H e a l t h , B i l t h o v e n , T h e N e t h e r l a n d s , f o r t h e i n t r o d u c t i o n t o t h e E L I S A m e t h o d . T h e a u t h o r s are also grateful to Dr. T h o m a s E. K w a s i g r o c h f o r h e l p f u l discussion and f o r reading of the manuscript. This w o r k was s u p p o r t e d b y grants f r o m t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a w a r d e d to t h e S o n d e r f o r s c h u n g s b e r e i c h 29 at t h e Freie Universit~it Berlin. REFERENCES Adelmann, B.C., G.J. Gentner and K. Hopper, 1973, J. Immunol. Methods 3,319. Andriopoulos, N.A., J.C. Bennett, J. Mestecky and E.J. Miller, 1975, Arthr. Rheum. 18, 384. Beil, W., H. Furthmayr and R. Timpl, 1972, Immunochemistry 9,779. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109,129. Engvall, E. and E. Ruoslahti, 1977, Int. J. Cancer 20, 1. Lindsley, H., M. Mannik and P. Bornstein, 1971, J. Exp. Med. 126, 1309. Menzel, J., 1977, J. Immunol. Methods 15, 77. Michaeli, D. and H.H. Fudenberg, 1974, Clin. Immunol. Immunopathol. 2, 153. Miller, E.J. and V.J. Matukas, 1974, Fed. Proc. 33, 1197. Rohde, H., H. Nowack, U. Becker and R. Timpl, 1976, J. Immunol. Methods 11, 135. Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi and J. Buys, 1976, J. Immunol. Methods 10, 67. Smith, B.D., G.R. Martin, E.J. Miller, A. Dorfmann and R. Swarm, 1975, Arch. Biochem. Biophys. 166, 181. Steffen, C., F. Schuster, G. Tausch, R. Timpl and J. Becket, 1968, Klin. Wschr. 46,976. Timpl, R., P.P. Fietzek, H. Furthmayr, W. Meigel and K. Kiihn, 1970, FEBS Lett. 9, 11. Voller, A., D.E. Bidwell and A. Bartlett, 1976, Bull. Wld Hlth Org. 53, 55. Von der Mark, H., K. Von der Mark and S. Gay, 1976, Develop. Biol. 48,237.
71
ENZYME-LINKED IMMUNOSORBENT MICROASSAY FOR QUANTIFICATION OF SPECIFIC ANTIBODIES TO COLLAGEN TYPE I, II, III
BIRGIT GOSSLAU and HANS-JURGEN B A R R ACH
Institu t fiir Toxikologie und Embryopharmakologie, Freie Universitiit Berlin, D-1000 Berlin 33, G.F.R. (Received 26 October 1978, accepted 28 February 1979) An enzyme-linked immunosorbent assay (ELISA) is described for determination of antibodies to collagen type I, II, III in serum or other body fluids. Polystyrene microplates are coated with either collagen I, II or III, and samples of body fluids being tested are incubated in the plates. Antibodies which attach specifically to the collagen are detected with anti-IgG antibody conjugated with peroxidase. Bound peroxidase is quantitatively estimated by the colour reaction produced with the substrate 5-aminosalicylic acid.
INTRODUCTION
Detection of anticollagen antibodies has been performed by various methods. Passive hemagglutination tests have been extensively used (Steffen et al., 1968; Timpl et al., 1970; Beil et al., 1972; Michaeli and Fudenberg, 1974; Andriopoulos et al., 1975). Radioimmunoassay has been demonstrated to be more sensitive for quantitation of antibodies to collagen (Lindsley et al., 1971; Adelmann et al., 1973; Rohde et al., 1976; Menzel, 1977). We expected the micro-sandwich ELISA to be a useful alternative, since it does not require isotopes. Engvall and Ruoslahti (1977) have used ELISA for binding tests. Collagen and other proteins were adsorbed to a plastic surface and a fibronectin solution was added. The bound fibronectin was detected using fibronectin antibodies. We use collagen coated microplates to identify antibodies to collagen type I, II and III in body fluids by an ELISA system adapted from the m e t h o d developed by Engvall and Perlmann (1972). The test is used routinely to determine the titer of antibodies to collagen type I, II and III in hyperimmune sera derived from rabbits and sheep and to examine the sera of patients with polyarthritis or collagen diseases (Gosslau and Barrach, in preparation). MATERIALS AND METHODS
Preparation of antigen Collagen type I and IIi were prepared by acetic acid extraction of the skin of mice of postnatal day 3--6 (Miller and Matukas, 1974; Von der Mark et
72 al., 1976). Separation of the two collagen types was performed by repeated sequential.salt precipitation at pH 7.5 (50 mM Tris buffer) with 1.2 M, 1.7 M (collagen type III) and 2.5 M NaC1 (collagen type I). Collagen type II was isolated from Swarm rat chondrosarcoma according to Smith et al. (1975). It was extracted with 200 mM NaC1, precipitated twice by 20% NaC1 at pH 7.6 and further purified by diethylaminoethyl cellulose chromatography.
Coating o f microplates with antigen 5 mg collagen type I (or type III) were dissolved in 100~ml coating buffer I containing 0.02% NAN3. 5 mg collagen type II were dissolved in 100 ml coating buffer II containing 0.02% NAN3. Coating buffer I (pH 7.5) contained 7 mM CH3COOH, 7 mM KzHPO4, 0.17 M NaC1. Coating buffer II (pH 8.3) contained 0.1 M NaHCO3, 0.5 M NaC1. The test was performed in polystyrene Microtiter Plates M29A (Greiner, Nfirtingen, G.F.R.). The trays were coated with 100 pl antigen solution per well for at least 1 day at 4 ° C. Routinely the plates were allowed to stand for 6 days. They were then washed 3 times. For each wash the plates were filled with 100 pl cold PBS pH 7.8 per well. After 5 min the PBS was removed and the plates were emptied and shaken dry. Then the plates were stored over P205 (Sicapent, Merck, Darmstadt, G.F.R.) in vacuum until use. They were kept in this way up to 4 months. PBS = 0.12 M NaC1, 0.01 M Na2HPO4, 0.004 M KH2PO4, pH 7.8.
Enzyme-linked antiglobulin conjugates The conjugates were horseradish peroxidase labeled goat antirabbit IgG (Miles Laboratories, Frankfurt, G.F.R.) and horseradish peroxidase labeled rabbit antisheep IgG (Nordic Immunological Laboratories, Dietzenbach, G.F.R).
Substrate solution 80 mg 5-aminosalicylic acid (Ferak, Berlin, G.F.R.) were dissolved in 100 ml distilled water at 70°C. The solution was cooled to room temperature. Before use the pH was adjusted to 6.0 by adding 1 N NaOH. 9 ml of this stock solution were mixed with 1 ml of 0.05% H202 (Voller et al., 1976).
ELISA The assay is a variation of the ELISA m e t h o d described by Engvall and Perlmann (1972) and modified by Ruitenberg et al. (1976). For antibodies against collagen type I, II or III the following procedure was used: serial dilutions of sera were made using PBS, pH 7.8. Of each serum dilution 100 ~l, containing 1% bovine serum albumin (BSA) fraction V (Sigma Chemie GmbH, Munich, G.F.R.), were transferred to the collagen coated wells. The trays were incubated for 45 min at room temperature on a Microshaker (Flow Laboratories GmbH, Bonn, G.F.R.). After washing 3 times with PBS,
73 pH 7.8, 100 pl of PBS-diluted conjugate containing 1% BSA were added. The microplates were again incubated on the Microshaker for 45 min at r o o m t e m p e r a t u r e and then washed 3 times with PBS pH 7.8. The a m o u n t of b o u n d peroxidase was det er m i ned by adding 100 pl of the substrate solution. After 1 h the reaction was stopped by 100 pl 1 N NaOH. 100 pl from each well was transferred into microcuvettes containing 900 pl distilled water. Th e absorbance of each sample was measured at 450 nm with distilled water as the blank. In all tests the following controls were included: ( 1 ) p o o l e d negative serum control, (2) pooled positive serum control, (3) conjugate control {i.e. instead o f serum, PBS pH 7.8 containing 1% BSA was added), (4) substrate c on tr o l (i.e. instead of serum and conjugate PBS pH 7.8 containing 1% BSA was added). Sera ex p ected to contain anticollagen antibodies were tested in serial dilutions. The results were expressed as titers, defined as serum dilutions which give higher absorbance than the pooled negative serum in the first dilution. Visual reading is also possible.
Passive hemagglutination test Passive hemagglutination test was p e r f o r m e d according to the m e t h o d of Beil et al. (1972) using 10 mg collagen per 1 ml h u m a n e r y t h r o c y t e s . RESULTS AND DISCUSSION Several experiments were p e r f o r m e d in order to determine optimal conditions for the assay. In all tests the same collagen t y p e I preparation and the same pooled positive and pooled negative rabbit sera were used. Only the variable to be investigated differed.
Determination o f optimal antigen concentration A 1 : 50 dilution of the pooled positive and negative sera was tested against various antigen concentrations. As shown in Fig. 1, 50 /~g/ml dilution of antigen provided consistent results. Effect o f conjugate dilution on the sensitivity o f the assay Conjugate dilutions of 1 : 500, 1 : 1500 and 1 : 4500 were tested against two-fold serial dilution (1 : 16 to 1 : 2048) of the pooled positive serum and c o m p a r e d to 1 : 16 diluted, pooled negative serum. The best discrimination b etween positive and negative serum was obtained with 1 : 1500 diluted conjugate (Fig. 2). Effect o f adding B S A during serum and conjugate incubation stages on the sensitivity o f the assay To decrease non-specific color d e v e l o p m e n t in conjugate and negative
74 E03 c
-,
p
j
z
., / f
<
~
filz o t~
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z
~
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~:
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/ /
/
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-
i
I
35
5
~
]
i
"
i
20 50 100 soc bg,m~ CONCE~T~AnON OF C O L L A S E ~ < ¥ P E
Fig. 1. D e t e r m i n a t i o n of optimal antigen concentration for coating. T h e plotted absorbance values express the differences b e t w e e n the absorbance values of pooled positive a n d negative rabbit sera diluted 1 : 50. T h e conjugate dilution w a s I : 1500. $025-,0~
S
I ] 0 ~ , IGA--E
2o 0 2 0 - ---
• - -
1
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1 500
i
4 500
-- - - * ' - - -
OIL
.
Q@~I
IV[LN
--
0
•
500
//
/,/'
015--
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~i////&! 0.10 - -
//
0.05 --
10
-L.
10-3
10-2
10-1 SERUM
DIL:
-~ i ! ,
Fig. 2. Effect o f conjugate dilution. The p l o t t e d absorbance values were o b t a i n e d by subtracting the absorbance o f the reference serum pool (1 : 16) from the absorbances o f the positive serum dilutions. The antigen c o n c e n t r a t i o n for coating was 2 0 / l g / m l .
75 serum controls, addition of BSA to serum and conjugate during incubation was necessary. Fig. 3 shows t hat the presence of BSA during incubation improves discrimination between positive and negative sera and lowers the absorbance o f t he conjugate control. It probabl y prevents non-specific adsorption o f serum protein to the plate. Omission of the BSA during incubation with the conjugate resulted in increased absorbance in the conjugate and negative serum controls (Fig. 4), probably due to non-specific attachm e n t o f conjugate to the microplates. No distinct difference was seen between the relative absorbances of positive and negative sera in the BSA c o n c e n t r a t i o n range (0.5--5%) tested. 1% BSA was a d o p t e d as a convenient working c o n c e n t r a t i o n in b o t h cases. Addition of Tween 20 to the washing buffer as suggested by Voller et al. (1976) p r o d u c e d no improvement.
Reproducibility We d eter mine d the titer of the pooled positive serum in a 4-fold serial dilution (1 : 8 to 1 : 512) and com pa r ed it with the absorbance of pooled negative serum diluted 1 : 8. Assays were carried out on 4 different days using plates coated in different batches. The resulting titer (1 : 128) was always the same (Table 1).
[] POSrIIVESERUM [] NEUAIIVESERUM • CONJUGAIECONTROL{[o)
I
i !
015
010
I
i
O05
1
3 -5 -7 -9
-11 -13 -15f0 2 1
0 % BSA
3
5
?
9
11 -13 -15C0 2-1
05 % BSA
-3
5 -7
-9 -11
I °£ BSh
13 -15C0 ~ 1
3 -5
-7
-9 -11 -13 -15[0
SERUMDILUIIOR 5 % 8Sh
Fig. 3. The i n f l u e n c e o£ a d d i n g B S A at the serum i n c u b a t i o n stage on the s e n s i t i v i t y o f t h e assay. Absorbanee values were read using the substrate c o n t r o l as the b l a n k . 1% B S A
was p r e s e n t during c o n j u g a t e i n c u b a t i o n . T h e antigen c o n c e n t r a t i o n for c o a t i n g was 20 p g / m l . T h e c o n j u g a t e d i l u t i o n was 1 : 500.
76 ~ 25 ~
m]
r
[] POSIIIVE SERUM D NEGAIIVE SERUM • CONJUGME CON]ROL (Co)
~030 < 0 Z5
020
015
i i
OlO
005~' 2-6
7
8
9
10 11 12 13(0 2-5 -7 -8 -9 -10 -11 -12 -13C0 2- 5 -7 -8 -9 -10 -11 -12 -13(0 ~-5 -7 -8 -9 -10 -11 -12 -13C0 SERUM DKUHON
o °/o BSA
0 5 °/o RSA
~ % BSA
S °/o BSA
Fig. 4. T h e i n f l u e n c e o f a d d i n g B S A at t h e c o n j u g a t e i n c u b a t i o n stage o n t h e s e n s i t i v i t y o f t h e a s s a y . A b s o r b a n c e v a l u e s w e r e r e a d u s i n g t h e s u b s t r a t e c o n t r o l as t h e b l a n k . 1% B S A w a s p r e s e n t d u r i n g s e r u m i n c u b a t i o n . A n t i g e n c o n c e n t r a t i o n for c o a t i n g w a s 50 p g / ml. C o n j u g a t e d i l u t i o n w a s 1 : 500.
Comparison of E L I S A with passive hemagglutination test We found ELISA more sensitive than the passive hemagglutination test. Titers in both tests did not always correlate well. This may be explained as suggested by Menzel (1977) since the passive hemagglutination test detects
TABLE 1 REPRODUCIBILITY OF ELISA F o u r d i f f e r e n t d e t e r m i n a t i o n s o n t h e s a m e s a m p l e s yield t h e s a m e t i t e r (1 : 128). A b s o r b a n c e was m e a s u r e d at 4 5 0 n m . Test no.
1 2 3 4
Serum
positive negative positive negative positive negative positive negative
Serum dilution ] :8
1 : 32
1:128
1 : 512
0.323 0.065 0.307 0.057 0.278 0.055 0.297 0.085
0.328
0.225
0.064
0.342
0.216
0.057
0.264
0.122
0.053
0.280
0.190
0.080
77 IgM a n t i b o d i e s m o r e r e a d i l y , w h e r e a s E L I S A d e t e c t s I g G a n t i b o d i e s . In this s t u d y we h a v e s h o w n t h a t E L I S A is a suitable m e t h o d f o r t h e r o u tine d e t e c t i o n o f a n t i b o d i e s t o collagen I, II a n d I I I in sera. T h e p e r f o r m a n c e o f t h e t e s t d o e s n o t r e q u i r e a n y special e q u i p m e n t a n d can be carried o u t within a few hours. Handling could be further facilitated by mechanization. ACKNOWLEDGEMENTS T h e a u t h o r s wish t o t h a n k Dr. P. Peters, Dr. F. van K n a p e n a n d Dr. A. H a g e n a a r s at t h e N a t i o n a l I n s t i t u t e o f Public H e a l t h , B i l t h o v e n , T h e N e t h e r l a n d s , f o r t h e i n t r o d u c t i o n t o t h e E L I S A m e t h o d . T h e a u t h o r s are also grateful to Dr. T h o m a s E. K w a s i g r o c h f o r h e l p f u l discussion and f o r reading of the manuscript. This w o r k was s u p p o r t e d b y grants f r o m t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a w a r d e d to t h e S o n d e r f o r s c h u n g s b e r e i c h 29 at t h e Freie Universit~it Berlin. REFERENCES Adelmann, B.C., G.J. Gentner and K. Hopper, 1973, J. Immunol. Methods 3,319. Andriopoulos, N.A., J.C. Bennett, J. Mestecky and E.J. Miller, 1975, Arthr. Rheum. 18, 384. Beil, W., H. Furthmayr and R. Timpl, 1972, Immunochemistry 9,779. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109,129. Engvall, E. and E. Ruoslahti, 1977, Int. J. Cancer 20, 1. Lindsley, H., M. Mannik and P. Bornstein, 1971, J. Exp. Med. 126, 1309. Menzel, J., 1977, J. Immunol. Methods 15, 77. Michaeli, D. and H.H. Fudenberg, 1974, Clin. Immunol. Immunopathol. 2, 153. Miller, E.J. and V.J. Matukas, 1974, Fed. Proc. 33, 1197. Rohde, H., H. Nowack, U. Becker and R. Timpl, 1976, J. Immunol. Methods 11, 135. Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi and J. Buys, 1976, J. Immunol. Methods 10, 67. Smith, B.D., G.R. Martin, E.J. Miller, A. Dorfmann and R. Swarm, 1975, Arch. Biochem. Biophys. 166, 181. Steffen, C., F. Schuster, G. Tausch, R. Timpl and J. Becket, 1968, Klin. Wschr. 46,976. Timpl, R., P.P. Fietzek, H. Furthmayr, W. Meigel and K. Kiihn, 1970, FEBS Lett. 9, 11. Voller, A., D.E. Bidwell and A. Bartlett, 1976, Bull. Wld Hlth Org. 53, 55. Von der Mark, H., K. Von der Mark and S. Gay, 1976, Develop. Biol. 48,237.