Antigen attachment in ELISA

Journal of Immunological Methods, 34 (1980) 61--70 © Elsevier/North-Holland Biomedical Press

61

A N T I G E N A T T A C H M E N T IN E L I S A

O.-P. LEHTONEN and M.K. VILJANEN Department of Medical Microbiology, Turku University, SF-20520 Turku, Finland

(Received 1 October 1979, accepted 3 December 1979)

The amount of antigen on the solid phase was studied during various steps of an enzyme-linked immunosorbent assay (ELISA) for chicken anti-bovine serum albumin (BSA) antibodies. Three materials for the solid phase were used: polystyrene, nylon and cyanogen bromide activated paper. There was noticeable leakage of antigen during the assay from both polystyrene (30%) and nylon (60%). This occurred during all steps of the assay: during incubation with the sample serum, incubation with the enzyme conjugated antibodies and during the washing procedures. During incubation with the sample serum detachment of antigen was quite rapid and could result in competition between bound and free antigen for antibodies. The presence of antigen-antibody complexes in reaction mixtures could also lead to erroneous results in the antibody assay. With covalent coupling of antigen to solid phase (cyanogen bromide activated paper), batch-to-batch variation in the surface concentration of antigen was strikingly less than in the assay using plastics. Detachment of antigen from activated paper during the assay was less than with polystyrene or nylon. ELISA with activated paper as the solid phase thus seems suitable as a reference method for standardisation of the antigen phase in ELISA systems. INTRODUCTION E n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) has b e c o m e a p o p u l a r m e t h o d f o r q u a n t i t a t i o n o f antibodies. It is applicable t o m a n y antigens (Schuurs a n d Van W e e m e n , 1 9 7 7 ) and is very simple and rapid t o p e r f o r m . In spite o f this w i d e s p r e a d use, s t a n d a r d i s a t i o n o f the m e t h o d has been s o m e w h a t neglected. F o r results f r o m d i f f e r e n t l a b o r a t o r i e s t o be c o m p a r able it is necessary f o r the reagents used f o r E L I S A t o be standardized. A p r i m a r y c o n c e r n is t h e a m o u n t a n d q u a l i t y o f t h e antigen in t h e assay as p o i n t e d o u t b y Bidwell et al. ( 1 9 7 7 ) and K a l m a k o f f et al. (1977). V a r i o u s materials have been used as t h e solid phase in ELI~ck (Schuurs a n d Van W e e m e n , 1 9 7 7 ) , especially plastics, p a r t l y o w i n g t o their c o m m e r c i a l availability. Antigens are a d s o r b e d t o plastic p o l y m e r surfaces b y weak, m a i n l y physical forces (Van Oss a n d Singer, 1 9 6 6 ) . The n a t u r e o f these forces is such t h a t d o u b t is cast on t h e stability o f t h e antigen phase d u r i n g t h e assay. In o r d e r t o d e v e l o p a q u a n t i t a t i v e E L I S A f o r c h i c k e n anti-bovine serum a l b u m i n (BSA) a n t i b o d i e s , we studied t w o c o m m e r c i a l l y p r o d u c e d solid phases a n d c o m p a r e d t h e m with a c o v a l e n t l y c o u p l e d antigen phase.

62 MATERIALS AND METHODS

Antigen Crystalline bovine serum albumin (fraction V from bovine plasma) from A r m o u r Pharmaceutical Co. Ltd., U.K. or Miles Laboratories Ltd., U.K. was used as antigen. 12SI-labelled BSA was prepared as described by H u n t e r and G r e e n w o o d (1962). Briefly, 2 mCi of [12SI]Na (The Radiochemical Centre, Amersham, U.K.), 50 pl BSA (5.0 mg/ml) and 50 pl chloramine-T (4.0 mg/ml) in phosphate-buffered saline, pH 7.4 (PBS), were rapidly mixed for 15 sec. The reaction was stopped by 100 ttl sodium metabisulphate (4.8 mg/ml) in PBS. The iodinated p r o d u c t was rinsed out o f the vial using PBS. Free iodide was separated b y dialysing 3 times against 1.0 litre PBS at 4 ° C. The specific activity obtained was 2--6 #Ci/gg BSA. O f the total iodide in the final preparation 95--97% was protein b o u n d as determined by precipitation with 20% trichloroacetic acid.

Solid phases Disposable p o l y s t y r e n e cuvettes (Finnpipette ® Labsystems, Helsinki, Finland) were initially studied. These cuvettes are com bi ned to form 9-cuvette blocks, adaptable to a 9-channel p h o t o m e t e r (FP-9 analyzer, Finnpipette ® Labsystems). Later, n y l o n rings designed to be inserted into the cuvettes for incubation and m eas ur em e nt were used. The rings were also produced by Finnpipette Labsystems. Activated paper for covalent coupling was prepared according to Ceska and Lundqvist (1972). A b o u t 400 cm 2 (both sides calculated) o f semitransp a r en t and semiglossy paper (Wulff, Helsinki, Finland) was allowed to swell for 2--5 min in distilled water on a reel for developing cinema film (Paterson, U.K.). Twenty-five grams cyanogen bromide (Eastman Kodak, Rochester, NY 14650, U.S.A.) was dissolved into 1.0 litre distilled water. The paper was p ut into the solution and the pH immediately raised to 10.5 with 1".0 M NaOH and kept at this level until 100 ml of NaOH was consumed. The paper was t hen washed 10 times with 0.005 M sodium bicarbonate. After washing twice with water the paper was d e h y d r a t e d in an increasing acetone gradient (20%, 40%, 60%, 80%, 100%) in volumes of 500 ml. The paper was dried and stored at 4 ° C.

Anti-BSA antisera To provide a standard pool o f anti-BSA antibodies White Leghorn chickens received 8 intraperitoneal injections o f 1.0 mg BSA at weekly intervals. The pooled sera were stored at --20°C in small aliquots. Control sera were obtained from normal 4-week-old chickens.

63

Preparation of an tiglo bulin Sheep anti-chicken-~-antibodies were prepared as described by Viljanen et al. (1975). Briefly, chicken immuniglobulins were precipitated with sodium sulphate. The precipitate was dissolved, dialyzed and applied to a Sephadex G-200 column (Pharmacia, Uppsala, Sweden). The IgG fraction thus obtained was dialysed and further purified by DEAE-chromatography. Sera from sheep injected with this material were absorbed by passage through Sepharose 4B columns (Pharmacia) with bound agammaglobulinemic chicken serum and purified chicken IgM. The absorbed antiserum was then passed through a Sepharose 4B column coupled with the chicken IgG used for immunisation. The eluted anti-chicken-~, was free from other antibodies and other serum proteins.

Preparation of enzyme-antiglobulin conjugate For labelling the anti-~' antibodies with alkaline phosphatase the single step m e t h o d described by Avrameas (1969) was used. An aliquot o f 0.5 ml o f alkaline phosphatase, Sigma type VII (Sigma Chemical Company, St. Louis., MO, U.S.A.), was centrifuged for 15 min at 10°C (350 Xg). After removing 0.2 ml o f the supernatant, 0.1 ml of the antiglobulin at a concentration of 5 mg/ml was added. The mixture was dialysed against PBS for 12 h. Glutaraldehyde (Merck) as a 4.2% solution was added in a volume of 10 pl and conjugation allowed to occur for 2 h at 20°C. The preparation was eluted with 1.0 ml PBS and dialysed again. The conjugate antibody was purified on a Sepharose 6B column (Pharmacia). Elution was with 0.05 M Tris-HC1 buffer (pH 8.0).

ELISA in polystyrene cuvettes BSA in PBS was placed in the cuvettes in volumes o f 1.0 ml. The corresponding coated area was 5.08 cm 2. Coupling was at room temperature for 16 h. 12SI-labelled BSA in the coupling solution had a specific activity o f 10-30 cpm/ng BSA. The coupling solution was then aspirated and the cuvettes washed with PBS. To reduce nonspecific attachment o f antibodies, 1.0 ml o f 0.1% gelatin in PBS (PBS-gelatin) was added to the cuvettes and incubated for 6 h at room temperature. The cuvettes were then washed with 0.9% NaCI containing 0.05% Tween-20 (NaC1-Tw). The last wash was with PBS. Sera diluted in PBS-gelatin were added and the cuvettes incubated for 16 h at room temperature. After washing twice with NaC1-Tw and once with PBS the conjugate was added. The conjugate was diluted in PBS-gelatin. The dilution was determined separately for each lot of conjugate and was usually 1 : 150--1 : 200. After washing as before, enzyme activity was measured by adding 1.0 ml p-nitrophenylphosphate (Sigma Chemical Company), 2.0 mg/ ml in diethanolamine-MgC12 buffer (Orion Diagnostica, Espoo, Finland).

64 T h e e n z y m e r e a c t i o n was carried o u t f o r 3 0 - - 6 0 m i n at 37°C, and was t h e n s t o p p e d b y a d d i t i o n o f 1.0 M N a O H . T h e i n t e n s i t y o f t h e c o l o u r e d end produ c t , p - n i t r o p h e n o l a t e , was m e a s u r e d in t h e c u v e t t e s w i t h t h e 9 - c h a n n e l p h o t o m e t e r ( F i n n p i p e t t e L a b s y s t e m s ) at a w a v e l e n g t h o f 4 0 5 n m . T h e absorb a n c e s w e r e c o r r e c t e d b y s u b t r a c t i n g t h e a b s o r b a n c e o f a b l a n k c u v e t t e containing the same amounts of substrate and NaOH. T h e [12sI] BSA in t h e c u v e t t e s was c o u n t e d w i t h a g a m m a scintillation c o u n t e r ( L K B U l t r o G a m m a 1280, Wallac, T u r k u , Finland). T h e b l o c k s w e r e c u t i n t o individual c u v e t t e s w i t h a h o t knife. T h e s a m e c u v e t t e s were c o u n t e d r e p e a t e d l y d u r i n g t h e E L I S A p r o c e d u r e , every c u v e t t e t h u s serving as its o w n c o n t r o l . In o r d e r to s t u d y t h e t i m e c o u r s e o f antigen leakage d u r i n g t h e p r i m a r y i n c u b a t i o n a c o n s t a n t d i l u t i o n o f 1 : 3 0 0 0 o f the i m m u n e s e r u m p o o l was used. T o s t u d y t h e a m o u n t o f d e t a c h e d antigen c o m p l e x e d with t h e p r i m a r y a n t i b o d y a v o l u m e o f 0.9 m l was p i p e t t e d o u t o f e a c h c u v e t t e a n d t r a n s f e r r e d t o glass t u b e s . T o equalise t h e globulin c o n t e n t o f each t u b e , 0.1 ml o f norm a l s h e e p s e r u m was a d d e d . T h e globulins w e r e p r e c i p i t a t e d b y adding 1.0 m l s a t u r a t e d a m m o n i u m s u l p h a t e at 0 ° C. A f t e r 30 m i n t h e p r e c i p i t a t e s were s p u n d o w n at 1 0 0 0 × g f o r 30 m i n at 4°C. T h e activities o f t h e p r e c i p i t a t e s were then counted. E L I S A o n n y l o n rings N y l o n rings, 200 at a t i m e , w e r e c o u p l e d w i t h BSA in a v o l u m e o f 2 0 0 ml PBS w i t h c o n s t a n t stirring f o r 9 h at 37 ° C. T h e rings were w a s h e d o n c e w i t h PBS a n d t h e n p l a c e d in PBS-gelatin f o r 14 h at 20°C. T h e r e a f t e r t h e y were w a s h e d 5 t i m e s w i t h NaC1-Tw a n d 3 t i m e s w i t h PBS. T h e rings w e r e p l a c e d in plastic t u b e s f o r E L I S A i n c u b a t i o n s w h i c h were similar to t h o s e in t h e p o l y s t y r e n e c u v e t t e s . F o r the e n z y m e r e a c t i o n t h e rings w e r e t r a n s f e r r e d t o p o l y s t y r e n e c u v e t t e s . This was d o n e b e c a u s e smaller v a r i a t i o n s b e t w e e n parallel d e t e r m i n a t i o n s w e r e o b s e r v e d t h a n w h e n p e r f o r m i n g t h e e n z y m e r e a c t i o n in t h e tubes. E L I S A on activated p a p e r An a r e a o f 200 c m 2 o f a c t i v a t e d p a p e r was p l a c e d in a b e a k e r c o n t a i n i n g 4 0 0 ml o f t h e BSA c o u p l i n g s o l u t i o n . PBS was used f o r c o u p l i n g b e c a u s e it was f o u n d to be as effective as b i c a r b o n a t e b u f f e r . A f t e r 8 - - 1 2 h at r o o m t e m p e r a t u r e , t h e p a p e r was w a s h e d w i t h PBS a n d t h e reactive g r o u p s b l o c k e d w i t h 0.1% h u m a n s e r u m a l b u m i n ( H S A ) or 0.05 M e t h a n o l a m i n e in 0.1 M NaHCO3 b u f f e r . All washes i n c l u d e d rinsing 3 t i m e s w i t h N a C I - T w a n d o n c e w i t h PBS. A f t e r drying, small discs w e r e p u n c h e d o u t w i t h p u n c h pliers (Maun, U.K.). I n c u b a t i o n o f discs w i t h s e r u m s a m p l e s a n d c o n j u g a t e t o o k p l a c e in v o l u m e s o f 1.0 ml in plastic t u b e s w i t h c o n s t a n t rolling at r o o m t e m p e r a t u r e . T h e i n c u b a t i o n p e r i o d in b o t h cases was 12 h. T h e s e r u m

65 s a m p l e a n d t h e c o n j u g a t e w e r e d i l u t e d in PBS-gelatin c o n t a i n i n g 0.1% T w e e n - 2 0 . T h e o p t i m a l d i l u t i o n o f t h e c o n j u g a t e was d e t e r m i n e d s e p a r a t e l y . F o r t h e e n z y m e r e a c t i o n t h e discs w e r e c a r e f u l l y t r a n s f e r r e d i n t o p o l y s t y r e n e c u v e t t e s a n d t h e s u b s t r a t e a d d e d in a v o l u m e o f 1.0 ml. Finally t h e discs w e r e p i c k e d o u t o f t h e c u v e t t e s w i t h a needle in o r d e r to a l l o w d i r e c t m e a s u r e m e n t o f t h e a b s o r b a n c e t h r o u g h t h e cuvettes. RESULTS 12SI-labelled BSA was u s e d t o s t u d y t h e a t t a c h m e n t o f BSA t o p o l y s t y r e n e c u v e t t e s . T h e a m o u n t o f b o u n d BSA was d e t e r m i n e d a f t e r each wash in t h e c o u p l i n g p r o c e d u r e . It was f o u n d t h a t t h e n u m b e r o f washes was critical w i t h regard to t h e final surface c o n c e n t r a t i o n . A n t i g e n d e t a c h m e n t o c c u r r e d t o a c o n s i d e r a b l e degree u p to t h e sixth wash (Table 1). It was also f o u n d t h a t s u f f i c i e n t w a s h i n g a l m o s t equalised t h e final surface c o n c e n t r a t i o n s o f antigen p r o d u c e d b y t w o d i f f e r e n t c o n c e n t r a t i o n s o f c o u p l i n g s o l u t i o n (10 m g / m l a n d 100 p g / m l ) . In general it was f o u n d t h a t t h e c u v e t t e s w e r e difficult to c o u p l e t o a pred i c t e d c o n s t a n t surface c o n c e n t r a t i o n d e s p i t e a d e q u a t e washing. F u r t h e r m o r e , clear d i f f e r e n c e s b e t w e e n various c o u p l i n g b a t c h e s w e r e o b s e r v e d . T h e s t a n d a r d d e v i a t i o n o f t h e antigen surface c o n c e n t r a t i o n s in t h e c u v e t t e s was 51% o f t h e m e a n surface c o n c e n t r a t i o n in 6 s e p a r a t e c o u p l i n g experiments. D u r i n g t h e c o m p l e t e E L I S A p r o c e d u r e antigen d e t a c h m e n t was s t u d i e d b y c o u n t i n g t h e r a d i o a c t i v i t y o f each c u v e t t e at various stages (Table 2). In t h e s e r u m i n c u b a t i o n stage d e t a c h m e n t was 3 - - 8 % o f t h e initial a m o u n t o f t h e antigen. S e r u m s e e m e d n o t t o be an essential f a c t o r in t h e leakage p h e n o m e n o n . On the c o n t r a r y , t h e r e was a slight decrease in t h e leakage at high s e r u m c o n c e n t r a t i o n s (1 : 40 a n d 1 : 160). In t h e first wash (A) t h e r e was a c o n s t a n t leakage o f a b o u t 7 - - 1 0 % , again e x c e p t at high s e r u m c o n c e n t r a -

TABLE 1 BSA DETACHMENT DURING WASHES OF POLYSTYRENE CUVETTES Number of washes 2 3 4 5 6

BSA concentration on the surface (ng/sq. cm) a 10 mg/ml b

100 pg/ml b

570 -+ 160 120-+ 20 85-+ 22 55 + 4.8 46-+ 5.4

130 -+ 23 58-+ 8.5 48-+ 9.4 46 + 7.9 32 -+ 7.8

a Measured in triplicate. Mean -+ S.D. are given. b Concentration of BSA in the coupling solution.

DURING

VARIOUS

STAGES

OF

ELISA

PROCEDURE

IN

BSA-COATED

POLYSTYRENE

2.87-+1.41 3.05 + 0 . 9 0 6.22 -+ 6 . 4 3 4.55_+1.76 3.95_+2.77 7.50_+6.05 6.47-+5.26 4.28 -+ 2.09 3.79 -+ 2.23 5.12 -+ 3.31

1:40 1 : 160 1 : 640 1:2560 1 : 10240 1:40960 1 : 163840 1 : 655 360 1 : 2 621 4 4 0 Buffer

a B S A 66 n g / c m 2.

Detached at t h e serum stage

Serum dilution

4.64-+3.61 5.05 _+ 2.05 7.12 + 2.96 10.1 + 2 . 0 4 6.75+2.16 7.29+2.96 7.93+2.09 7.24 -+ 2.31 8.33 -+ 1.28 8.07 + 1.56

Detached at t h e first w a s h (A) 15.0 + 1 7 . 7 9.05 + 10.5 9 . 7 2 -+ 1 0 . 0 6.37-+ 7 . 0 3 3.69+ 1.51 2.76-+ 0 . 4 5 5.01- + 3.46 5.99 -+ 4.21 3.64 -+ 0.97 3.12 -+ 1.55

Detached at t h e conjugate stage 12.2 8.11 7.28 11.9 11.1 10.9 13.1 8.12 8.92 9.00

+7.73 + 2.26 -+ 4.08 +3.49 -+7.02 -+4.41 -+9.08 -+ 3.56 __%2.55 + 4.04

Detached with the following w a s h (B)

21.4+7.23 19.8 + 4.87 25.2 -+ 7.67 28.8_+3.37 24.2+4.80 26.8+6.93 27.5+6.48 21.5 --%3.33 23.1 -+ 5.00 24.0 + 4 . 4 6

Total detachment

0.380+0.048 0.580 + 0.081 0.739 + 0.176 0.715-+0.093 0.617-+0.061 0.301-+0.066 0.118+0.019 0 . 0 4 5 -+ 0 . 0 0 9 0 . 0 1 0 -+ 0 . 0 0 2 0 . 0 0 5 -+ 0 . 0 0 8

Absorbance p e r 0.5 h

Figures r e p r e s e n t p e r c e n t a g e o f t h e initial a m o u n t b e f o r e a d d i n g t h e s e r u m d i l u t i o n ( e x c e p t f o r last c o l u m n ) . T h e m e a n + S.D. o f 7 parallel d e t e r m i n a t i o n s are given.

ANTIGEN DETACHMENT VETTES a

TABLE 2 CU-

67 tions. F u r th er leakage occurred during secondary incubation with the conjugate and again in the following wash (B). The total d e t a c h m e n t varied b e tween 20 and 29%. Leakage during the primary serum incubation was studied using different times o f incubation (Table 3). A b o u t half o f the total leakage had taken place within 15 min. After 2 h almost three-quarters of the total leaking antigen was detached. The assay eluate in the secondary (conjugate) incubation, where the same a m o u n t o f conjugate had been added to each cuvette, was analysed by a m m o n i u m sulphate precipitation (Table 4). The detached a m o u n t of the antigen was almost equal (3.5--4.9%) at all dilutions of the primary a n t i b o d y studied. However, 62.8% of the detached antigen was precipitable at the highest c o n c e n t r a t i o n o f the primary antibody. Only 15.3% was precipitable when only b u f f e r was present in the primary incubation. The a t t a c h m e n t and d e t a c h m e n t o f BSA was studied also with nyl on and with cyanogen bromide activated paper (Table 5). On nylon rings surface concentrations of 150 and 210 ng/cm 2 were achieved by using coupling concentrations o f 10 and 100 pg/ml, respectively. From one ring to a n o t h e r the a m o u n t coupled varied slightly less than for the pol yst yrene cuvettes. However, the total d e t a c h m e n t was considerable (60 and 48%). On the cyanogen bromide activated paper BSA could be coupled t o a surface c o n c e n t r a t i o n o f 140 and 540 ng/cm 2 by using coupling concentrations o f 1 and 10 pg/ml. The variation between individual discs was small: the standard deviations were 5.2 and 4.3% of the mean values. The standard deviation o f surface concent r a t i ons on discs was 15% of the mean value in 5 separate coupling experiments. D e t a c h m e n t during ELISA was 13% in bot h concentrations and its variation between discs was low (S.D.s 4.2 and 5.3% o f mean values). In the ELISA with activated paper, high background values were initially obtained. Ethanolamine was f ound to be superior to HSA in blocking the

TABLE 3 EFFECT OF TIME ON ANTIGEN LEAKAGE DURING PRIMARY INCUBATION STAGE Time from adding the serum

Amount of antigen (percentage of initial amount) a

15min 30min lh 2h 10h 14h

2.4±2.7 1.8±0.7 2.6±0.8 3.3±1.6 4.6±2.8 4.6±1.8

a Mean + S.D. of 5 parallel determinations.

68 TABLE 4 PRECIPITATION ANALYSIS OF THE ASSAY ELUATE FROM THE SECONDARY INCUBATION STAGE Serum dilution

Detached at conjugate stage a

Percentage precipitable by 50% ammonium sulphate b

1 : 40 1 : 160 1 : 640 1 : 2,560 1 : 10,240 Buffer

4.68-+0.82 5.35-+0.62 4.44 + 0.49 3.52 + 0.46 3.37+0.26 3.52-+0.49

6 2 . 8 + 7.10 5 5 . 8 + 3.71 48.0 -+ 4.49 30.3 -+ 11.4 1 9 .0 + 8.74 15.3-+ 4.38

a Expressed as percent of the initial amount before adding the serum; mean + S.D. of 9 parallel determinations. b Expressed as percentage of the whole amount detached with conjugate; mean + S.D. of 5 parallel determinations.

unreacted groups on the paper. Furthermore, addition of 0.1% Tween-20 to t h e b u f f e r u s e d in t h e p r i m a r y a n d s e c o n d a r y i n c u b a t i o n s i n c r e a s e d a b s o r bance values by about 150% and decreased the background absorbances of normal sera by about 60%. To compare the performances of polystyrene and paper solid phases, the ratio of the maximum absorbance by the optimal dilution of the positive s e r u m t o t h e a b s o r b a n c e b y t h e n o r m a l s e r u m c o n t r o l in t h e s a m e d i l u t i o n was determined. The ratios were 10.4 for polystyrene and 22.4 for paper.

TABLE 5 COMPARISON OF T H R E E SOLID PHASE MATERIALS FO R ANTIGEN LEAKAGE DURING ELISA ASSAY

Polystyrene c Nylon CNBr-activated paper

Antigen concentration in the coupling solution (pg/ml)

Antigen surface concentration a

Detached during ELISA a

Mean (ng/cm 2 )

S.D. b (percentage of the mean)

Mean (%)

S.D. b (percentage of the mean)

100 100 10 100 1 10

66 150 150 210 140 540

15 20 11 5.2 5.2 4.3

25 37 60 48 13 13

31 15 4.3 14 4.2 3.4

a Five parallel determinations. b Standard deviation. c Two batches of cuvettes.

69 The standard deviations of absorbancies of 6 parallel determinations were 24 and 13% of the mean values, respectively. DISCUSSION In the ELISA m e t h o d the antigen coupled solid phase is exposed to extensive washing procedures and it is therefore necessary to ascertain its stability during the assay. The present work shows that it is important to wash the solid phase carefully after antigen coupling. This can also be deduced from the data o f Christensen et al. (1978). As m a n y as 6 washes were necessary to remove all loose antigen from polystyrene surfaces especially when the antigen concentration during the coupling was high (10 mg/ml). This may provide one explanation for the poor performance of polystyrene antigen phases obtained at high coupling concentrations of antigen as described by Engvall and Perlmann (1972) and also by other investigators. Although m a n y washes after coupling leave no loosely bound antigen on polystyrene, leakage taking place during subsequent incubations and washings is not excluded. Engvall et al. (1971) reported a total leakage of 20--30% during the assay and our results are in accordance with this. However, Cantarero et al. (1979) reported stable coupling to polystyrene in an amplified ELISA system. This contradiction may be due to differences in the properties of the polystyrene used. With nylon as the solid phase, detachm e n t of antigen was at least of the same order of magnitude as with polystyrene, but it is possible that there are some types of plastic that bind antigen firmly. Polyvinyl seems promising in this respect because Zollinger et al. (1976) observed a total leakage o f only 6--8% of meningococcal antigen in a radioimmunoassay with polyvinyl microtitre plates as the solid phase. Frankel and Gerhard (1979) have also successfully used polyvinyl plates for quantitation of h y b r i d o m a proteins. In the present study detachment during primary incubation was found to be independent of the presence of specific antibodies, as reported by Zollinger et al. (1976). This indicates that the binding of antibody to the antigen is n o t responsible for the detachment. Detachment was observed to occur quite quickly during primary incubation. Thus considerable concentrations of free antigen appear in the liquid phase before the reaction between the primary antibody and the antigen on the solid phase has reached completion. Although the a m o u n t o f detached antigen appears to be too low to compete with solid phase antigen, one must bear in mind that all the antigen on the solid phase is not immunoreactive as pointed out by Tosi and Celada (1974) and Ansari et al. (1978). Analysis of assay eluates during the second incubation shows that antigen-antibody complexes are also detached. This effect seems to be related to the a m o u n t of bound specific antibody. An important aspect requiring future investigation is the ratio between detached complexed antigen and free antigen. Differences in this parameter between

70 d i f f e r e n t s a m p l e s m a y lead to e r r o n e o u s results in m e a s u r i n g the a m o u n t and avidity o f a n t i b o d i e s . P e r h a p s t h e g r e a t e s t b a r r i e r t o s t a n d a r d i s a t i o n o f E L I S A is t h e large variat i o n b e t w e e n d i f f e r e n t c o u p l i n g b a t c h e s w h e n p o l y s t y r e n e is used. A l t h o u g h we used a c a r e f u l l y s t a n d a r d i s e d p r o c e d u r e f o r coupling, t h e b a t c h - t o - b a t c h s t a n d a r d d e v i a t i o n o f surface c o n c e n t r a t i o n o f antigen was a b o u t 50% o f t h e m e a n . T h e r e a s o n f o r this p o o r p r e d i c t a b i l i t y o f surface c o n c e n t r a t i o n o f antigen r e m a i n s to be clarified. In this r e s p e c t c y a n o g e n b r o m i d e a c t i v a t e d p a p e r was s u p e r i o r t o p o l y s t y r e n e a n d n y l o n . T h e v a r i a t i o n b e t w e e n diff e r e n t c o u p l i n g b a t c h e s o f a c t i v a t e d p a p e r was o n l y a b o u t 15%. Also the degree a n d v a r i a t i o n o f leakage f r o m p a p e r d u r i n g t h e assay w e r e less t h a n with t h e plastics. A c t i v a t e d p a p e r has also a large b i n d i n g c a p a c i t y a n d suffic i e n t surface c o n c e n t r a t i o n s are a c h i e v e d w i t h quite l o w c o n c e n t r a t i o n s o f antigen in t h e c o u p l i n g s o l u t i o n . This is an a d v a n t a g e w h e n o n l y small quantities o f antigen are available. H o w e v e r , t h e assay w i t h a c t i v a t e d p a p e r involves m a n y l a b o r i o u s m a n i p u l a t i o n s a n d is n o t suitable as a r o u t i n e method. I f t h e E L I S A m e t h o d is u s e d f o r q u a n t i t a t i v e m e a s u r e m e n t o f a n t i b o d i e s t h e surface c o n c e n t r a t i o n a n d leakage o f t h e antigen h a v e t o be c o n t r o l l e d a n d s t a n d a r d i s e d . O u r results s h o w t h a t v a r i a t i o n s in t h e c o n c e n t r a t i o n and leakage o f antigen are so great in E L I S A using p o l y s t y r e n e or n y l o n t h a t reliable s t a n d a r d i s a t i o n is a l m o s t i m p o s s i b l e . It is, t h e r e f o r e , r e c o m m e n d e d t o use a c o v a l e n t c o u p l i n g o f antigen to t h e solid p h a s e in r e f e r e n c e m e t h o d s for quantitative antibody detection. ACKNOWLEDGEMENTS This w o r k has b e e n s u p p o r t e d b y t h e R e s e a r c h a n d Science F o u n d a t i o n o f L~i~ike, T u r k u , F i n l a n d and t h e P a u l o F o u n d a t i o n , Helsinki, Finland. We wish t o t h a n k Mrs. Soile N i i t t o a h o f o r p r e p a r i n g c o n j u g a t e s o f g o o d quality. REFERENCES Ansari, A.A., L.M. Bahuguna and H.V. Malling, 1978, J. Immunol. Methods 23, 219. Avrameas, S., 1969, Immunochemistry 6, 43. Bidwell, D.E., A. Bartlett and A. Voller, 1977, J. Infect. Dis. 136, $274. Cantarero, L.A., J.E. Butler and J.W. Osborne, 1979, Fed. Proc. 38, 1012. Ceska, M. and U. Lundqvist, 1972, Immunochemistry 9, 1021. Christensen, P., A. Johansson and V. Nielsen, 1978, J. Immunol. Methods 23, 23. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109, 129. Engvall, E., K. Jonsson and P. Perlmann, 1971, Biochim. Biophys. Acta 251,427. Frankel, M.E. and W. Gerhard, 1979, Mol. Immunol. 16, 101. Hunter, W.M. and C. Greenwood, 1962, Nature 194,495. Kalmakoff, J., A.J. Parkinson, A.M. Crawford and B.R.G. Williams, 1977, J. Immuno|. Methods 14, 73. Schuurs, A.H.W.M. and B.K. Van Weemen, 1977, Clin. Chim. Acta 81, 1. Tosi, R.M. and F. Celada, 1974, Immunology 27, 941. Van Oss, C.J. and J.M. Singer, 1966, J. Reticuloendoth. Soc. 3, 29. Viljanen, M.K., K. Granfors and P. Toivanen, 1975, Immunochemistry 12,699. Voller, A., A. Bartlett and D.E. Bidwell, 1978, J. Clin Pathol. 31, 507. Zollinger, W.D., J.M. Dalrymple and M.S. Artenstein, 1976, J. Immunol. 117, 1788.