Journal o f Immunological Methods, 31 (1979) 101--107 © Elsevier/North-Holland Biomedical Press

101

DIFFUSION IN GEL-ENZYME LINKED IMMUNOSORBENT ASSAY SIMPLE METHOD FOR QUANTITATION OF CLASS-SPECIFIC ANTIBODIES

(DIG-ELISA): A

HANS ELWING 1 and HAKAN NYGREN 2 I Institute o f Medical Microbiology and 2 Department o f Histology, University o f GSteborg, G6teborg, Sweden

(Received 23 April 1979, accepted 3 Juli 1979)

A new method for quantifying class-specific antibodies is presented. The method has been named Diffusion-In-Gel-Enzyme-Linked-ImmunoSorbentAssay (DIG-ELISA), and is briefly as follows. Antiserum is allowed to diffuse from wells in a gel layered over an antigen-coated plastic surface. The gel is then removed and the preparation is incubated with enzymeconjugated anti-immunoglobulin. The enzyme is then visualised in situ by a colour reaction produced by pouring a substrate-containing gel over the plastic surface. Bovine serum albumin and rabbit-anti-BSA were used as a model system, and horseradish peroxidase or alkaline phosphatase as enzymes for visualization.

INTRODUCTION Diffusion-in-gel t h i n - l a y e r - i m m u n o - a s s a y ( D I G - T I A ) (Elwing et al., 1 9 7 6 ) a n d e n z y m e - l i n k e d - i m m u n o a d s o r b e n t - a s s a y ( E L I S A ) (Engvall a n d P e r l m a n n , 1 9 7 2 ; G u e s d o n a n d A v r a m e a s , 1 9 7 7 ) are t w o m e t h o d s w h i c h have b e e n used f o r q u a n t i f y i n g a n t i b o d i e s . In b o t h m e t h o d s , antigen is a d s o r b e d t o a solid p h a s e , usually a plastic surface, a n d t h e t e s t a n t i b o d i e s are t h e n a l l o w e d t o b i n d t o t h e antigen. V i s u a l i z a t i o n a n d q u a n t i f i c a t i o n o f b o u n d a n t i b o d i e s are, h o w e v e r , p e r f o r m e d in d i f f e r e n t w a y s in t h e t w o m e t h o d s . In D I G - T I A , a n t i b o d i e s are q u a n t i f i e d b y t h e i r ability t o f o r m a d i f f u s i o n g r a d i e n t in a gel l a y e r e d o v e r t h e a n t i g e n - c o a t e d surface. T h e a n t i g e n - - a n t i b o d y r e a c t i o n is t h e n visualized b y c o n d e n s a t i o n o f w a t e r o n t h e plastic surface. In E L I S A , t e s t a n t i b o d i e s are first a l l o w e d t o b i n d t o t h e i m m o b i l i s e d antigen, a n d an e n z y m e - c o n j u g a t e d a n t i - i m m u n o g l o b u l i n is t h e n b o u n d to t h e a n t i b o d y . Visualization a n d q u a n t i f i c a t i o n o f t h e p r i m a r y a n t i g e n - - a n t i b o d y r e a c t i o n is t h e n p e r f o r m e d i n d i r e c t l y b y an e n z y m e s u b s t r a t e r e a c t i o n , yielding a c o l o u r e d r e a c t i o n p r o d u c t w h e r e t h e c o l o u r i n t e n s i t y is p r o p o r t i o n a l to t h e a m o u n t o f a n t i b o d y b o u n d to i m m o b i l i s e d antigen. Correspondence to: Dr. Hans Elwing, Institute of Medical Microbiology, Guldhedsgatan 10, S-413 46 GSteborg, Sweden.

102

In the present paper, a simple m e t h o d for visualizing and quantifying immunospecific antibodies is described. The m e t h o d is based on a combination of the DIG-TIA and ELISA techniques. The diffusion-in-gel of antibodies over an antigen-coated surface is performed as in DIG-TIA, and visualization of the antigen--antibody reaction is by enzyme-conjugated antiimmunoglobulin as in ELISA. This combination is made possible by performing the enzymatic reaction in an immobilised gel, rather than in a free solution. MATERIAL AND METHODS

Antigen and antibodies Crystallized and lyophilized bovine serum albumin (BSA) was obtained from Sigma Chem. Co., St. Louis, MO, U.S.A. Anti-BSA was prepared in rabbits as earlier described (Elwing et al., 1977). The antiserum contained 1.4 mg specific a n t i b o d y / m l as determined by quantitative precipitation according to Gitlin et al. (1949).

Conjugates The immunoglobulin fractions of goat-anti-rabbit IgG and rabbit-antihuman-IgA, IgG or IgM were conjugated with alkaline phosphatase or peroxidase with glutaraldehyde as cross-linking agent according to the methods of Holmgren and Svennerholm (1973) and Nygren et al. (1979).

Agar and agarose Special Noble agar was obtained from Difco laboratories, Detroit, MI, U.S.A. Agarose was obtained from Marine Colloids Inc., Biomedical Systems, Rocklande, ME, U.S.A. The agarose, lot No. 37844, had a gelling temperature below 37.5°C and contained less than 0.15% sulfate.

Polystyrene petri dishes These dishes, 8.5 cm in diameter, were obtained from NUNC A/S, Roskilde, Denmark. The critical surface tension (CST) of the inner surface was 36.6 d y n / c m 2 as determined by the m e t h o d of Fox and Zisman (1950).

Diffusion-in-gel A schematic drawing of the principle of the DIG-ELISA m e t h o d is shown in Fig. 1. Antigen-coating of polystyrene petri dishes and the diffusion-in-gel m e t h o d for quantitation of antibodies were performed according to Elwing et al. (1977). Briefly, the insides of the dishes were washed with 95% ethanol, and the dishes dried and filled with 18 ml 0.15 M NaC1. BSA, dissolved in 0.15 M NaC1 was added to the dish to the final coating concentration indicated in the figures. After incubation for 60 min at room temperature, the plastic surfaces were washed with 0.15 M NaC1. Fifteen ml of 1% agar in 0.15 M NaC1 were then poured into each dish. When the agar had set, 3-mm

103

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Fig. 1. The principle of the DIG-ELISA method. A; A plastic surface is coated with antigen, and a gel is poured over the surface; B: Antibodies are allowed to diffuse from wells in the gel; C: The gel is removed and the plate is incubated with enzyme-conjugated anti-immunoglobulin; D: A substrate-containing gel is poured over the surface and the reaction zones indicating antigen--antibody reactions are measured.

d i a m e t e r wells were p u n c h e d in t h e gel. T h e wells w e r e filled to t h e level o f t h e agar surface w i t h d i l u t i o n s o f a n t i - B S A or n o r m a l r a b b i t s e r u m ( N R S ) , a n d t h e dishes i n c u b a t e d in a m o i s t a t m o s p h e r e f o r 40 h. T h e gel was t h e n r e m o v e d b y rinsing t h e dishes w i t h distilled water.

Alkaline phosphatase visualization system A f t e r r e m o v a l o f t h e gel, t h e dishes w e r e i n c u b a t e d f o r 5 m i n w i t h p h o s p h a t e - b u f f e r e d saline ( p H 7.0) c o n t a i n i n g 0 . 0 5 % T w e e n 20 ( P B S - T w e e n ) , f o l l o w e d b y 10 m l P B S - T w e e n c o n t a i n i n g alkaline p h o s p h a t a s e - c o n j u g a t e d i m m u n o g l o b u l i n at suitable c o n c e n t r a t i o n . A f t e r 2 h at r o o m t e m p e r a t u r e , t h e dishes w e r e rinsed w i t h 0.15 M NaC1. Agarose was m e l t e d in 0 . 0 5 M c a r b o n a t e b u f f e r , p H 8.9, a n d c o o l e d to 45°C. E n z y m e s u b s t r a t e (p-nitrop h e n y l p h o s p h a t e ) was dissoved in t h e a g a r o s e t o a final c o n c e n t r a t i o n o f 15 g/1. T h e s u b s t r a t e - c o n t a i n i n g agarose (15 ml) was t h e n p o u r e d i n t o each dish. A f t e r 45 m i n at r o o m t e m p e r a t u r e , t h e dishes w e r e p l a c e d on a p h o t o g r a p h i c p a p e r u n d e r red safelight c o n d i t i o n s , a n d t r a n s i l l u m i n a t e d w i t h a light s o u r c e e q u i p p e d w i t h an i n t e r f e r e n c e filter at 4 0 3 n m . T h e p r i n t was d e v e l o p e d a c c o r d i n g t o r o u t i n e p r o c e d u r e s . T h e d i a m e t e r o f t h e circular c o l o u r e d areas was m e a s u r e d o n t h e p h o t o g r a p h s w i t h a ruler t o w i t h i n 0.5 m m .

Horseradish peroxidase visualization system A f t e r r e m o v a l o f t h e gel, the dishes w e r e i n c u b a t e d w i t h P B S - T w e e n f o r 5 m i n , f o l l o w e d b y i n c u b a t i o n in 10 m l o f P B S - T w e e n c o n t a i n i n g p e r o x i d a s e - c o n j u g a t e d i m m u n o g l o b u l i n . A f t e r 2 h at r o o m t e m p e r a t u r e , t h e dishes w e r e rinsed in 0.15 M NaC1. Agarose was m e l t e d in 0.1 M p h o s p h a t e b u f f e r , p H 7.0, a n d c o o l e d t o 4 5 ° C . S u b s t r a t e ( p - p h e n y l e n e d i a m i n e , 1 g/l, a n d 0 . 0 1 % h y d r o g e n p e r o x i d e )

104 was a d d e d and the s u b s t r a t e - c o n t a i n i n g agarose s o l u t i o n (15 ml) was p o u r e d into each o f the dishes. A f t e r 20 min o f i n c u b a t i o n , the c o l o u r e d areas were m e a s u r e d with a ruler as described above. The dishes were p h o t o g r a p h e d as described above, b u t w i t h o u t the use o f an i n t e r f e r e n c e filter. RESULTS

Visualization of test antibody with alkaline phosphatase-conjugated antiimrnunoglobulin Yellow circular areas, representing e n z y m e substrate p r o d u c t , were f o r m e d in the agarose gel a b o u t 1 min after a d d i t i o n o f the substratec o n t a i n i n g gel. T h e y e l l o w areas were l o c a t e d c o n c e n t r i c a l l y a r o u n d the f o r m e r well positions, and their d i a m e t e r increased with increasing incubation time. The y e l l o w circular areas had f u z z y margins, and c o n t r a s t e d b a d l y against d i f f e r e n t b a c k g r o u n d s b u t p h o t o g r a p h i c prints s h o w e d g o o d c o n t r a s t (Fig. 2A) and the d i a m e t e r o f z o n e s c o u l d be d e t e r m i n e d w i t h o u t difficulty. F u r t h e r i n c u b a t i o n o f the dishes with substrate up to several h o u r s resulted in c o n t i n u e d radial g r o w t h o f the c o l o u r e d zones, with the margins b e c o m i n g increasingly f u z z y . N o f u r t h e r increase in sensitivity was a t t a i n e d by incubat i o n b e y o n d 45 min. No r e a c t i o n z o n e s were f o r m e d a r o u n d wells c o n t a i n i n g NRS.

Fig. 2. Dishes were coated with BSA (10 mg/l) for 60 min. Experimental procedure was as indicated in Fig. 1. Four-fold dilution series of anti-BSA were placed in the wells. Normal rabbit serum was applied at locations indicated by arrow. Diffusion time 40 h. A: Alkaline phosphatase-conjugated anti-immunoglobulin. Incubation with substrate for 45 min. The plate was photographed by light of wavelength 403 rim. B: Peroxidaseconjugated anti-immunoglobulin. Incubation with substrate for 45 rain. Photographic copy was made by trans-illuminating with white light.

105

Visualization with peroxidase-conjugated anti-immunoglobulin Brown circular areas appeared ar ound the location of the form er application basins within a few sec after adding the substrate-containing gel. The diameter o f the zones increased with increasing incubation time. The margins o f the zones were well demarcated (Fig. 2B) and the brown colour contrasted well to a white background, allowing measurements o f the zone diameter directly on the plates. Measuring 10 areas t o o k a b o u t 1 min, and during this time, the radial growth of the zones was negligible. After 1 h of incubation it was n o t e d t h at the peroxidase substrate converted spontaneously, giving rise to a background staining. However, if the g e l was removed, and the dishes re-filled with gel containing fresh substrate, the reaction could be r e p r o d u c e d w i t h o u t any apparent loss of sensitivity. NRS did n o t give rise to a ny reaction zones.

Dose--response diagram The diameters of the reaction zones described above were pl ot t ed against the logarithm of serum dilutions (Fig. 3). The diameter of zones showed a linear relationship to the logarithm of anti-BSA concentration. The sensitivity o f DIG-ELISA for measurements of anti-BSA antibodies was 100 pg/1 in b o th visualization systems used.

Applicability Several serological systems of diagnostic relevance were successfully tested with the HRP visualization system. Hu man antibodies against thyroglobulin were determined in sera from patients with thyroiditis. Human IgG and IgM antibodies against extracts f r o m Schistosoma mansoni and Entamoeb¢t histolytica were measured in sera from patients with schistosomiasis and amoebiasis, respectively. Rheu-

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Fig. 3. Diagram showing the dose--response plot. The diameters of the reaction areas in the HRP- and AP-visualization s y s t e m s are linear with the logarithm o f the a n t i b o d y concentration.

106 matoid factor was detected by using surfaces coated with human IgG. Finally, anti-LPS antibodies in rabbit serum were detected against LPS from four different bacterial stains. DISCUSSION The DIG-ELISA m e t h o d has several advantages, as compared to the traditional ELISA-technique. In DIG-ELISA, it is possible to quantitate antibody w i t h o u t making dilutions of antisera, and w i t h o u t the use of a spectrophotometer. The interpretation of test results is also very simple in DIG-ELISA, since only one factor, namely the diameter of zones, has to be considered. Furthermore, the petri dishes used for immobilisation of antigens are easy to handle during washing and coating and several sera may be assayed in one dish. This simplicity and efficacy is not achieved in the usual ELISA where the antigen is immobilized in test tubes or micro trays. These advantages of DIG-ELISA make the technique useful for screening large numbers of sera for antibody content. Visualization of the antigen--antibody reaction in DIG-ELISA is not as simple as in DIG-TIA. However, in systems where the antigen has high wettability, or when the antigen is crude and heavily contaminated, DIG-ELISA may serve as a valuable complement to DIG-TIA. Crude antigens are often used in the field of parasitology, and in this field there is a need for simple serological techniques like DIG-TIA and DIG-ELISA. Two enzymatic visualization systems, horseradish peroxidase (HRP) and alkaline phosphatase (AP) were used in the DIG-ELISA method. The HRPsystem has some theoretical and practical advantages as compared to the AP system. Theoretically, conjugates of HRP and IgG prepared by the two-step m e t h o d with glutaraldehyde as cross-linker give reproducible conjugates with a homogeneous molecular weight and well preserved enzymatic activity and antigen binding capacity (Nygren et al., 1979). In contrast, conjugation of IgG with AP by the one-step m e t h o d gives a wider variation in quality of the conjugates obtained. In practice, the circular areas of HRP products were readily detectable with the naked eye and the zones had sharp margins, permitting accurate determinations of size. Similar accuracy was obtained in the AP system only by use of a photographic technique with light of the wavelength of the absorption maximum of the enzyme substrate product. The sensitivity for detecting anti-BSA with DIG-ELISA was about the same for both the HRP- and AP-visualization system. Our results indicate that the DIG-ELISA technique may be more sensitive than DIG-TIA when the antigens are heavily contaminated with serologically irrelevant material. ACKNOWLEDGEMENTS This study was supported by grants from the faculties of Medicine and Odontology, University of GSteborg. The authors wish to thank Dr. L.-A. Nilsson for critical reading of the manuscript.

107 REFERENCES Elwing, H., L.-A. Nilsson and C). Ouchterlony, 1976, Int. Arch. Allergy Appl. Immunol. 51,757. Elwing, H., L.-~,. Nilsson and O. Ouchterlony, 1977, J. Immunol. Methods 17, 131. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109, 129. Fox, H.W. and W.A. Zisman, 1950, J. Colloid Sci. 5 , 5 1 4 . Guesdon, I.L. and S. Avrameas, 1977, Immunochem. 14,443. Holmgren, J. and A.M. Svennerholm, 1973, Infect. Immun. 7, 759. Nygren, H., H.-A. Hansson and S. Lange, 1979, Med. Biol. 57, 187.