Journal o f Immunological Methods, 26 (1979) 61--67

61

© Elsevier/North-Holland Biomedical Press

A MICROPLATE I M M U N O E N Z Y M E A S S A Y F O R A N T I - I N F L U E N Z A ANTIBODIES

CLAUDE LAMBRE I and KUPPUSWAMY NAIDU KASTURI Laboratoire de Physiologie Cellulaire, Universitd Paris 6, and Unitd d 'Ecologie Virale, Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris Cedex 15, France

(Received 20 July 1978, accepted 7 September 1978)

An enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase is described for the detection and quantitation of anti-influenza virus antibodies. Compared with complement fixation and hemagglutination inhibition tests, ELISA is far superior with respect to sensitivity and reliability. Non-specific viral inhibitors present in sera do not affect the titer in ELISA. Its sensitivity, close to that of radioimmunoassay permits detection of small amounts of antibodies in pulmonary secretions and supernatants from in vitro spleen cell cultures.

INTRODUCTION Anti-influenza antibodies are usually assayed e i t h e r b y h e m a g g l u t i n a t i o n i n h i b i t i o n (HAI) (Fazekas de St. G r o t h and Webster, 1 9 6 6 ) or b y complem e n t f i x a t i o n (CF) tests. H o w e v e r , these tests lack sensitivity and p r e s e n t some disadvantages. T h e presence o f non-specific inhibitors in sera interferes in the H A I test. T h e trypsin p e r i o d a t e t r e a t m e n t (Jensen, 1961) does n o t c o m p l e t e l y d e s t r o y these inhibitors. The CF test is even less sensitive t h a n HAI. M o r e o v e r it is n o t suitable f o r the d e t e c t i o n o f IgA, the p r e d o m i n a n t class o f a n t i b o d i e s present in nasal and b r o n c h o p u l m o n a r y secretions a f t e r i n f l u e n z a virus i n f e c t i o n (Alford et al., 1967; S c o t t and Walker, 1976). In the last few years, an 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 been d e v e l o p e d t o measure a n t i b o d i e s (Avrameas and Guilbert, 1971; Engval and P e r l m a n n , 1971). This m e t h o d has already been successfully applied f o r the d e t e c t i o n o f a variety o f antibodies and antigens (Leinikki and Pfissilfi, 1 9 7 6 ; Voller and Bidwell, 1 9 7 6 ; Wolters et al., 1976; Bishai and Galli, 1977; P r e v o t and G u e s d o n , 1 9 7 7 ; Svensori and Larsen, 1977). In this p a p e r we describe a m i c r o m e t h o d which is suitable f o r the q u a n t i t a t i o n o f antii n f l u e n z a virus antibodies. This m e t h o d is m o r e sensitive and reliable t h a n HAI and CF tests. Reprint requests: Dr. Claude Lambre, Unit6 d'Ecologie Virale, Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris Cedex 15, France.

62 MATERIAL AND METHODS

Virus Egg-adapted A/Hong Kong/1/68 (H3N2) strain of influenza virus was grown in 11-day-old embryonated chicken eggs for 48 h. Infective allantoic fluids were clarified by centrifugation at 5000 X g for 15 rain and the supernatant was centrifuged at 65,000 X g for 30 rain. The pellet containing the virus was resuspended in phosphate-buffered saline (PBS) pH 7.2, layered on a 3 0 - 6 0 % linear sucrose gradient and centrifuged at 100,000 X g for 16 h. The opalescent virus band was recovered, diluted with PBS and pelleted at 65,000 × g. Immunizatio~ Young adult male rabbits (Red Burgundy) weighing 2.5--3 kg were immunized by intratracheal instillation of live influenza vires after light ether anesthesia. 107 egg-infective doses of virus were given in 5 ml PBS both for primary and secondary immunizations. Animals were boosted 15 days after priming and bled 1 week later. Some rabbits were inoculated intradermally with 1200 hemagglutinin (HA) units of formalin-inactivated virus in 0.1 ml physiological saline and bled 6 weeks later. Lung washings Lung washings were obtained with 70 ml of PBS according to the procedure described by Myrvik et al. (1961). Cells and particulate material were removed by centrifuging at 1500 X g for 15 min. In vitro culture of spleen cells Spleens from either control or immune animals were homogenized in a Potter tissue homogenizer and the cells were washed twice with RPMI 1640 medium (Gibco) by centrifuging at 800 X g for 10 rain. The cells were resuspended to 1,5 X 107/ml in fresh medium containing 5% heat-inactivated fetal calf serum. Ten ml samples of the cell suspension were transferred into tissue culture flasks (Falcon Ref. 3024) and incubated for 24, 48 and 72 h at 37°C. The cell-free culture media were recovered by centrifugation and assayed for the presence of antiviral antibodies by HAI test and ELISA. The cultures at time zero treated in the same manner were used as control. Anti-hemagglutinin antibody assay Sera were treated with trypsin and periodate to inactivate non-specific viral inhibitors (Jensen, 1961) prior to the assay. This treatment was omitted for the lung washings since lung washings from control rabbits did not contain detectable level of inhibitors. Hemagglutination-inhibiting antibody titers were determined by the method described by Fazekas de St. Groth and Webster (1966).

63

Complement-fixing antibody titration The complement fixation test was carried out using whole virus as antigen (2500 HA U/ml) according to the micromethod described by Lemieux et al., 1974). Results were expressed as the reciprocal of the final dilution showing partial or complete inhibition of lysis. Enzyme-linked immunosorbent assay ELISA was performed using virus-coated microtitration plates (Cooks microtitration plates M 220-24A). Antigen was diluted in 0.1 M carbonate buffer, pH 9.6, containing 0.01% sodium azide. 150 pl of antigen suspension was added to each well and the plates were incubated overnight at 4 °C. They were then washed thrice with buffered saline (PBS, pH 7.2, containing 0.1% Tween 20), air-dried and either used at once or stored until use at 4°C. Serial dilutions of antibody-containing samples (in PBS containing 0.5% bovine serum albumin (BSA) and 0.1% Tween 20) were distributed in 100 pl volumes in duplicates in antigen-coated microplates and incubated for 2 h at room temperature in humid chambers. The wells were washed 5 times with buffered saline and 100 pl of 1/500 diluted conjugate (peroxidase-conjugated sheep anti-rabbit immunoglobulin sera; obtained from Pasteur Institute, Garches) in PBS containing 4% BSA and 0.1% Tween 20 was added to each well. After 2 h of incubation, the plates were washed thoroughly and the enzyme activity was determined by incubating with 100 pl of 0.04% orthophenylene diamine (Sigma Chemical Co., St. Louis, MO) dissolved in 1/10,000 diluted hydrogen peroxide (110 vol) in 10 mM citric acid-phosphate buffer, pH 6. After 30 rain in the dark at room temperature, the reaction was stopped by the addition of 25 pl 0.5 N citric acid. These o p t i m u m incubation conditions were established by preliminary experiments. The yellow reaction product was diluted with 0.1 N citric acid and the absorption (A) was measured at 454 nm. Under these conditions, the peroxidasecatalyzed reaction product was stable for at least 24 h. RESULTS

Determination of optimum antigen concentration required for coating the microplates used in ELISA Serial dilutions of control and immune sera were assayed in microplates coated with antigen suspension containing 0.1, 1, 5, 10, 20 or 50 pg of viral protein/ml. The results are shown in Fig. 1. Microplates coated with 10, 20 or 50 pg protein/ml gave the same ELISA values for immune sera. However, in the case of control sera, the plates coated with 20 and 50 pg gave higher background values than those with 10/~g protein/ml. Antigen at concentrations lower than 10 pg/ml gave much less background, but the sensitivity of the assay also diminished markedly. Therefore we chose 10 pg viral protein/ ml as optimum for coating microplates for further experiments.

64 A . 454 nm

0e

04

/

02

0,1

i

i

II

50ug

lOpg

~ug

olpg

Fig. 1, Determination of optimum antigen concentration for coating microplates used in ELISA. Optical absorption at 454 nm of two-fold dilutions (10 -s to 6.4 10 -6) of an immune serum and 10 -3 dilution of a control serum at antigen concentrations from 0.1 to 50 pg of viral protein/ml.

Comparison o f the sensitivity o f CF and HA1 tests with E L I S A Titration o f sera. C o n t r o l r a b b i t sera applied t o the plates at dilutions higher t h a n 1 / 1 0 0 0 were negative while i m m u n e sera gave d e t e c t a b l e optical d e n s i t y (OD) even at dilutions higher t h a n o n e million. E L I S A titers were expressed as t h e ratio b e t w e e n the reciprocal o f the d i l u t i o n o f i m m u n e and the reciprocal o f c o n t r o l sera giving an OD o f 0.05 at 4 5 4 n m . The dilutions c o r r e s p o n d i n g to an OD o f 0.05 were calculated f r o m the curves o b t a i n e d f r o m serial t w o - f o l d dilutions. T h e results s u m m a r i z e d in Table 1 s h o w t h a t (1) E L I S A is m o r e sensitive t h a n H A I and CF tests, and (2) there is a g o o d c o r r e l a t i o n b e t w e e n the diff e r e n t assays (r = 0.80 b e t w e e n E L I S A and H A I ; r = 0.70 b e t w e e n E L I S A and CF). H o w e v e r it s h o u l d be n o t e d t h a t the ratios b e t w e e n E L I S A and H A I values are always higher with s t r o n g l y i m m u n e sera t h a n with w e a k e r ones. This is due to the presence o f non-specific inhibitors. The e f f e c t o f these inhibitors is less p r o n o u n c e d in the case o f s t r o n g i m m u n e sera since t h e y get highly diluted in the assay. These inhibitors d o n o t interfere in E L I S A since either s t r o n g or weak i m m u n e sera did n o t s h o w a n y significant

65 TABLE 1 C O M P A R I S O N B E T W E E N CF, HAI AND E L I S A T I T E R S O F R A B B I T S E R A R a b b i t No.

CF titer

HAI titer a

E L I S A titer

41 55 32 31 2581 2586 19 57 2580 2583 2599 2582

2 8 80 80 320 320 320 320 2560 2560 5120 5120

148 563 650 830 1610 1910 3090 4470 14150 14150 12500 20000

7 10 30 130 40 90 1000 560 2080 5020 1630 3340

b

a N o r m a l r a b b i t sera gave a m e a n t i t e r o f 138. reciprocal o f the d i l u t i o n o f i m m u n e s e r u m giving an a b s o r p t i o n o f 0.05 b ELISA titer = 300 (300 = m e a n o f reciprocal o f t h e d i l u t i o n o f c o n t r o l sera giving an a b s o r p t i o n o f 0.05).

decrease in ELISA values after trypsin periodate treatment. Titration of bronchopulmonary washings. Bronchopulmonary washings from control rabbits gave an OD of only 0.006-+ 0.001 at 1/10 dilution. Therefore ELISA titer is expressed as the reciprocal of the dilution giving an OD of 0.050. TABLE 2 C O M P A R I S O N B E T W E E N CF, HAI AND E L I S A T I T E R S O F B R O N C H O P U L M O N A R Y W A S H I N G S F R O M R A B B I T S I M M U N I Z E D VIA D I F F E R E N T R O U T E S R a b b i t No.

Inoculation a

CF t i t e r

HAI t i t e r

ELISA titer b

78 79 80 81 35 37 01 05 2580 2581 04

ID ID ID ID IT IT IT IT IT IT IT

0 0 0 0 0 0 4 4 16 16 32

0 0 0 0 40 40 56 56 128 128 224

36 34 83 31 780 1350 3390 4670 7410 7900 25120

a ID, i n t r a d e r m a l ; IT, intratracheal. b E L I S A t i t e r = reciprocal o f the d i l u t i o n o f lung washing giving an a b s o r p t i o n o f 0.05.

66 TABLE 3 TITRATION Hours of culture

0 24 48 72

OF SPLEEN CELL CULTURE HAI titer

SUPERNATANTS

BY HAI AND ELISA

ELISA titer a

Control

Immune

Control

Immune

10 10 10 20

12 52 56 60

0 0 0 10

0 320 580 600

a ELISA titer = reciprocal of the dilution of supernatant giving an absorption of 0.05.

It can be seen from the Table 2 that ELISA permitted the det ect i on of small quantities of antibodies present in b r o n c h o p u l m o n a r y washings which were undetectable by HAI and CF tests. Here again, the correlation between the results obtained by the different tests is highly significant (r = 0.83 between ELISA and HA/; r = 0.97 between ELISA and CF). There is a close parallelism between HAI titers and ELISA values i n d e p e n d e n t of the strength of the sample. This confirms our earlier observation that nonspecific inhibitors interfere in HAl reaction in the case o f weakly immune sera. ELISA is thousand-fold more sensitive than CF reaction for measuring secretory antibodies. This is n o t surprising since IgA, the p r e d o m i n a n t class o f antibodies present in secretions are no t revealed by CF test whereas all classes o f antibodies are revealed in ELISA. Titration o f antibodies secreted by spleen cells in culture. Spleen cell culture supernatants were assayed by HA1 and ELISA in the same way as for b r o n c h o p u l m o n a r y washings. The results are shown in Table 3. Spleen cells from immune animals when cultured in vitro continued to secrete antibodies. These antibodies were detectable both by HAl and ELISA, but the sensitivity of ELISA is several times superior to that of HAI reaction. The rise in a n t i b o d y titer was linear only during the first 48 h in culture. DISCUSSION

We have d e m o n s t r a t e d the advantages of the enzyme-linked immunosorbent assay (ELISA) compared with HAI and CF tests in measuring antiinfluenza virus antibodies. Influenza virus adsorbs easily on plastic microtitration plates. The antigen-coated plates were stable in the cold for at least 6 months. By using Tween 20 in washings and incubation buffers the background could be minimized. The residual background could be diminished fu r th er (OD o f 0.05 at 1/10 dilution for control sera) by using anti-heavy chain specific antibodies instead of anti-whole immunoglobin sera. The reagents used in peroxidase assay are stabilized by citric acid-phosphate buffer. Addition of citric acid to stop the e nz ym e reaction also prevents artifactual

67 f o r m a t i o n o f t h e y e l l o w p r o d u c t . It is t h u s possible t o assay several s a m p l e s s i m u l t a n e o u s l y . T h e sensitivity o f E L I S A is c o m p a r a b l e to t h a t o f radioi m m u n o a s s a y (close t o 1 ng o f specific a n t i b o d y ) (Braciale et al., 1 9 7 6 ) b u t E L I S A d o e s n o t r e q u i r e as m u c h e q u i p m e n t as r a d i o i m m u n o a s s a y . T h u s it is possible to d e m o n s t r a t e b y E L I S A the p r e s e n c e o f a n t i b o d i e s even in pulm o n a r y secretions. T h e high sensitivity o f t h e assay also p e r m i t s t h e detection o f small q u a n t i t i e s o f a n t i b o d i e s s e c r e t e d b y in v i t r o c u l t u r e s o f spleen cells. This sensitive assay c o u l d be specially suitable f o r screening a n t i b o d y secreting clones a m o n g h y b r i d o m a cell lines ( K o p r o w s k i et al., 1 9 7 7 ) prod u c e d in v i t r o f o r t h e s t u d y o f antigenic v a r i a t i o n a m o n g i n f l u e n z a viruses. ACKNOWLEDGEMENT T h e a u t h o r s wish to t h a n k Dr. J.L. G u e s d o n f o r h e l p f u l discussions. REFERENCES Alford, R.H., R.D. Rossen, W.T. Butler and J.A. Kasel, 1967, J. Immunol. 98,724. Avrameas, S. and B. Guilbert, 1971, C.R. Acad. Sci. (Paris) 273, 2705. Bishai, F.R. and R. Galli, 1977, Lancet ii, 696. Braciale, T.J., W. Gerhard and N.R. Klinman, 1976, J. Immunol. 116, 827. Engval, E. and P. Perlmann, 1971, Immunochemistry 8,871. Fazekas de St. Groth, S. and S. Webster, 1966, J. Exp. Med. 124, 331. Jensen, K.E., 1961, Am. Rev. Resp. Dis. 83, 120. Koprowski, H., W. Gerhard and C.M. Groce, 1977, Proc. Natl. Acad. Sci. U.S.A. 74, 2985. Leinikki, P. and S. P/~ssil~, 1976, J. Clin. Pathol. 29, 1116. Lemieux, S., S. Avrameas and A.E. Bussard, 1974, Immunochemistry 11,261. Myrvik, Q.N., E.S. Leake and B. Fariss, 1961, J. Immunol. 86, 128. Prevot, J. and J.L. Guesdon, 1977, Ann. Microbiol. (Inst. Pasteur) 1288, 531. Scott, G.H. and J.S. Walker, 1976, Infect. Immun. 13, 1525. Svenson, S.B. and K. Larsen, 1977, J. Immunol. Methods 17,249. Voller, A. and D.E. Bidwell, 1976, Brit. J. Exp. Pathol. 57, 243. Wolters, G., L. Kuijpers, J. Kacaki and A. Schurs, 1976, J. Clin. Pathol. 29,873.

A microplate immunoenzyme assay for anti-influenza antibodies.

Journal o f Immunological Methods, 26 (1979) 61--67 61 © Elsevier/North-Holland Biomedical Press A MICROPLATE I M M U N O E N Z Y M E A S S A Y F O...
353KB Sizes 0 Downloads 0 Views