An improved dot ELISA to detect fowl adenovirus type-1 antigen

Comp. lmmun. MicrobioLinfect. Dis. Vol. 16, No. 3, pp. 245-250, 1993 Printed in Great Britain. All rights reserved

0147-9571/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

SHORT COMMUNICATION A N I M P R O V E D D O T E L I S A TO D E T E C T F O W L A D E N O V I R U S TYPE-1 A N T I G E N N . K . MAITI, S. S. SAINI, R. SINGH, M . S. OBEROI a n d S. N . SHARMA Department of Veterinary Bacteriology and Virology, Punjab Agricultural University, Ludhiana-141 004, India

(Received for publication 19 January 1993) Abstract--An improved dot immunobinding assay to detect fowl adenovirus type-1 is described. The method consists of spotting of antigen on nitrocellulose membrane sheet, blocking with either 5% acetic acid or with 5% defatted milk powder and fixation of either antigen or antigen-antibody complex, with either 50% methanol or 0.25% glutaraldehyde or 0.2% tannic acid. The results revealed that fixation of both antigen and antigen-antibody complex resulted in 4-fold increase in sensitivity when acetic acid was used as blocking agent. The use of two substrates simultaneously resulted in more colour intensity and clarity than using both substrates separately.

Key words: Improved, dot ELISA, detection, fowl adenovirus. Rtsumt---L'amtlioration du dosage per immunobinding pour dttecter l'adtnovirus de volatile type-1 est ici dtcfite. La mtthode consiste fi d6poser l'antigtne sue une feuille de membrane de nitrocellulose saturte avec une solution d'acide acttique fi 5% ou de poudre de lait dtgraiss6 ~t 5%, et fixer l'antigtne seul ou le complexe antigtne-anticorps avec une solution de mtthanol ~i 50% ou de glutaraldhyde fi 0.25% ou d'acide tannique ~i 0.2%. Les rtsultats ont rtvt16 que la fixation de l'anti#ne seul et du complexe anti#ne-anti¢orps a entrain6 une augmentation de la sensibilit6 de la mtthode d'un facteur 4 lorsqu¢ l'acide acttique est utilis6 comme agent saturant. L'utilisation siraultan~e de deux substrats a entraln6 une augmentation de l'intensit~ de la coloration et une plus grande lisibilit6 qu¢ rutilisation stparte de deux substrats.

Mots-clefs: Amtlioration, du ELISA, dttection, l'adtnovirus de volaille.

INTRODUCTION

Dot immunobinding has become an important solid phase immunoassay since its development by Hawkes et al. [1] to detect either antigen or antibody. The test has got a number of advantages over the other immunoassays in that it is cheap and simple to perform. But high sensitivity of the test is essential to detect very low concentration of antigen present in various secretions of chronically infected animals. Hence, we report a dot ELISA of higher sensitivity to detect fowl adenovirus typed antigen.

MATERIALS AND METHODS

Antigen F o w l a d e n o v i r u s type-1 g r o w n in 9 - 1 0 d a y o l d c h i c k e n e m b r y o s w a s c o n c e n t r a t e d b y 245

246

Short C o m m u n i c a t i o n Table 1. Protocol and results of dot ELISA when antigen is fixed Fixation of antigen at 4°C for 15 min with: 0.25% Glutaraldehyde 0.2% Tannic acid 50% Methanol

Blocking agents (1) 5% Acetic acid at 4°C for 15 min (2) 5% Milk powder at 40°C for 30 rain

Incubation with antibody

Increase in sensitivity

40°C for 30 min

4-fold

40°C for 30 min

2-fold

precipitation with 55% saturation of ammonium sulphate. Normal allantoic fluid received similar treatment and was used as control antigen.

Antiserum Antiserum against virus was raised in 10 week old chickens as per the method of Yates

et al. [2]. Fixatives Glutaraldehyde (0.25%) in phosphate buffer saline (PBS), aqueous solution of tannic acid (0.2%) and methanol (50%) in PBS were used to fix either antigen or antigen-antibody complex.

Blocking agents Defatted milk powder (5%) in Tris buffer saline (pH 7.2) and 5% acetic acid in PBS were used as blocking agents.

Fixation of antigen Approximately I pl of antigen diluted 2-fold serially in normal saline was spotted onto nitrocellulose membrane (NCM) sheets. After drying at room temperature, the antigen was fixed with different fixatives at 4°C for 15 min (Table 1). The strips were then washed once with PBS, blocked either with 5% acetic acid at 4°C for 15 min or with 5% milk powder at 4°C for 30 min and incubated with 1: 50 dilution of antiserum diluted in PBS containing 0.05% tween-20 (PBST) at 4°C for 30 min (Table 1).

Fixation of antigen-antibody complex The antigen spotted NCM sheets, after drying were blocked and incubated with antibody (as above) and washed thrice with PBST. The bound antibody was fixed with different fixatives at 4°C for 15 min (Table 2). Finally the sheets were washed once with PBS. Table 2. Protocol and results of dot ELISA when antigen-antibody complex is fixed Blocking of antigen No. (1) (2)

Agent 5% Acetic acid

Temperature and time 4°C for 15min

Incubation with antibody at: 40°C for 30rain

5% Milk powder 40°C for 30rain 40°C for 30rain

Fixation of antigen-antibody complex at 4°C for 15 rain with: 0.25% Glutaraldehyde 0.2% Tannic acid 50% Methanol 0.25% Glutaraldehyde 0.2% Tannic acid 50% Methanol

Increase in sensitivity 4-fold 2-fold

Fig. 1. Blocking of non-specific binding sites with acetic acid. Lanes B, D, F, H, J, L and P: spotted with normal allantoic fluids; lanes C and D, antigen fixed with glutaraldehydes; lanes E and F, antigen fixed with methanol; lanes G and H, antigen fixed with tannic acid. In lanes K and L, M and N, O and P antigen-antibody complexes were fixed with glutaraldehyde, methanol and tannic acid, respectively. In lanes A, B, I and J, no fixation was done. Initial protein content of both virus and control antigen were 1 mg/ml.

Fig. 2. Blocking with milk powder. Other details are the same as those described in Fig. 1.

Fig. 3. Showing the affect of multiple substrates. Lanes A, C, E and G were spotted with viral antigen and lanes B, D, F and H with control antigen. Lanes A and B, colour was developed with alfa-chloronaphthol (0.06%); lanes C and D, colour was developed with alfa-chloronaphthol (0.03%) and lanes G and H, colour was developed with both 3-Y-diaminobenzidine (0.012%) and alfa-chloronaphthol (0.015%). Initial protein content of the sample was 5 mg/ml.

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Short Communication Table 3. Results of using two enzyme substrates alone and in combination No, (1) (2) (3) (4)

Intensity and rapidity of reaction

Substrate(s) 3-Y-diaminobenzidine alfa-chloronaphthol 3-Y-diaminobenzidine + alfa-chloronaphthol 3-Y-diaminobenzidine + alfa-chloronaphthol

0.05% 0.06% 0.05%

+ + + + + + ++++

0.03 % 0.012% ++++ 0.015%

Immunological detection of antigen-antibody complex The NCM sheets were incubated sequentially with 1:1000 dilution of HPRO conjugated with rabbit raised antichicken IgG (Sigma) at 40°C for 30 min and substrate either 3-Y-diaminobenzidine (0.05%) in PBS or alfa-chloronaphthol (0.06%) or both containing different concentrations of 3-Y-diaminobenzidine (0.025-0.012%) and alfa-chloronaphthol (0.03-0.015%) for 10-15 min at room temperatue (Table 3). The reaction was stopped by adding cold water. RESULTS The dot binding assay carried out using different fixatives and blocking agents are shown in Figs 1 and 2. When neither antigen nor antigen-antibody complex was fixed, the positive reaction appeared only in undiluted antigen spot (lanes A and I in Figs 1 and 2). But fixation of antigen as well as antigen-antibody complex resulted in 4-fold increase in the sensitivity with all the fixatives when acetic acid was used as blocking agent (Fig. 1) but only 2-fold increase in the sensitivity was observed with milk powder based blocking system (Fig. 2, Tables 1 and 2). The effect of simultaneous use of two substrates on the sensitivity of the assay is shown in Fig. 3 and Table 3. The use of two substrates in combination resulted in more rapid development of blue colour spot to the use of two substrates alone (lanes C and G in Fig. 3) with both the blocking buffer systems. The use of acetic acid as blocking agent had less background and similar to that obtained with milk powder blocking system. DISCUSSIONS The main problem encountered in dot ELISA is that antigen is solubilized during processing thus resulting in lower sensitivity. Our aim was to overcome these problems by using various fixatives. It was evident that the use of fixatives increased the sensitivity of the assay by 4-fold. Natsukari et al. [3] obtained similar increase in the sensitivity by fixing the antigen with formaldehyde vapour. However, they did not observe any increase in the sensitivity when glutaraldehyde, isopropyl alcohol and acetic acid were used to fix the antigen. These are in contrast with the present findings indicating the differences in the native antigens used. The significance of the present findings is the superiority of acetic acid as a blocking agent over the milk powder in that the time and cost of the assay could be reduced without loss of sensitivity. However, milk powder seemed to lower the antigen-antibody reaction. There have been reports that protein based blocking agents somewhat lower the reactivity

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of antigen and antibody. This may be due to the steric hindrance caused by some milk protein which bind the antigen and mask the epitope from reaction of antibody [4] or due to the non-selective loss of protein from NCM [5]. In the present study, we also obtained similar results. Generally 3-3'-diaminobenzidine gives higher background compared to alfachloronaphthol, but intensity of colour developed seemed to be less in case of alfachloronaphthol (lane A, Fig. 3). To compromise these facts, we used two substrates simultaneously and as a result colour developed quickly and more intensely. The use of multiple substrates in combination offers the advantage of time saving and clarity. Similar observations were also obtained by Lee et al. [6]. The findings of the present study indicate that weak or doubtful reactions in conventional dot ELISA can be reevaluated by using the present improved dot ELISA to arrive at a definite conclusion. REFERENCES 1. Hawkes R., Naiday E. and Gordon J. A dot immunobinding assay for monoclonal and other antibodies. Analyt. Biochem. 119, 142 (1982). 2. Yates V. J., Rhee Y. O., Fry D. E., Mishad A. M. E. I. and McCromick K. J. The presence of avian adenoviruses and adenoassociated viruses in healthy chickens. Avian Dis. 20, 146-152 (1976). 3. Natsukari N., Ohta H. and Fujta M. Simple immunoassay for semiquantitative determination of calmodulin (CAM) and anti CaM: application of affinity column chromatography of anti CaM antibodies and estimation of CaM in synaptosomal membranes from rat cerebral cortex. J. Immunol. Meth. 125, 159-166 (1989). 4. Mohammad K. and Esen A. A blocking agent and a blocking step are not needed in ELISA. Immunostaining dot-blots and Western blots. J. Immunol. Meth. 117, 141-145 (1989). 5. Denttollander N. and Befus D. Loss of antigen from immunoblotting membranes. J. Immunol. Meth. 122, 129-135 (1989). 6. Lee N. I., Zhang Sun-Qu and Testa D. A rapid multicolour Western blot. J. lmmunol. Meth. 106, 27-30 (1989).