Bioscience Reports, Vol. 8, No. 5, 1988

A Sensitive and Rapid Immunochemical Method for Quantitation of Proteins M. R. Mawai, Y. R. Mawal, S. N. Ranadive ~ and P. K. Ranjekal "~ Received November, 1987 A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also. KEY WORDS: ELISA; storage proteins; globulins; pigeon pea (Cajanus ca]an)

INTRODUCTION Immunoassays have become an important tool in biomedical research and continuous efforts are being made to increase convenience, specificity and sensitivity of such assays (1). The conventional methods that afford the requisite sensitivity include radiolabels, enzyme labels and fluorescence or phosphorescence labels (2-8). These assays are elaborate, time consuming and require the use of sophisticated instruments. One of the ways to reduce assay time is to use the recognizing antibody and the marker label in the same step. This would require coupling of marker label to antibody itself. However, it has been shown that there is a loss or reduction in binding of antibodies to antigen when the former are coupled to other proteins or marker enzymes (16). An alternative to this would be to have the recognizing antibody and marker label coupled separately to a different matrix at the same time, so that the whole matrix as such may be used in the assay. In this context, it is interesting to note that coupling of bleomycin (BLM), a potent glycopeptide anticancer antibiotic, and murine monoclonal anti-HLA IgG antibody (H-l) to dextran, has been reported to be Division of Biochemical Sciences National Chemical Laboratory, Pune-411 008, india. I Tata Research Development and Design Centre, 1 Mangaldas Road, Pune 411 008, India: Present address: National Institute of Virology, Pune-411 001. 2 To whom correspondence should be addressed. 427

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Mawal, Mawal, Ranadive and Ranjekar

without significant loss in antibody activity (13, 14). We therefore thought of using this approach to couple antibodies and a marker enzyme to dextran and to use this conjugate in an ELISA. In this paper, we report an ELISA procedure for assaying pigeon pea (Cajanus cajan) globulins using pigeon pea globulin antibodies bound to dextran along with the enzyme horseradish peroxidase.

MATERIALS AND METHODS Materials

Pigeon pea seeds (cv T-21) were obtained locally. Mutated pigeon pea varieties TT-5, T-F-6 and TAT-10 (obtained by neutron bombardment) were from Bhabha Atomic Research Centre, Bombay. Bovine serum albumin (BSA), goat antirabbit IgG peroxidase conjugate and dextran T-40 were obtained from Sigma Chemical Company (MO, USA). Sephadex G-25 and Sephadex G-200 were obtained from Pharmacia Company (Uppsala, Sweden). All other chemicals were purchased locally and were of Analar grade. Generation of Antibodies

Pigeon pea (Cajanus cajan) globulins from T-21, Tr-5, TT-6 and TAT-10 were isolated and purified according to Krishna et al. (9). Antibodies were raised in New Zealand white strain rabbits against pigeon pea globulin (cv T-21) and affinity purified according to Mawal et al. (10). Binding of Antibody and Peroxidase to Dextran

Dextran T-40 (mol. wt. 40,000) was oxidized to polyaldehyde dextran (PAD) by Malaprade reaction (11-13). In this reaction, dextran T-40 was mixed with sodium periodate (0.33g in 200 ml distilled water) and was stirred well for 24 hrs at 4~ The resultant mixture was lyophilized, dissolved in a minimum volume of distilled water and dialyzed extensively against distilled water. The dialyzate was desalted over a Sephadex G-25 column (12cm x 1 cm) and the eluate was redialyzed against water prior to lyophilization. To 100 mg of PAD, 10 ml of PBS (0.1 M KzHPO4, 0.1 M KH2PO4, 0.85% NaC1, pH 7.2) was added, the solution stirred well for 2 hrs at 4~ and centrifuged for 5 minutes at 2800 rpm in a Sorval SS-34 rotor. To the supernatant, 1 mg of horseradish peroxidase and 2 mg of affinity purified pigeon pea globulin antibodies were added, and the mixture incubated overnight at 4~ with slow stirring. The Schiff bases thus formed were reduced by incubation with 0.3 ml of sodium borohydride solution (5 mg/ml in PBS) for 30 minutes at 4~ (15) and the mixture was then dialyzed extensively against water overnight. The dialyzate was centrifuged at 12,000 rpm for 20 min at 4~ in a Sorval SS-34 rotor and the supernatant was then chromatographed on a Sephadex G-200 column (23cm x 1.5cm) using PBS as an eluent. The void volume fractions were pooled and concentrated to a 2ml volume using a

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collodion bag (Sartorius, 10,000 cut off). Preimmune antibodies (2mg) were coupled to peroxidase (1 rag) via dextran by the above method and were used as a negative control in all the experiments. Affinity purified pigeon pea globulin antibodies (2 rag) were coupled to horseradish peroxidase (1 rag) by the periodate oxidation conjugation method according to the procedure of Hurn et al. (8) and the conjugate was concentrated to a 2ml volume. Using the same method, preimmune IgG (2 rag) was also coupled to horseradish peroxidase (1 mg) and the conjugate was concentrated to a 2 ml volume.

ELISA Comparative ELISA Using Goat Antirabbit IgG Conjugate and Pigeon Pea Globulin IgG-Dextran Conjugate The rapidity and feasibility of our modified ELISA was compared with (a) conventional ELISA using goat antirabbit IgG peroxidase conjugate and (b) control ELISA using pigeon pea globulin peroxidase conjugate. BSA and preimmune serum were used as internal negative controls. The ELISA plate was divided into seven parts. To part A various amounts of BSA (0-1000 ng) were added while in parts B to G various amounts of pigeon pea globulins (0-1000 rig) were added. In the second step the additions were done as follows: A & F: Pigeon pea globulin antibody-dextran-peroxidase conjugate (1:1000 dilution). B & C: Pigeon pea globulin antibody (0.2 #g). D: Preimmune antibody-peroxidase conjugate (1 : 1000 dilution) E: Pigeon pea globulin antibody-peroxidase conjugate (1 : 1000 dilution). G: Preimmune antibody-dextran-peroxidase conjugate (1 : 1000 dilution). In step three, goat antirabbit IgG horseradish peroxidase conjugate (1:1000 dilution) was added to part B, and 2% BSA (100 f~l) was added to the remaining parts A, C to F. Peroxidase was assayed with 5-amino salicylic acid (0.2 rag) and H202 (0.01%). Absorbance of the color developed in the assay was measured at 450 nm. Experiments were repeated in triplicate and best fit value of A450 nm was plotted versus antigen amount.

ELISA for Quantitation of Pigeon Pea Globulin In order to further confirm the sensitivity of our method, modified ELISA was employed for quantitation of pigeon pea globulins to as low a level as possible. In this experiment various amounts (0-1000 ng) of pigeon pea globulins were added to each well and incubated with various dilutions (1:1000; 1:3000; 1:5000 and 1:7000) of pigeon pea globulin antibody-dextran-peroxidase conjugate. Preimmune antibody peroxidase conjugate was used as internal negative control. The peroxidase was assayed with 5-amino salicylic acid and H 2 0 2 a s above.

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RESULTS A N D DISCUSSION

The results of peroxidase assay using goat antirabbit IgG conjugate and dextran conjugate are depicted in Fig. 1. It can be seen from the figure that the goat antirabbit IgG horseradish peroxidase conjugate can detect pigeon pea globulin down to 1 ng when the dilution of 1:1000 is used. In contrast, at the same dilution pigeon pea globulin IgG-peroxidase conjugate can detect 0.34 ng, while the dextran conjugate detects 0.1 ng of pigeon pea globulin. In fact, for measuring 1 ng pigeon pea globulin, a 1 : 7000 dilution of the dextran conjugate is sufficient. These results indicate that the dextran conjugate has a 10-fold greater efficiency of quantitating pigeon pea globulin than the commercial goat antirabbit IgG conjugate and a 3-fold greater efficiency than the pigeon pea globulin IgG peroxidase conjugate. From Fig. 1, it is further evident that as the dilutions of the dextran conjugate increase, the detectable amount of pigeon pea globulin decreases. Based on these studies, we decided that a dilution of 1:7000 was sensitive enough to quantitate pigeon pea globulins in the range of 0-1000 ng. These results are depicted in Fig. 2. Moreover, the dextran conjugate did not show any decrease in activity during storage at 4~ 0.10 0

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PROTEtN AMOUNT(ng) Fig. 1. ELISA of pigeon pea globulins using various dilutions of pigeon pea globulin antibody dextran-peroxidase conjugate. The antigen was adsorbed on the microtiter plate well and ELISA performed as described in Materials and Methods. (11) 1:1000 dilution of dextran conjugate; (| 1 : 3000 dilution of dextran conjugate; (A) 1 : 5000 dilution of dextran conjugate; ( ~ ) 1 : 7000 dilution of dextran conjugate; (&) 1 : 1000 dilution of goat antirabbit IgG peroxidase conjugate; (E]) bovine serum albumin; (• 1:1000 dilution of pigeon pea globulin antibodies peroxidase conjugate and (O) 1 : 1000 dilution of preimmune antibodies peroxidasc conjugate.

To further confirm the efficiency of our method, we compared the time required to complete one assay. This comparison is schematically represented in Fig. 3. It is clear from the figure that while the conventional ELISA using goat antirabbit IgG conjugate takes about 3 hours 40 minutes for completion, the ELISA using dextran conjugate is completed in just 1 hour and 50 minutes, thereby achieving a time saving of half the duration of a conventional ELISA. The feasibility of our method was confirmed by screening pigeon pea globulins from variety T-21 along with neutron-bombarded varieties TF-5, T-I?-6

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10 !00 1000 ANTIGEN AMOUNT(ng) Fig. 2. ELISA of pigeon pea globulins. The antigen was adsorbed on microtiter plate wells and pigeon pea globulin antibody-dextran conjugate (1 : 1000) was allowed to bind to these antigens. The bound conjugate was quantitated. (O) 1:1000 dilution of dextran conjugate; (0) bovine serum albumin; ( | ) 1 : 1000 dilution of preimmune antibody peroxidase conjugate and ([]) 1 : 1000 dilution of pigeon pea globulin-peroxidase conjugate. Conventional ELISA Protein (pigeon pea globulin) bound to solid matrix.

Dextran-conjugate ELISA Protein (pigeon pea globulin) bound to solid matrix.

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50 min, 42~ Aspirate and wash thrice with 10 mM Tris-saline buffer pH 7.2 containing 0.01% Tween-20 with each wash of 20 rain duration. 1 hour Assay using suitable buffer (HaO 2 and 5-amino salicyclic acid) and read absorbance at 450 nm. Total assay time = 220 minutes. Total assay time = 110 minutes Fig. 3. Schematic representation for comparison between conventional ELISA and ELISA using pigeon pea globulin antibody-dextran-peroxidase conjugate.

432

Mawal, Mawal, Ranadive and Ranjekar

and TAT-10 for globulin content per seed. Table 1 shows the amount of globulin present in seeds of these varieties and the amount of globulin required for 50% binding of the labelled antigen (cv T-21) to dextran-pigeon pea globulin IgG conjugate. It can be seen from Table 1 that 53, 71 and 88 ng of competing TT-5, TT-6 and TAT-10 respectively are required for 50% binding of T-21 globulin to dextran-conjugated globulins. The corresponding value for T-21 globulin is 48 ng. Table 1. ELISA of globulins of different varieties of pigeon pea using dextran-conjugate

Variety

Globulin detected by ELISA (ng/mg of seed meal)

50% binding (ng)

T-21 TF-5 TF-6 TAT-10

1.01 :k 0.05 0.84 + 0.03 0.76 5:0.04 0.56 + 0.06

48 :t: 0.82 53 5:0.71 71 5:0.41 81 + 0.86

Each value is an average of experiments done in triplicate.

Thus in conclusion it may be stated that our method employing dextran conjugate is more sensitive and rapid. Furthermore, since the use of a second antibody is avoided, our method will prove to be economical also. An additional advantage of our method is that it is simple, requiring no elaborate calibrations, no radioactive labels and no expensive equipment, and it uses stable reagents. The only prerequisite for our method is the availability of antibody against the protein to be quantitated. Any unknown protein can, therefore, be quantitated to as low an amount as 0.1 ng once antibodies against the same protein are obtained. A certain background is always seen with normal sera in this kind of assay. This background may be due to non-specific binding of IgG in normal serum and probably to other unknown protein-protein interactions as well. The non-specific reaction can be minimized by using affinity purified antibodies and also by inclusion of non-ionic detergents such as Triton 3(-100 or Tween 20. The latter do not interfere with the antigen-antibody reaction and prevent the formation of new hydrophobic interactions between added protein and solid phase. Neither do they disrupt the hydrophobic bonds already formed between the previously adsorbed protein and the plastic surface (2). In our method, the optimal concentration of antigen as suggested for ELISA is between 0.1 and 200 ng. Although a higher concentration of antigen leads to increased adsorption, the actual percentage of adsorbed antigen decreases. Furthermore, a high concentration of protein during coating leads to increased desorption during incubation with immunoreactants. This sometimes gives rise to undesirable prozone phenomena (2). Therefore, in order to design an assay with optimal sensitivity, it is essential to (a) choose an antiserum of highest possible affinity; (b) use incubation times that allow equilibration between antigen and antibody; (c) use a lower concentration of antigen and antibody and keep for a longer time for color development. In conclusion, it is suggested that if affinity

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purified a n t i b o d i e s a r e u s e d a n d 1 : 7 0 0 0 d i l u t i o n d e x t r a n c o n j u g a t e is t a k e n , t h e sensitivity o f E L I S A assay will b e c o m p a r a b l e to t h a t of r a d i o i m m u n o a s s a y a n d such assays will o p e n a n e w a r e a o f t e c h n o l o g y f o r e n z y m e i m m u n o a s s a y s .

ACKNOWLEDGEMENTS T h e a u t h o r s a r e g r a t e f u l to D r s ( M r s ) V i d y a G u p t a a n d Shirish R a n a d e f o r t h e i r v a l u a b l e a d v i c e , d i s c u s s i o n a n d h e l p f u l c o m m e n t s in p r e p a r i n g t h e m a n u script. T h e a w a r d o f r e s e a r c h f e l l o w s h i p s to M . R . M a w a l a n d Y. R . M a w a l b y t h e C o u n c i l o f Scientific a n d I n d u s t r i a l R e s e a r c h , N e w D e l h i is g r a t e f u l l y a c k n o w l e d g e d . T h i s w o r k is N C L C o m m u n i c a t i o n N o . 4188.

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