Single-chain fragment variable antibody against the capsid protein of bovine immunodeficiency virus and its use in ELISA

Journal of Virological Methods 167 (2010) 68–73

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Single-chain fragment variable antibody against the capsid protein of bovine immunodeficiency virus and its use in ELISA S. Bhatia ∗ , Rakhi Gangil, Devyani S. Gupta, Richa Sood, H.K. Pradhan, S.C. Dubey High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anandnagar, Bhopal, Madhya Pradesh, 462021 MP, India

a b s t r a c t Article history: Received 26 September 2009 Received in revised form 5 March 2010 Accepted 10 March 2010 Available online 19 March 2010 Keywords: BIV Capsid ScFv gag Recombinant antibody

Recombinant antibody specific for the capsid (CA) protein of bovine immunodeficiency virus (BIV) was generated in the form of single-chain fragment variable (ScFv) using the phage display technique for affinity selection. The variable heavy (VH ) and variable light (VL ) chain gene fragments were recovered from cells of CA-specific hybridoma (9G10) described previously. The VH and VL DNA fragments were assembled through a flexible linker DNA to generate ScFv fragment which was cloned in a phagemid expression vector to express ScFv protein. The specific reactivity of the expressed ScFv to the CA antigen was confirmed by Western blot, and the ScFv fragment was used to develop a competitive inhibition ELISA for detection of antibodies to BIV in cattle and buffalo. The recombinant antibody was shown to be more than four times sensitive than its parent monoclonal antibody (MAb, 9G10) by testing of spiked samples of reference positive sera. The improved sensitivity of the recombinant antibody-based ELISA was confirmed by the detection of a larger proportion of animals with BIV antibody by it than by the MAb-based ELISA. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Bovine immunodeficiency virus (BIV) is a member of the Lentivirinae subfamily of the Retroviridae family and is similar morphologically, genetically and antigenically to the human immunodeficiency virus (HIV) and other lentiviruses (Van der Maaten et al., 1972; Gonda et al., 1987, 1994). BIV causes a persistent infection in cattle (Gonda et al., 1994) and secondary bacterial infection (Mc Nab et al., 1994; Snider et al., 1996). In India, BIV infection was reported in cattle by the detection of BIV proviral DNA in peripheral blood leukocytes (Patil et al., 2003) and subsequently by a capsid (CA) protein-based indirect ELISA (Bhatia et al., 2008a) and monoclonal antibody (MAb)-based competitive inhibition ELISA (Bhatia et al., 2008b). In a previous study, the diagnostic usefulness of MAb (9G10) was established by the development of a specific and sensitive competitive inhibition ELISA for serological diagnosis of BIV (Bhatia et al., 2008b). In the present study, an attempt has been made to develop recombinant antibodies in the form of ScFv (single-chain fragment variable) protein derived genetically from the CA-specific hybridoma (9G10) by the application of the phage display technique for affinity selection. The phage display technique was first described by McCafferty et al. (1990). This method relies on a phage display system in

∗ Corresponding author. Tel.: +91 755 2750647; fax: +91 755 2758842. E-mail address: bhatia [email protected] (S. Bhatia). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.03.012

which fragments of antibodies are expressed as fusion proteins displayed on the phage surface. The recombinant antibodies obtained from bacteria are cheaper than the hybridoma derived MAbs. It is also easier to maintain the prokaryotic clones of recombinant antibodies than the hybridoma clones. Hence, it is reasonable to obtain recombinant antibodies from hybridoma clones of MAbs with well-proven diagnostic use of their application in diagnostic assays as a cheaper substitute for MAb. The present study was conducted with the aim of generating recombinant antibodies from the BIV-CA-specific hybridoma clone and to develop a recombinant antibody-based competitive inhibition ELISA for serological detection of BIV infection in cattle and buffalo. A comparative study of the sensitivity of the recombinant antibody and the MAb-based method by competitive inhibition is also described.

2. Material and methods 2.1. Expression of recombinant capsid (CA) protein The cloning and subsequent expression of the gag region (750 bp) of BIV in pQE32 vector (Qiagen GmbH, Hilden, Germany) was described elsewhere (Bhatia et al., 2008a). The expressed 6×-His tagged CA protein was purified by the Ni-NTA column (Amersham Biosciences, NJ, USA) and was refolded using Protein refolding kit (Novagen, Madison, USA).

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2.2. Generation of ScFv DNA fragment from hybridoma cells Briefly, hybridoma cells (clone-9G10) were centrifuged at 150 × g for 10 min. The mRNA was isolated from 5 × 106 viable cells using the Quick Prep mRNA purification kit (Amersham Biosciences, NJ, USA). The cDNA was generated from the mRNA by reverse transcription in the presence of random primers. The variable heavy (VH ; 340 bp) and light chain (VL ; 325 bp) gene fragments were amplified from the cDNA according to the manufacturer’s instructions (RPAS ScFv module, Amersham Biosciences, NJ, USA). After purification and quantitation, the VH and VL DNA products were assembled into a single gene using a DNA linker fragment coding for a (Gly4 -Ser)3 peptide (provided in the RPAS ScFv module). In a second PCR, the restriction sites were added using restriction site primers (RS primers) with either Sfi 1 or Not 1 restriction sites. The amplified ScFv DNA with restriction sites was gel purified and quantified by comparing the band intensities with the standard ScFv DNA on agarose gel. 2.3. Cloning and expression of ScFv The ScFv DNA was digested by Sfi 1 and Not 1 enzyme and then ligated into the phagemid vector pCANTAB 5E (Amersham Biosciences, NJ, USA). The E. coli TG1 cells were transformed with the ligation mixture by standard method (42 ◦ C for 2 min). The transformed cells were grown in 2× YT-G (glucose) medium for 1 h followed by addition of the helper phage M13KO7 (6 × 1010 pfu) and ampicillin (100 ␮g/ml) and further growth of the culture at 37 ◦ C for 1 h with shaking at 250 rpm. The culture was centrifuged and the cell pellet was re-suspended in 10 ml of 2× YT-AK (2× YT with ampicillin and kanamycin) and was incubated further at 37 ◦ C for overnight with shaking at 250 rpm. The recombinant phage was recovered from the overnight culture by precipitation with PEG 8000 followed by filtration (0.45 ␮m). The recombinant phage displaying CA-specific ScFvs were selected by three rounds of panning with the CA antigen coated on a 25 cm2 tissue culture flask. A fresh E. coli TG1 culture was inoculated with the enriched pool of the CA-specific phage followed by incubation at 37 ◦ C for 1 h. Tenfold dilutions of this culture were inoculated on the super optimal broth (Hanahan, 1983) agarose medium supplemented with ampicillin and glucose. After overnight incubation at 30 ◦ C, individual well-isolated colonies on plates were transferred to separate tubes containing 2× YT-AG and were incubated overnight at 30 ◦ C with shaking at 250 rpm. These cultures were used as master cultures. Fresh cultures were seeded in 5 ml of 2× YT-AG from master cultures of 15 clones and were incubated at 30 ◦ C with shaking at 250 rpm. After 2 h of incubation, the medium was replaced by 2× YT-AI (2× YT with ampicillin and IPTG) and the cultures were further incubated with the same conditions for 24 h. The supernatants of the expression cultures were collected in tubes and stored at 4 ◦ C until screening by ELISA. 2.4. ELISA screening of ScFv antibodies The supernatants from the 15 clones were tested by indirect ELISA for the presence of CA-specific E-tagged ScFvs (pCANTAB 5 E also contains a sequence encoding for a peptide tag-‘E-tag’ and thus yields E-tagged ScFv). The binding of E-tagged recombinant antibodies was detected by anti-E-tag HRPO conjugate (Amersham Biosciences, NJ, USA). Briefly, 50 ␮l of the purified BIV-CA antigen (50 ng/well) was coated on ELISA plate (MaxisorpTM from Nunc, Germany) in antigen coating buffer (Carbonate-bicarbonate buffer) and was incubated at 4 ◦ C overnight. After washing once with PBS, 50 ␮l of each supernatant (diluted 1:2 in blocking buffer-PBS with 1% non-fat dry milk) was added to the corresponding wells in the ELISA plate. The plate was incubated at 37 ◦ C for 2 h. After incuba-

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tion, the plate was washed 3 times with PBS-T. Fifty microliters of the anti-E-tag conjugate diluted 1:8000 in the blocking buffer was added to each well and the plate was incubated for 1 h for 37 ◦ C. After washing 3 times, 50 ␮l of TMB substrate (Tetra methyl benzedine) was added to each well and kept in dark for 15 min. A stop solution was added (50 ␮l per well) and reading was taken in ELISA reader (Tecan, Austria) at 450 nm. 2.5. Preparation and purification of BIV-CA-specific recombinant antibodies One of the phage clones which reacted by ELISA with the BIVCA antigen was selected and its phagemid was transferred to the E. coli HB2151 strain (Amersham Biosciences, NJ, USA) for bulk production of anti-CA-specific recombinant antibodies (soluble ScFvs). Briefly, the log phase culture of the E. coli HB2151 was inoculated with 25 ␮l of the recombinant phage (culture supernatant stored at 4 ◦ C and selected positive in ELISA screening) and incubated with gentle shaking for 30 min at 37 ◦ C followed by streaking on super optimal broth agarose supplemented with ampicillin (100 ␮g/ml), Nalidixic acid (100 ␮g/ml) and glucose. The plates were incubated overnight at 30 ◦ C. Colonies were transferred from the plate to 1 ml of freshly prepared 2× YT-AG medium and incubated overnight at 30 ◦ C with shaking at 250 rpm. A 500 ␮l aliquot of the overnight culture was added to 5 ml of freshly prepared 2× YT-AG medium and was incubated for 1 h at 30 ◦ C with shaking at 250 rpm. The culture was centrifuged at 1500 × g for 20 min at room temperature and the supernatant was removed carefully. The pellet was re-suspended in 5 ml of freshly prepared 2× YT-AI medium (no glucose) and was incubated for 24 h at 30 ◦ C with shaking at 250 rpm. The culture was centrifuged again as in the previous step and the cell pellets obtained were re-suspended in 40 ␮l of ice cold 1× TES (0.2 M Tris–HCl, 0.5 mM EDTA, 0.5 M sucrose, pH 8.0). Then 60 ␮l of ice cold 1/5× TES was added in each tube and mixed gently. The suspension was incubated on ice for 30 min and then centrifuged at 10,000 × g for 10 min at 4 ◦ C. The supernatant was transferred carefully to a separate tube and stored at 4 ◦ C until further use. The soluble recombinant antibodies were screened by ELISA as described in Section 2.4. The recombinant antibody (ScFv) was prepared in bulk from a positive clone of E. coli HB2151 and was purified from the periplasmic extract using the anti-E-tag affinity column (Amersham Biosciences, NJ, USA) as per the manufacturer’s protocol. The purified ScFv was checked for its purity and concentration by SDS-PAGE. 2.6. Western blot The expression of ScFv was confirmed by Western blot of Etagged ScFv with the anti-E-tagged HRPO conjugate. Briefly, the periplasmic extracts of the uninduced and the induced culture of the recombinant clone in E. coli HB2151 were run on the 12.5% SDSPAGE and transferred to a nitrocellulose membrane using semi-dry transfer system (from Hoefer, Holliston, USA). The membrane was blocked with 3% bovine serum albumin (BSA) in Tris buffered saline (TBS; 10 mM Tris–Cl, 150 mM NaCl, pH 7.5) at room temperature for 1 h with gentle shaking. After blocking, the membrane was washed 3 times with TBS and 3 times with TBS-Tween (5 min each). The membrane was incubated with the anti-E-tag HRPO conjugate (diluted 1:3000 in blocking buffer) for 1 h at room temperature with gentle shaking. After washing 3 times with TBS-Tween, the blot was developed with DAB (diaminobenzedine). To confirm the reactivity of the expressed ScFv with the BIVCA antigen, the antigen was blotted with ScFv and was detected subsequently by the anti-E-tagged HRPO by the same procedure as described above. Briefly, the cell pellet of the uninduced and the induced culture of pQE32-CA clone (in E. coli M15 cells) and the

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purified CA protein were run on 12.5% SDS-PAGE along with the molecular weight marker and were transferred to a nitrocellulose membrane. Membrane was blocked with the 3% BSA as described previously. After washing, the membrane was incubated with 1:20 dilution of ScFv for 1 h. The Membrane was again washed and incubated with the anti-E-tag HRPO conjugate (diluted 1:3000 in blocking buffer) with shaking for 1 h and developed subsequently with DAB. 2.7. Recombinant antibody-based competitive ELISA for testing serum for antibodies against BIV The concentration of the purified CA antigen and dilution of the recombinant antibody in C-ELISA was optimized through a preliminary checkerboard titration of antigen vs. recombinant antibody. The CA antigen (62.5 ng/well) was coated on an ELISA plate and incubated at 37 ◦ C for 1 h. After washing once with PBS, 40 ␮l of diluent was added in the wells followed by the addition of 10 ␮l of the test serum in duplicate wells. Fetal calf serum (10 ␮l) was added in two wells as negative control. Fifty microliters of the purified recombinant antibody (1:20) was added in all the wells and the plate was shaken gently to mix the contents. After 2 h incubation at 37 ◦ C, the plate was washed thrice with PBS-T and 50 ␮l of the antiE-tag HRPO conjugate diluted at 1:8000 was added to each well and incubated at 37 ◦ C for 1 h. After 3 times washing, 50 ␮l of TMB substrate was added and kept in dark at room temperature for 15 min. The stop solution was added 50 ␮l per well to stop the reaction. The plate was read at 450 nm and the mean percent inhibition value for each test serum was calculated by the formula given as under: Percent Inhibition (%) = 1 −



Mean OD of test serum Mean OD of the negative control



× 100

2.8. Testing of sera by competitive ELISA A total of 132 serum samples from cattle (103) and buffalo (29) were collected randomly and were tested by the recombinant antibody-based competitive ELISA as described above. The same panel of sera was also tested by the MAb-based competitive ELISA using the parent MAb (9G10) as described previously (Bhatia et al., 2008b). A cut-off value of 40% was used as a positive/negative reading of sera tested by both the ELISAs as described previously (Bhatia et al., 2008b). Two rabbits (8 weeks old) were immunized with 100 ␮g of the purified CA antigen per rabbit with equal volume of Freund’s Complete Adjuvant (FCA) by intramuscular route followed by boosters of 100 ␮g antigen per rabbit with Freund’s Incomplete Adjuvant (FIA) at weekly intervals up to 7 weeks. The sera of immunized rabbits were collected on 4th (I bleed) and 7th week (II bleed) and were titrated by the recombinant antibody-based ELISA as well as the MAb-based ELISA for detection of anti-CA antibodies. In another experiment, a known negative serum (0 day rabbit serum) was spiked with serial dilutions (1:2, 1:4 to 1:512) of known positive serum (hyper-immune rabbit sera). The spiked sera were tested by both the recombinant antibody and the MAb-based competitive ELISA as described above.

Fig. 1. SDS-PAGE analysis of anti-BIV-CA recombinant antibody (ScFv) after affinity purification from periplasmic extract. M - Molecular wt. marker, lane 1 - Crude periplasmic extract, lane 2 - purified recombinant antibody (30 kDa).

and was cloned subsequently to express recombinant E-tagged ScFv. Out of fifteen recombinants screened for the expression of anti-CA ScFv, only one clone 9G10.9 was found to be positive (OD450 − 0.937) while other clones showed OD of less than 0.2 (cut-off). The cut-off was calculated by 3 times multiplying the average OD of the negative control well (Antigen + supernatant from uninduced culture of clones + Anti-E-tag HRPO). The positive clone (9G10.9) identified by the screening ELISA was used to produce E-tagged recombinant antibodies in HB2151 strain of E. coli. The periplasmic extracts of the induced cultures of all the five recombinants screened showed positive reaction with the BIVCA in ELISA (OD450 − 2.5 to 2.7). One of the clones was used to prepare anti-CA ScFv in bulk and was purified by the anti-E-tag column using FPLC system (Pharmacia Biotech, Uppsala, Sweden). The SDS-PAGE analysis of the purified and the unpurified ScFv revealed a single band of approximately 30 kDa for the purified ScFv in comparison to the multiple bands in crude periplasmic extract (Fig. 1).

3. Results 3.1. Expression of anti-CA ScFv from hybridoma clone

3.2. Immunological characterization of anti-CA ScFv (recombinant antibody)

The heavy chain (VH ) and light chain (VL ) genes were amplified from the cDNA derived from the mRNA extracted from the BIV-CA-specific hybridoma clone (9G10) and were visualized on 1.5% agarose gel as 340 bp and 325 bp bands, respectively. A 750bp ScFv DNA was produced by joining VH and VL DNA fragments

Western blot analysis of the E-tagged ScFvs with the anti-Etagged HRPO conjugate showed a ∼30 kDa band visible in the positive clones but not in the negative control (Fig. 2). The CA antigen was blotted as 29 kDa band in the induced cell pellet and the purified antigen with E-tagged ScFv (Fig. 3).

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Table 1 Comparison of MAb and recombinant antibody-based competitive ELISA using a panel of field sera. State

Cattle

Buffalo

Total Cattle and Buffalo

Tested

+ve by MAb ELISA

+ve by recombinant antibody ELISA

Tested

+ve by MAb ELISA

+ve by recombinant antibody ELISA

Tested

+ve by MAb ELISA

+ve by recombinant antibody ELISA

M.P. Tamilnadu Himanchal Pradesh

55 – 48

35 – 8

38 – 10

– 29 –

– 11 –

– 13 –

55 29 48

35 11 8

38 13 10

Total

103

43

48

29

11

13

132

54 (41%)

61 (46.2%)

Fig. 2. Confirmation of expression of E-tagged ScFv (recombinant antibody) by Western blot. NC - total cell pellet of uninduced culture (negative control), lane 1–5 - total cell pellets of induced culture.

Fig. 5. Titration of spiked samples (dilutions of reference positive in known negative serum) by recombinant antibody and MAb-based ELISA showed greater sensitivity of the recombinant antibody than the MAb.

Fig. 3. Western blot of BIV-CA antigen (29 kDa) with anti-BIV-CA recombinant antibody (E-tagged ScFv). M - molecular wt. marker, lane 1 and 2 - total cell pellet (TCP) of uninduced and induced culture of pQE32-CA clone, respectively, lane 3 - purified CA antigen.

3.3. Recombinant antibody-based competitive ELISA for detection of antibodies against recombinant CA antigen of BIV in field sera Out of the 132 sera (103 from cattle and 29 from buffalo) tested, 61 sera (46.2%) were positive by the recombinant antibody-based

ELISA and 54 sera (41%) were positive by the MAb-based ELISA (Table 1). The CA-specific hyper-immune sera of rabbit and a known positive cattle serum had higher percent inhibition by the recombinant antibody-based ELISA (75–96%) than by the MAb-based ELISA (44–82%) indicating a greater sensitivity of the recombinant antibody than MAb in detecting CA-specific antibodies and a higher signal-to-noise ratio in the ELISA test (Fig. 4). In the titration of spiked samples, the cut-off point inhibition (40%) was extrapolated at a point very close to 1:160 dilution of spiked sample in case of the recombinant antibody while the 40% point was below 1:40 dilution in case of the MAb-based ELISA (Fig. 5) indicating that the recombinant antibody-based competitive ELISA was at least four times more sensitive (1/40 vs. 1/160) than the MAb-based ELISA.

Fig. 4. Difference in the PI values obtained by recombinant antibody and MAb-based ELISA for a panel of reference positive sera indicating higher signal amplification in recombinant antibody-based ELISA resulting in better resolution between negative and positive samples.

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4. Discussion The phage display method represents one of the most powerful tools for production and selection of recombinant antibodies and has been recognized as a valuable alternative for the preparation of antibodies of a desired specificity. Using the phage display technique, the recombinant antibodies can be prepared more rapidly with reduced consumption of laboratory animals in comparison to the MAbs. The Phage display was first introduced by Smith (1985) more than 20 years ago and was used initially for studying protein–protein interactions. Since then, the technique has gained wide acceptance as an effective method for producing large amounts of diverse proteins, including antibody fragments and peptides (Holt et al., 2000; Willats, 2002). The development of a competitive ELISA for BIV using the CA-specific MAb (9G10) has been reported previously (Bhatia et al., 2008b). In the present study, a recombinant antibody (CAspecific) was developed in the ScFv format by expression of VH and VL genes derived from mRNA extracted from a hybridoma clone (9G10). For the purpose of expressing the antibody genes of hybridoma cells into prokaryotic system, mRNA is an enriched source of expressed and spliced antibody genes. Since an antibody producing cell expresses the heavy and light chain genes for a single antibody, it represents therefore the most abundant and available source from which antibody genes can be cloned. Synthesis of the first-strand cDNA from mRNA was done with reverse transcriptase and random hexamers. The use of random hexamers eliminates the need for immunoglobulin-specific primers or oligo (dT) primers which would require the synthesis of a long cDNA, encoding the entire heavy chain or light chain. Using random hexamers, the resulting cDNAs are of sufficient length to clone the V regions from both the heavy and light chain genes. Since these genes represent a very small fraction of the total cDNA, they must be amplified first to generate sufficient DNA for cloning using primers specific to mouse Ig gene. The primers that have been used to generate heavy and light chain DNA from cDNA were specific for the mouse immunoglobulin gene and were derived from the framework region of the fragment variable. The concentration of VH and VL was determined very carefully in order to maintain amounts constant which was critical for the success of the subsequent assembly reaction. The most accurate measure was obtained by running gel electrophoresis and correlating the intensity of bands with known quantities on the DNA ladder (results not shown here). A 15-amino acid linker (Gly4 -Ser)3 was incorporated in the assembly reaction to create the fusion molecule in VH -linker-VL order. The assembled ScFv was amplified with a set of oligonucleotide primers that introduce restriction sites (Sfi 1 and Not 1) for cloning into the expression vector pCANTAB 5E. These particular restriction sites occur with very low frequency in antibody genes and thus allow cloning of most genes as a single Sfi 1/Not 1 fragment. The recombinant antibody 9G10.9 derived from the MAb 9G10 (IgG isotype) was equivalently reactive with the CA antigen as its parent MAb (OD450 − 2.5 to 3.00). Two earlier reports, one of ScFv to FMDV type O strain (Sheng Feng et al., 2003) and another on ScFv to BTV (Nagesha et al., 2001) showed that the expressed ScFvs retained the binding characteristics of the parent MAb. Presence of E-tag fused with ScFv molecule proved to be very effective in its purification through anti-E-tag Ab conjugated affinity column. The affinity purification could remove all the “in-house” bacterial proteins from crude periplasmic extract preparation of recombinant antibody as shown in SDS-PAGE analysis (Fig. 1). However, the unpurified periplasmic extract itself could be used satisfactorily without any non-specific reaction in downstream applications such as the competitive ELISA. The competitive ELISA standardized with the anti-CA recombinant antibody (9G10.9) was compared with the MAb-based

competitive ELISA, standardized earlier (Bhatia et al., 2008b). In the testing of 132 cattle and buffalo sera, the recombinant antibody detected additional positive sera (61) than its parent MAb (54). Since the sample size of the 132 sera tested was much smaller in the present study than the 672 sera tested previously (Bhatia et al., 2008b), the present report may not be considered as an update of the previous study on the sero-prevalence of BIV in India. The purpose of this screening was solely to compare the sensitivity of the two formats of ELISA (recombinant antibody and MAb-based). The lack of a “gold-standard” or a reference test has been a problem in evaluating the sensitivity and specificity of serological tests for BIV. Although, Western blot of BIV antigens (CA or TM-transmembrane) has been used as a reference test (Abed et al., 1999), it is subject to incorrect readings due to personal biases in recording results. In absence of a reference test for calculating and comparing the diagnostic sensitivity, the data on a large number of positive samples by the recombinant antibody-based ELISA does not alone prove the improved diagnostic sensitivity of the assay. However, it is supported by the other findings of the study, e.g. spiking which demonstrated greater (4 times) sensitivity of the recombinant antibody than its parent MAb in C-ELISA. Although diagnostic sensitivity of the two tests could not be ascertained due to the lack of a reference test for BIV antibody, the above findings indicate better performance of the recombinant antibody as diagnostic reagents as compared to MAbs. When positive sera (rabbit hyper-immune sera and cattle serum) were tested by the two assays, a higher percentage inhibition (75–96%) was observed with the recombinant antibody than the MAb-based assay (44–82%) indicating higher signal amplification resulting in better resolution between negative and positive samples. In another experiment, the recombinant antibody used in the assay was shown to be specific for BIV-CA protein by Western blot. In conclusion, a recombinant antibody (ScFv) was generated against the recombinant CA protein of BIV and was shown to react specifically with the antigen (CA). The study describes a recombinant antibody-based ELISA for detection of BIV antibodies which was found to be more sensitive than the MAb-based ELISA. The study also demonstrated the usefulness of ScFvs as an immunodiagnostic reagent and showed that the specificity of hybridoma clones could be preserved in the ScFv form as an alternative and cheaper source of specific antibodies. Acknowledgements This work was supported by a project grant of Department of Biotechnology, Ministry of Science and Technology, Government of India. The infrastructure and other facilities were provided by High Security Animal Disease Laboratory, Indian Veterinary Research Institute in Bhopal of Indian Council of Agricultural Research. References Abed, Y., St-Laurent, G., Zhang, H., Jacobs, R.M., Archambault, D., 1999. Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane envelope proteins expressed from recombinant baculovirus. Clin. Diagn. Lab. Immunol. 6, 168–172. Bhatia, S., Patil, S.S., Sood, R., Dubey, R., Bhatia, A.K., Pattnaik, B., Pradhan, H.K., 2008a. Prokaryotic expression of a 750-bp capsid region of bovine immunodeficiency virus gag gene and development of a recombinant capsid (p26) protein based immunoassay for seroprevalence studies. Indian J. Biotechnol. 7 (1), 50–55. Bhatia, S., Sood, R., Bhatia, A.K., Pattnaik, B., Pradhan, H.K., 2008b. Development of a capsid based competitive inhibition enzyme linked immunosorbent assay for detection of bovine immunodeficiency virus antibodies in cattle and buffalo serum. J. Virol. Methods 148, 218–225. Gonda, M.A., Braun, M.J., Carter, S.G., Kost, T.A., Bess Jr., J.W., Arthur, L.O., Van Der Maaten, M.J., 1987. Characterization and molecular cloning of a bovine lentivirus related to human immunodeficiency virus. Nature 330, 388–391. Gonda, M.A., Luther, D.G., Fong, S.E., Tobin, G.J., 1994. Bovine immunodeficiency virus: molecular biology and virus–host interactions. Virus Res. 32, 155–181.

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