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International Journal of Current Microbiology and Applied Sciences (IJCMAS)
IJCMAS is now DOI (CrossRef) registered Research Journal. The DOIs are assigned to all published IJCMAS Articles.
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National Academy of Agricultural Sciences (NAAS)
NAAS Score: *5.38 (2020)
[Effective from January 1, 2020]
For more details click here

ICV 2018: 95.39
Index Copernicus ICI Journals Master List 2018 - IJCMAS--ICV 2018: 95.39
For more details click here

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Original Research Articles

PRINT ISSN : 2319-7692
Online ISSN : 2319-7706
Issues : 12 per year
Publisher : Excellent Publishers
Email : editorijcmas@gmail.com / submit@ijcmas.com
Editor-in-chief: Dr.M.Prakash
Index Copernicus ICV 2018: 95.39
NAAS RATING 2020: 5.38

Int.J.Curr.Microbiol.App.Sci.2017.6(7): 4401-4411
DOI: https://doi.org/10.20546/ijcmas.2017.607.459


Production of Diagnostic Kit to Detect Cry 2B Antigen by Use of scFv Monoclonal Antibody
Shashi Kumar1, Narayan Moger2*, Chidanand A. Rabinal3,P.U. Krishnaraj4 and K.N. Chandrashekara5
1Department of Biotechnology, 2Department of Microbiology, University of Agriculutral Sciences, Dharwad, Karnataka, India
3Department of Plant Physiology and Biotechnology UPASI TRI TRF, Valparai,Tamil Nadu, India
*Corresponding author
Abstract:

The single chain fragment antibody (scFv) antibodies were raised against CRY2B antigen by phage display technology. In antigen-antibody affinity selection the scFv antibody displaying phages concentration was increased (4.54 × 1010 to 2.3 ×1011) and fourth round phages were higher in concentration and affinity (1.06) to antigen revealed by ELISA. The ELISA reading of randomly selected forty-five scFv monoclonal antibodies were almost similar except two clones viz., pscFvCry2B15 and pscFvCry2B29. The ELISA reading of clone pcFvCry2B15 was 1.40 and for pscFvCry2B29 was 1.260. The sequence result had indicated both the clones were identical and size was about 448 bp. Both the sequences were shown homology to Homo sapiens Ig kappa variable chain. The developed scFv gene was cloned in to pQUANTa body vector by sfiI and NotI restriction enzymes to produce the secondary antibody conjugated scFv antibody. The raised antibody conjugates were cross checked with other than Cry2B proteins and found no cross reactivity ensuring the developed antibodies were specific to CRY2B protein. The minimum concentration of antigen to detect by developed scFv antibody was checked by spotting the different concentration antibody on NC membrane and found 0.8 µg/ml of antigen is enough to detect by conjugated scFv antibody.


Keywords: Biopanning, Single chain fragment antibody, cry2B, Tomlinson library, ELISA.
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How to cite this article:

Shashi Kumar, N., Narayan Moger, Chidanand A. Rabinal, P.U. Krishnaraj and Chandrashekara, K.N. 2017. Production of Diagnostic Kit to Detect Cry 2B Antigen by Use of scFv Monoclonal Antibody.Int.J.Curr.Microbiol.App.Sci. 6(7): 4401-4411. doi: https://doi.org/10.20546/ijcmas.2017.607.459