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Development of Immuno-Diagnostic Assay to Detect Cry2B Protein

KrishiKosh

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Title Development of Immuno-Diagnostic Assay to Detect Cry2B Protein
 
Creator Shashi Kumara N.
 
Contributor Narayan Mogeri
 
Subject Plant Biotechnology
 
Description Cry2B as an antigen was used to produce single chain fragment variable (scFv)
antibody using phage display technology. M13K07 helper phage was used to infect the
Tomlinson scFv library which enabled the display of scFv on their surfaces and this
constitutes the phage scFv library. These phages were screened against immobilized cry2B
for specific binders. The unbound phages are getting rid of through the process of
biopanning. Four rounds of biopanning were conducted, during each biopanning the higher
specific binders (phages) were enriched. After fourth round of biopan the scFv specific to
cry2B was recovered by infecting phages to E.coli K12. Randomly 45 colonies were selected
for monoclonal ELISA, two colonies showed highest reading viz., pSNM15 and pSNM29.
The selected clones were sequenced with LMB3 forward and pHEN reverse primer and
characterized. Both the clones have similar conserved domains in Ig super family. The
homology search with BLASTn algorithm indicated 93% homology over Homo sapiens
chromosome 2 genomic contig and BLASTp algorithm indicated 98% homology over anti-
TREM-like transcript-1 antibody [synthetic construct].
Single chain antibody fragments of pSNM15 and pSNM29 were subcloned into
pQUANTabody expression vector which enabled transcriptionally fuse with scFv
monoclonal antibody fragment and the gene coding for bacterial alkaline phosphatase (PhoA)
enzyme. Upon expression of clone, it has produced fusion protein of ALP along with
pSNM15 and pSNM29 monoclonal antibody. The scFv-ALP conjugate has been produced
against Cry2B protein. Using this antibody conjugate, a detection assay was developed. The
assay was efficient to detect to the cry2B protein at the concentration of 800 ng/ml and it was
also cross checked with other proteins to evaluate the specificity of monoclones raised
against Cry2B.
 
Date 2016-11-21T19:00:07Z
2016-11-21T19:00:07Z
2012
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/86842
 
Format application/pdf
 
Publisher UAS, Dharwad