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Cloning and expression of Epstein-Barr viral antigens in Escherichia coli : purification of recombinant EBV antigens for diagnostic application
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Type
Thesis
Author
Fones-Tan, Angela
Supervisor
Gan, Yik Yuen
Chan, Soh Ha
Li, Benjamin
Abstract
One of the factors associated with the etiology of nasopharyngeal carcinoma (NPC) is the Epstein-Barr virus (EBV). Ever since IgA antibodies towards the viral capsid antigen (VCA) and early antigen (EA) showed a high correlation with this disease, indirect immunofluorescence assays (IFAs) to detect these antibodies have conventionally been used to diagnose for NPC. However, this test needs skill to interpret and is not suitable for large-scale screening of populations. ELISA tests are, in contrast, easily automated, quick to perform, and does not involve much skill. However, this test has a high sensitivity which could easily lead to false positivity. Therefore, to maintain specificity, individual EBV antigens have to be purified and coated on the microtitre plates.
The IgA anti-EBV immunological assays utilized before the advent of monoclonal antibody technology and gene cloning have in general measured antibodies to the viral capsid antigen (VCA), membrane antigen (MA), early antigen (EA) and nuclear antigen (EBNA) complexes. It is now known that these assays measure antibodies to a variety of different polypeptides associated with each complex. It is possible that antibodies to individual polypeptides might be more sensitive and specific for diagnosing or monitoring patients than antibodies directed against the whole complex.
We have developed an easy way to clone and screen for Epstein-Barr virus antigens significant in the diagnosis of NPC. Using cDNA libraries, two proteins, cloned and expressed as fusion proteins in lambda gt11 have been identified to be expressed from 661 bp of the BORF2 and from 607 bp of the BKRF4 reading frames. Western blotting studies on these proteins using sera from 16 NPC and 16 normal healthy individuals showed that 15 NPC patients had either IgG or IgA antibodies towards either protein whereas only 2 normal individuals were positive, hence IgG and IgA antibodies towards these two antigens are of diagnostic value for NPC.
The 661 bp of cDNA coding for the carboxyl end of the large subunit of EBV ribonucleotide reductase was cloned into the pMal plasmid expression vector. Purified recombinant protein was tested in IgG and IgA ELISAs. For the IgG assay, 81 out of 100 IgA-IFA positive NPC patients tested positive (sensitivity = 81%) whereas for the IgA assay, 60 of them tested positive (sensitivity = 60%). Among 100 normal individuals, 1 tested positive for the IgG assay (specificity = 99%) and 9 tested positive for the IgA assay (specificity = 91%). The IgG assay was also detected 5 out of 20 NPC sera which were IFA-VCA and IFA-EA negative for IgA antibodies. Hence the recombinant ribonucleotide reductase ELISA has good potential as a diagnostic test for NPC or as a complementary test to IFA. The ELISA was also able to differentiate NPC from other head and neck diseases.
Titre studies using the IgG ELISA showed close correlation between the titres obtained from NPC sera with their IgA-IFA/EA titres. Thus titre assays detecting IgG antibodies towards ribonucleotide reductase might also be useful in the monitoring of the recovery of NPC patients.
Sensitivity and specificity was optimised and an ELISA kit for the diagnosis of NPC was developed to be commercially produced.
The IgA anti-EBV immunological assays utilized before the advent of monoclonal antibody technology and gene cloning have in general measured antibodies to the viral capsid antigen (VCA), membrane antigen (MA), early antigen (EA) and nuclear antigen (EBNA) complexes. It is now known that these assays measure antibodies to a variety of different polypeptides associated with each complex. It is possible that antibodies to individual polypeptides might be more sensitive and specific for diagnosing or monitoring patients than antibodies directed against the whole complex.
We have developed an easy way to clone and screen for Epstein-Barr virus antigens significant in the diagnosis of NPC. Using cDNA libraries, two proteins, cloned and expressed as fusion proteins in lambda gt11 have been identified to be expressed from 661 bp of the BORF2 and from 607 bp of the BKRF4 reading frames. Western blotting studies on these proteins using sera from 16 NPC and 16 normal healthy individuals showed that 15 NPC patients had either IgG or IgA antibodies towards either protein whereas only 2 normal individuals were positive, hence IgG and IgA antibodies towards these two antigens are of diagnostic value for NPC.
The 661 bp of cDNA coding for the carboxyl end of the large subunit of EBV ribonucleotide reductase was cloned into the pMal plasmid expression vector. Purified recombinant protein was tested in IgG and IgA ELISAs. For the IgG assay, 81 out of 100 IgA-IFA positive NPC patients tested positive (sensitivity = 81%) whereas for the IgA assay, 60 of them tested positive (sensitivity = 60%). Among 100 normal individuals, 1 tested positive for the IgG assay (specificity = 99%) and 9 tested positive for the IgA assay (specificity = 91%). The IgG assay was also detected 5 out of 20 NPC sera which were IFA-VCA and IFA-EA negative for IgA antibodies. Hence the recombinant ribonucleotide reductase ELISA has good potential as a diagnostic test for NPC or as a complementary test to IFA. The ELISA was also able to differentiate NPC from other head and neck diseases.
Titre studies using the IgG ELISA showed close correlation between the titres obtained from NPC sera with their IgA-IFA/EA titres. Thus titre assays detecting IgG antibodies towards ribonucleotide reductase might also be useful in the monitoring of the recovery of NPC patients.
Sensitivity and specificity was optimised and an ELISA kit for the diagnosis of NPC was developed to be commercially produced.
Date Issued
1994
Call Number
QR400.2.E68 Fon
Date Submitted
1994