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Cloning and expression of Epstein-Barr viral antigens for the diagnosis of nasopharygeal carcinoma
Author
He, Yan Xin
Supervisor
Gan-Yap, Yik Yuen
Abstract
Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs is prevalent among Chinese in southern China, and in places wherever these Chinese migrate to. Studies on factors affecting the prognosis of NPC have revealed that early diagnosis and treatment are very important in disease management. One of the factors associated with the etiology of NPC is the Epstein-Barr virus (EBV), and antibodies towards this viral antigen have commonly been used in the diagnosis of this disease.
Various antigen complexes of EBV which are useful as serological markers in the diagnosis of NPC are broadly categorized as viral capsid antigen (VCA), diffuse early antigen (EA-D), restricted early antigen (EA-R), membrane antigen (MA) and Epstein-Barr nuclear antigen (EBNA). Each of these antigens has several polypeptides. For the diagnosis of NPC using the whole antigen complex extracted from the cell lines, immunofluorescence assay (IFA) has been shown to be the most sensitive and specific assay compared to other techniques. Enzyme-linked immunosorbent assay (ELISA) is less sensitive but has the advantage of being more suitable for screening populations at high risk for NPC. This test is easily automated, quick to perform and does not involve much skill. The sensitivity and specificity of using ELISA for the detection of NPC can be improved by using gene cloning technology to obtain pure polypeptides from the EBV antigen complex. Polypeptides are used to coat microtiter plates for ELISA.
The cDNA inserts, which were confirmed to have high potential for NPC diagnosis by immunoblotting, were recloned in the plasmid expression vector, pMAL-C2, and over-expressed in E. coli. The fusion protein was easily purified by affinity chromatography on an amylose column. However, out of the eight cDNA inserts studied, only the cDNA inserts from clones R29-3, R29-2, B19-4 and P24-1 were successfully expressed in large quantities. Large-scale purification of the MBP-fusion proteins was carried out and ELISA tests were done using the polypeptides as target antigens.
DNA sequencing indicated that the EBV antigens expressed from clone R29-3 and R29-2 belonged to the large subunit of the EBV ribonucleotide reductase, which was encoded by the carboxyl-terminal end of the EBV BORF2 reading frame. The recombinant protein p28 expressed from clone R29-3 was 28.1 kDa in length, which was encoded by 759 bp of DNA. The recombinant protein p15 from clone R29-2 was 14.9 kDa in length, which was encoded by 405 bp of DNA. In addition, a 13.1 kDa EA-D antigen p13, which was expressed from clone B19-4, was encoded by the 352 bp carboxyl-terminal end of the BMRF1 reading frame of the EBV genome. Recombinant antigen, p18, expressed from clone P24-1, was a thymidine kinase (TK) coded by the carboxyl-terminal end of the EBV BXLF1 open reading frame. This recombinant antigen was 17.6 kDa in length and was encoded by 480 bp of DNA.
A total of 156 sera from patients with NPC and 100 sera from healthy donors were tested for IgA and IgG antibodies to native VCA and EA, as well as recombinant polypeptides p28, p15, p13 and p18 by ELISA. IgA-VCA antibodies were detected in 80% (125/156) of the NPC samples, whilst only 4/100 (4%) of the sera from healthy donors were positive for IgA to VCA. In the IgA-EA assay, however, sensitivity was calculated as 64% (100/156), but this test was more specific with a specificity of 99% (1/100 positive).
In the diagnosis of NPC, detection of anti-EBV RR IgG antibodies had a higher sensitivity of 80% by p28-ELISA and 69% by p15-ELISA. The detection of anti-EBV RR IgA antibodies had a sensitivity of 60% by p28-ELISA and 50% by p15-ELISA. However, it was more specific for the detection of IgA anti-EBV RR antibodies with a specificity of 100%. The detection of IgG anti-EBV RR antibodies had a specificity of 98%.
Results showed that IgG-ELISA test with recombinant p13 (EA-D) could detect 71% of NPC patients' sera, whereas only 1% of normal individuals gave positive results. IgA-ELISA was less sensitive with a sensitivity of 67% but more specific with a specificity of 100% compared with IgG-ELISA.
The TK-IgG ELISA showed a sensitivity of 57% and a specificity of 91%. The Tk-IgA Elisa, however, was not as good and gave a low sensitivity of 43%, but showed a higher specificity of 100%.
We have also tried to test the suitability of combining different recombinant antigens as target antigens in ELISA for the diagnosis of NPC. Recombinant antigen p28 belonging to EA-R and the recombinant antigen p13 belonging to EA-D were combined at a ratio of 2:1 and coated onto ELISA plates. It was found that 133 out of 156 NPC patients (85%) had IgG antibodies to the combined antigens, whilst only 2 out of 100 normal individuals (2%) had such antibodies. 111 samples from the 156 NPC patients (71%) had IgA antibodies against the combined antigens whereas only 1 out of 100 normal samples (1%) had such antibodies. The ELISA using combined antigens was more sensitive than the ELISA in which individual recombinant antigens as well as the native antigens were used separately.
The IgG ELISA with combined antigens also detected 9 out of 49 NPC sera which were IFA-VCA and IFA-EA negative for IgA antibodies. These results implied that combined antigens might be a good complementary diagnostic test to IFA. Based on the results of 72 patients with other diseases, some of them were having early primary symptoms of NPC, 18 cases were tested positive indicating EBV infection. A follow up study of these samples to see whether the EBV positive cases would finally lead to any EBV related carcinomas would be very useful in ensuring early detection and early treatment.
Conventionally, IgA was the antibody used in both IFA and ELISA for the diagnosis of NPC and this is supported by many research publications. In our study, when pure recombinant protein of different antigen complexes (recombinant ribonucleotide reductase (p28 and p15), EA-D (p13) and thymidine kinase (p18)) were analysed systematically by ELISA, we found that in fact IgG against the different recombinant antigens is much better than IgA in the diagnosis of NPC. In comparison, use of IgG as serological markers was not recommended when the conventional method of native VCA and EA, extracted from the whole cell lines, were used.
The IgG antibody titers against the combined antigens determined by ELISA were found to correlate well with the IgA anti-EA titers determined by IFA. Hence this assay might also be suitable for use in monitoring the recovery of NPC patients after radiation or other therapies. In addition, there was also a positive and significant correlation between the values of the OD readings of IgG anti-p28/p13 combined antigens determined by ELISA and the IgA titers against EA (r = 0.37) and VCA (r = 0.33) determined by IFA. Therefore the ELISA IgG test using combined antigens could be considered as a complementary screening assay to the IgA-IFA for the detection of NPC. Using the p28/p13 combined polypeptides as target antigen, there was also a significant correlation (r = 0.34) between the ELISA IgG antibody titers and the dOD readings determined by ELISA.
In summary, for the diagnosis of NPC, it is strongly recommended to use a mixture of several recombinant antigens in detecting antibodies to EBV by ELISA. Sensitivity is greatly improved. The ELISA with combined antigens p28/p13 is recommended for rapid and inexpensive serodiagnosis of populations with high risk of NPC. The combined recombinant antigens anti-EBV ELISA described here represent an innovative assay system for the diagnosis of NPC.
Various antigen complexes of EBV which are useful as serological markers in the diagnosis of NPC are broadly categorized as viral capsid antigen (VCA), diffuse early antigen (EA-D), restricted early antigen (EA-R), membrane antigen (MA) and Epstein-Barr nuclear antigen (EBNA). Each of these antigens has several polypeptides. For the diagnosis of NPC using the whole antigen complex extracted from the cell lines, immunofluorescence assay (IFA) has been shown to be the most sensitive and specific assay compared to other techniques. Enzyme-linked immunosorbent assay (ELISA) is less sensitive but has the advantage of being more suitable for screening populations at high risk for NPC. This test is easily automated, quick to perform and does not involve much skill. The sensitivity and specificity of using ELISA for the detection of NPC can be improved by using gene cloning technology to obtain pure polypeptides from the EBV antigen complex. Polypeptides are used to coat microtiter plates for ELISA.
The cDNA inserts, which were confirmed to have high potential for NPC diagnosis by immunoblotting, were recloned in the plasmid expression vector, pMAL-C2, and over-expressed in E. coli. The fusion protein was easily purified by affinity chromatography on an amylose column. However, out of the eight cDNA inserts studied, only the cDNA inserts from clones R29-3, R29-2, B19-4 and P24-1 were successfully expressed in large quantities. Large-scale purification of the MBP-fusion proteins was carried out and ELISA tests were done using the polypeptides as target antigens.
DNA sequencing indicated that the EBV antigens expressed from clone R29-3 and R29-2 belonged to the large subunit of the EBV ribonucleotide reductase, which was encoded by the carboxyl-terminal end of the EBV BORF2 reading frame. The recombinant protein p28 expressed from clone R29-3 was 28.1 kDa in length, which was encoded by 759 bp of DNA. The recombinant protein p15 from clone R29-2 was 14.9 kDa in length, which was encoded by 405 bp of DNA. In addition, a 13.1 kDa EA-D antigen p13, which was expressed from clone B19-4, was encoded by the 352 bp carboxyl-terminal end of the BMRF1 reading frame of the EBV genome. Recombinant antigen, p18, expressed from clone P24-1, was a thymidine kinase (TK) coded by the carboxyl-terminal end of the EBV BXLF1 open reading frame. This recombinant antigen was 17.6 kDa in length and was encoded by 480 bp of DNA.
A total of 156 sera from patients with NPC and 100 sera from healthy donors were tested for IgA and IgG antibodies to native VCA and EA, as well as recombinant polypeptides p28, p15, p13 and p18 by ELISA. IgA-VCA antibodies were detected in 80% (125/156) of the NPC samples, whilst only 4/100 (4%) of the sera from healthy donors were positive for IgA to VCA. In the IgA-EA assay, however, sensitivity was calculated as 64% (100/156), but this test was more specific with a specificity of 99% (1/100 positive).
In the diagnosis of NPC, detection of anti-EBV RR IgG antibodies had a higher sensitivity of 80% by p28-ELISA and 69% by p15-ELISA. The detection of anti-EBV RR IgA antibodies had a sensitivity of 60% by p28-ELISA and 50% by p15-ELISA. However, it was more specific for the detection of IgA anti-EBV RR antibodies with a specificity of 100%. The detection of IgG anti-EBV RR antibodies had a specificity of 98%.
Results showed that IgG-ELISA test with recombinant p13 (EA-D) could detect 71% of NPC patients' sera, whereas only 1% of normal individuals gave positive results. IgA-ELISA was less sensitive with a sensitivity of 67% but more specific with a specificity of 100% compared with IgG-ELISA.
The TK-IgG ELISA showed a sensitivity of 57% and a specificity of 91%. The Tk-IgA Elisa, however, was not as good and gave a low sensitivity of 43%, but showed a higher specificity of 100%.
We have also tried to test the suitability of combining different recombinant antigens as target antigens in ELISA for the diagnosis of NPC. Recombinant antigen p28 belonging to EA-R and the recombinant antigen p13 belonging to EA-D were combined at a ratio of 2:1 and coated onto ELISA plates. It was found that 133 out of 156 NPC patients (85%) had IgG antibodies to the combined antigens, whilst only 2 out of 100 normal individuals (2%) had such antibodies. 111 samples from the 156 NPC patients (71%) had IgA antibodies against the combined antigens whereas only 1 out of 100 normal samples (1%) had such antibodies. The ELISA using combined antigens was more sensitive than the ELISA in which individual recombinant antigens as well as the native antigens were used separately.
The IgG ELISA with combined antigens also detected 9 out of 49 NPC sera which were IFA-VCA and IFA-EA negative for IgA antibodies. These results implied that combined antigens might be a good complementary diagnostic test to IFA. Based on the results of 72 patients with other diseases, some of them were having early primary symptoms of NPC, 18 cases were tested positive indicating EBV infection. A follow up study of these samples to see whether the EBV positive cases would finally lead to any EBV related carcinomas would be very useful in ensuring early detection and early treatment.
Conventionally, IgA was the antibody used in both IFA and ELISA for the diagnosis of NPC and this is supported by many research publications. In our study, when pure recombinant protein of different antigen complexes (recombinant ribonucleotide reductase (p28 and p15), EA-D (p13) and thymidine kinase (p18)) were analysed systematically by ELISA, we found that in fact IgG against the different recombinant antigens is much better than IgA in the diagnosis of NPC. In comparison, use of IgG as serological markers was not recommended when the conventional method of native VCA and EA, extracted from the whole cell lines, were used.
The IgG antibody titers against the combined antigens determined by ELISA were found to correlate well with the IgA anti-EA titers determined by IFA. Hence this assay might also be suitable for use in monitoring the recovery of NPC patients after radiation or other therapies. In addition, there was also a positive and significant correlation between the values of the OD readings of IgG anti-p28/p13 combined antigens determined by ELISA and the IgA titers against EA (r = 0.37) and VCA (r = 0.33) determined by IFA. Therefore the ELISA IgG test using combined antigens could be considered as a complementary screening assay to the IgA-IFA for the detection of NPC. Using the p28/p13 combined polypeptides as target antigen, there was also a significant correlation (r = 0.34) between the ELISA IgG antibody titers and the dOD readings determined by ELISA.
In summary, for the diagnosis of NPC, it is strongly recommended to use a mixture of several recombinant antigens in detecting antibodies to EBV by ELISA. Sensitivity is greatly improved. The ELISA with combined antigens p28/p13 is recommended for rapid and inexpensive serodiagnosis of populations with high risk of NPC. The combined recombinant antigens anti-EBV ELISA described here represent an innovative assay system for the diagnosis of NPC.
Date Issued
1997
Call Number
QR400.2.E68 He
Date Submitted
1997