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[Preparation of human phage antibodies specific for SSA/Ro antigen and its sequence analysis].
Zhonghua Yi Xue Za Zhi. 2004 Nov 17; 84(22):1904-8.ZY

Abstract

OBJECTIVE

To prepare monoclonal antibodies (mAbs) specific for SSA/Ro antigen from the phage-displayed human single-chain Fv (scFv) antibody library constructed previously, and analyze its gene sequence.

METHODS

Frozen strain of Escherichia coli TG1 containing pHEN2-scFv was defrosted and infected by helper phage to produce scFv phage antibody library. 10 random clones from the primary library were checked for the presence of inserts by PCR screening. Four clones containing exogenous gene with correct length were selected randomly to undergo gene sequencing. The diversity of the library was monitored by digesting the PCR products of 8 different phagemids encoding scFv genes. The phage antibody library was biopanned for 3 cycles, using purified SSA/Ro antigen. After biopanning, the binding characterization and the specificity of the enriched library to SSA/Ro antigen was assayed by ELISA. mAbs were obtained from this enriched library and ELISA was used to detect the binding characterization and the specificity of these mAbs, using different purified antigens. The variable region of the monoclonal antibody undergoes sequencing.

RESULTS

A phage scFv library with the titer of 1.6 x 10(12) cfu/ml was established. Sample screening showed that eight of the ten clones contained scFv fragments. The insertion and recombination rate of exogenous gene was 80%. These 8 clones were also analyzed by restriction enzyme analysis to assess the diversity of the library. Each of the 8 clones showed a distinct restriction pattern. Sequencing results demonstrated that the genes of scFv were the human antibody genes, and the gene ligating and cloning were correct and successful. The library was subjected to 3 rounds of rescue and panning. A secondary phage antibody library against SSA/Ro was generated and the interest phages were obviously enriched. The 3rd panned library showed good reaction to the SSA/Ro antigen with higher OD value than unenriched library demonstrated by ELISA. The result of ELISA showed that the enriched library reacted specifically to SSA/Ro and had no cross-reactivity to the other autoantigen. 5 mAbs specific for SSA/Ro antigen could be selected from 52 clones, and the gene sequences of their VH and VL were coded by VH1, VH3, VH4, Vkappa1, Vkappa2, and Vkappa3 family genes with somatic mutations.

CONCLUSION

The primary scFv phage antibody library fits the need for later operations, and this library was enriched successfully. Enriched scFv phage antibody library is specific for SSA/Ro antigen, from which 5 monoclonal anti-SSA/Ro phage antibodies can be obtained successfully. The sequences of these mAbs show somatic mutations comparing with germline. It may help understand the pathogenesis of some antibody-mediated diseases.

Authors+Show Affiliations

Department of Rheumatology and Immunology, Peking Union Hospital of PUMC, Beijing 100370, China.No affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

15631804

Citation

Li, Ya-jie, et al. "[Preparation of Human Phage Antibodies Specific for SSA/Ro Antigen and Its Sequence Analysis]." Zhonghua Yi Xue Za Zhi, vol. 84, no. 22, 2004, pp. 1904-8.
Li YJ, Peng JM, Zhang FC. [Preparation of human phage antibodies specific for SSA/Ro antigen and its sequence analysis]. Zhonghua Yi Xue Za Zhi. 2004;84(22):1904-8.
Li, Y. J., Peng, J. M., & Zhang, F. C. (2004). [Preparation of human phage antibodies specific for SSA/Ro antigen and its sequence analysis]. Zhonghua Yi Xue Za Zhi, 84(22), 1904-8.
Li YJ, Peng JM, Zhang FC. [Preparation of Human Phage Antibodies Specific for SSA/Ro Antigen and Its Sequence Analysis]. Zhonghua Yi Xue Za Zhi. 2004 Nov 17;84(22):1904-8. PubMed PMID: 15631804.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Preparation of human phage antibodies specific for SSA/Ro antigen and its sequence analysis]. AU - Li,Ya-jie, AU - Peng,Jin-min, AU - Zhang,Feng-chun, PY - 2005/1/6/pubmed PY - 2006/8/22/medline PY - 2005/1/6/entrez SP - 1904 EP - 8 JF - Zhonghua yi xue za zhi JO - Zhonghua Yi Xue Za Zhi VL - 84 IS - 22 N2 - OBJECTIVE: To prepare monoclonal antibodies (mAbs) specific for SSA/Ro antigen from the phage-displayed human single-chain Fv (scFv) antibody library constructed previously, and analyze its gene sequence. METHODS: Frozen strain of Escherichia coli TG1 containing pHEN2-scFv was defrosted and infected by helper phage to produce scFv phage antibody library. 10 random clones from the primary library were checked for the presence of inserts by PCR screening. Four clones containing exogenous gene with correct length were selected randomly to undergo gene sequencing. The diversity of the library was monitored by digesting the PCR products of 8 different phagemids encoding scFv genes. The phage antibody library was biopanned for 3 cycles, using purified SSA/Ro antigen. After biopanning, the binding characterization and the specificity of the enriched library to SSA/Ro antigen was assayed by ELISA. mAbs were obtained from this enriched library and ELISA was used to detect the binding characterization and the specificity of these mAbs, using different purified antigens. The variable region of the monoclonal antibody undergoes sequencing. RESULTS: A phage scFv library with the titer of 1.6 x 10(12) cfu/ml was established. Sample screening showed that eight of the ten clones contained scFv fragments. The insertion and recombination rate of exogenous gene was 80%. These 8 clones were also analyzed by restriction enzyme analysis to assess the diversity of the library. Each of the 8 clones showed a distinct restriction pattern. Sequencing results demonstrated that the genes of scFv were the human antibody genes, and the gene ligating and cloning were correct and successful. The library was subjected to 3 rounds of rescue and panning. A secondary phage antibody library against SSA/Ro was generated and the interest phages were obviously enriched. The 3rd panned library showed good reaction to the SSA/Ro antigen with higher OD value than unenriched library demonstrated by ELISA. The result of ELISA showed that the enriched library reacted specifically to SSA/Ro and had no cross-reactivity to the other autoantigen. 5 mAbs specific for SSA/Ro antigen could be selected from 52 clones, and the gene sequences of their VH and VL were coded by VH1, VH3, VH4, Vkappa1, Vkappa2, and Vkappa3 family genes with somatic mutations. CONCLUSION: The primary scFv phage antibody library fits the need for later operations, and this library was enriched successfully. Enriched scFv phage antibody library is specific for SSA/Ro antigen, from which 5 monoclonal anti-SSA/Ro phage antibodies can be obtained successfully. The sequences of these mAbs show somatic mutations comparing with germline. It may help understand the pathogenesis of some antibody-mediated diseases. SN - 0376-2491 UR - https://www.unboundmedicine.com/medline/citation/15631804/[Preparation_of_human_phage_antibodies_specific_for_SSA/Ro_antigen_and_its_sequence_analysis]_ L2 - http://journal.yiigle.com/LinkIn.do?linkin_type=pubmed&issn=0376-2491&year=2004&vol=84&issue=22&fpage=1904 DB - PRIME DP - Unbound Medicine ER -