Abstract | BACKGROUND: OBJECTIVE: To produce a La/ SS-B antigen for use in ELISA that can be obtained easily and inexpensively without the need for extensive purification (including affinity chromatography). STUDY DESIGN: The antigen was produced as a fusion protein of the minor coat protein of M13 bacteriophage and used in this phage-associated form in an ELISA. La/SS-B cDNA derived from Hep-2 cells was cloned into the phagemid, pCANTAB-5E, and transfected to Escherichia coli. Phage clones selected for the presence of insert both by gene and antigenic analyses were used in the ELISA to detect anti-La/SS-B antibodies from patients with Sjogren's syndrome and SLE. RESULTS: A phage clone was obtained which contained a La/SS-B cDNA fragment truncated at the C-terminal end (after base-pair 631). The phage-displayed antigen derived from this clone was obtained by precipitation of the phage particles from the bacterial culture supernatant with polyethylene glycol. Used in the ELISA, this antigen detected 27 of 28 precipitin-positive sera and was negative for 50 control sera. The soluble (phage-free) form of the antigen was obtained from a nonsuppressor host as a cell lysate which could not be used in this form in an ELISA for antibody detection. It was useable, however, in Western blot analysis which confirmed the reactivity of the recombinant antigen. CONCLUSION: Phage-displayed antigens may be used in place of soluble forms of these antigens in detection assays which have the advantage that they are easy and inexpensive to produce.
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Authors | V K Poon, Y L Chui, P L Lim |
Journal | Immunotechnology : an international journal of immunological engineering
(Immunotechnology)
Vol. 3
Issue 2
Pg. 155-9
(Jun 1997)
ISSN: 1380-2933 [Print] Netherlands |
PMID | 9237100
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Autoantigens
- Indicators and Reagents
- Recombinant Fusion Proteins
- Ribonucleoproteins
- SS-B antigen
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Topics |
- Autoantigens
(chemistry)
- Bacteriophage M13
(chemistry, genetics, immunology)
- Blotting, Western
- Cloning, Molecular
- Enzyme-Linked Immunosorbent Assay
- Humans
- Indicators and Reagents
- Lupus Erythematosus, Systemic
(diagnosis, immunology)
- Recombinant Fusion Proteins
(chemistry, immunology, isolation & purification)
- Ribonucleoproteins
(chemistry)
- Sjogren's Syndrome
(diagnosis, immunology)
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