Phage-displayed La/SS-B antigen as a diagnostic reagent.
BACKGROUND
The detection of antibodies to La/SS-B, a nuclear RNA-binding protein in mammalian cells, aids in the diagnosis of Sjogren's syndrome and systemic lupus erythematosus (SLE). This is performed conventionally by immunoprecipitation using a crude splenic extract and more recently, by the more sensitive and rapid enzyme-linked immunosorbent assay (ELISA) which uses a purified La/SS-B antigen. The latter antigen is obtained from cellular extracts of the antigen or from bacterial cell lysates containing the recombinant antigen usually by affinity chromatographic method.


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).


METHODS
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.


CONCLUSIONS
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.
Poon
V K
VK
Clinical Immunology Unit, Chinese University of Hong Kong, Prince of Wales Hospital.
Chui
Y L
YL
Lim
P L
PL
D001324:Autoantigens / Q000737:chemistry*; D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007202:Indicators and Reagents; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000737:chemistry*; D012859:Sjogren's Syndrome / Q000175:diagnosis / Q000276:immunology; D000097585:SS-B Antigen
D001324:Autoantigens; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen