[Expression of fusion proteins in beta(2)GP I gene-transfected HEp-2 cells and its clinical application].
OBJECTIVE
To establish an indirect immunofluorescent test so as to improve the sensitivity and specificity of examination of antibodies to beta(2)-glycoprotein.


METHODS
Full-length beta(2)GP cDNA was obtained from human hepatocellular cancer cell line HepG2 by RT-PCR and cloned into the mammalian expression vector pEGFP-C1. The recombinant plasmid pEGFP-beta(2)GP was transfected into HEp-2 cells. RT-PCR, immunoblotting (IBT), confocal fluorescence microscopy, and indirect immunofluorescent test (IIF) were used to confirm the expression, localization, and antigenicity of fusion protein of green fluorescent protein (GFP). Serum specimens from 19 patients suspected as with secondary antiphospholipid syndrome (APS), 1 patient diagnosed as with primary APS, and 10 normal persons were detected with IIF-IgG-beta(2)GP1, ELISA-IgG-ACL, and ELISA-IgG-beta(2)GP I simultaneously.


RESULTS
(1) The HEp-beta(2)GP I cells thus obtained retained their ability of expression of beta(2)GP-I-GFP for more than ten generations. This beta(2)GP-I-GFP showed the antigenicity of beta(2)GP-I with a characteristic feature. (2) Seven of the 20 serum specimens from APS patients showed characteristic immunofluorescent pattern. No serum specimen from normal persons showed immunofluorescent staining. The comparison of results of the three methods showed that the concordance between IIF-IgG-beta(2)GP I and ELISA-IgG-beta(2)GP I was the most perfect (Kappa = 0.886). (3) HEp-beta(2)GP I retained the immunofluorescent property of HEp-2 cell.


CONCLUSIONS
As a new kind of substrate of IIF, beta(2)GP I transfectant can be used to detect anti-beta(2)GP-I antibodies. Transfeted HEp-2 cells keep the immunofluorescent property of HEp-2 cells in IFANA test and can be used as substrate for routine IFANA detection.
Lu
Liangjing
L
Department of Rheumatology, Renji Hospital, Shanghai Seceond Medical University, Shanghai Immunology Institute, Shanghai 200001, China.
Chen
Shunle
S
Gu
Yueying
Y
Shen
Nan
N
Bao
Chunde
C
Wang
Yuan
Y
Yang
Chengde
C
Ye
Ping
P
Yu
Chongzhao
C
D017152:Antibodies, Antiphospholipid / Q000097:blood*; D016736:Antiphospholipid Syndrome / Q000097:blood* / Q000175:diagnosis / Q000276:immunology; D001053:Apolipoproteins / Q000235:genetics / Q000276:immunology*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D015870:Gene Expression; D005818:Genetic Engineering; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D053482:beta 2-Glycoprotein I
D017152:Antibodies, Antiphospholipid; D001053:Apolipoproteins; D006023:Glycoproteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D053482:beta 2-Glycoprotein I; D049452:Green Fluorescent Proteins