Construction of ELISA system to quantify human ST2 protein in sera of patients.
The human ST2 gene can be specifically induced by growth stimulation in fibroblastic cells, and can also be induced by antigen stimulation in Th2 cells. The gene encodes a soluble secreted protein, ST2, and a transmembrane protein, ST2L, which are closely related to the interleukin-1 receptor. To gain insight into the biological roles of the ST2 gene, three monoclonal antibodies (MAbs) against human ST2 gene products were obtained. To obtain these antibodies, immunization was carried out using two different immunogens: purified soluble human ST2 protein (hST2), and COS7 cells, which express the extracellular portion of human ST2L. 2A5 and FB9 MAbs were derived from the immunization with soluble hST2, and HB12 was derived from the COS7 cell immunization. All three antibodies were shown to detect native forms of the human ST2 gene products by immunoprecipitation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In the competitive ELISA using biotinylated and nonlabelled MAbs, neither FB9 nor HB12 affected the binding of 2A5 to ST2 gene products. Based on this result, we constructed a sandwich ELISA system using 2A5 and FB9 to measure the concentration of soluble hST2 in sera. The ELISA, combined with the flow cytometry using these antibodies, will be a useful tool for elucidating the functions of human ST2 gene products in individuals.
Kuroiwa
K
K
Department of Biochemistry, Jichi Medical School, Tochigi, Japan.
Li
H
H
Tago
K
K
Iwahana
H
H
Yanagisawa
K
K
Komatsu
N
N
Oshikawa
K
K
Sugiyama
Y
Y
Arai
T
T
Tominaga
S I
SI
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001249:Asthma / Q000097:blood; D019921:Biotinylation; D015153:Blotting, Western; D019556:COS Cells; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D000072179:Interleukin-1 Receptor-Like 1 Protein; D008565:Membrane Proteins; D011233:Precipitin Tests; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; C079241:IL1RL1 protein, human; D000072179:Interleukin-1 Receptor-Like 1 Protein; D008565:Membrane Proteins; D011506:Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins