Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining.
Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells that werw stably transfected with IL-32 and in the sera of stomach cancer patients, by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32alpha was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean+/-SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1alpha, hIL-1beta, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL- 32 in stomach cancer patients.
Seo
Eun-Hee
EH
Department of Bioscience and Biotechnology,Konkuk University, Seoul 143-701, Korea.
Kang
Jeongwoo
J
Kim
Ki-Hong
KH
Cho
Min-Chul
MC
Lee
Sojung
S
Kim
Hee-Jong
HJ
Kim
Jung-Hee
JH
Kim
Eun-Jin
EJ
Park
Dong-Ki
DK
Kim
Soo-Hyun
SH
Choi
Yang Kyu
YK
Kim
Jin Man
JM
Hong
Jin Tae
JT
Yoon
Do-Young
DY
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis; D007150:Immunohistochemistry / Q000379:methods*; D007378:Interleukins / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D020014:K562 Cells; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013274:Stomach Neoplasms / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C496971:IL32 protein, human; D007074:Immunoglobulin G; D007378:Interleukins; D011994:Recombinant Proteins