In vitro induction and detection of human B cells producing HLA antibodies
10.3760/cma.j.issn.0254-5101.2009.06.018
- VernacularTitle:体外诱导和检测人类B细胞产生抗HLA抗体的研究
- Author:
Aihua LIAO
;
Liping LIU
;
Wei HUANG
;
Dong CHEN
;
Yan WANG
;
Ling ZHANG
- Publication Type:Journal Article
- Keywords:
ELISPOT;
B lymphocyte;
HLA antibody;
Pokeweed mitogen
- From:
Chinese Journal of Microbiology and Immunology
2009;29(6):559-563
- CountryChina
- Language:Chinese
-
Abstract:
Objective To induce and detect human B cells producing HLA antibodies in vitro and by an ELISPOT assay. Methods Human peripheral blood mononuclear cells (PBMC) from 7 healthy vol-unteers were isolated by density gradient centrifugation, and cultured with the stimulation of PWM alone or PWM + SAC for 5 d. The lg levels in culture supernatants and the number of Ig producing B cells were de-termined by ELISA and ELISPOT assay, respectively. The optimal stimulation protocol for PBMC to produce optimal Ig in vitro was anticipated to be obtained. Using the optimal pre-cuhure and ELISPOT conditions, PBMC from 9 alloimmunized subjects were analyzed for the presence of B cells secreting HLA antibodies. Results There was a tendency towards more viable cells following the activation with PWM and SAC vs PWM alone in a 5-day culture (P =0. 052). The IgG levels in the supernatants were found to be comparable for the two culture conditions whereas the lgM levels were increased ( P = 0.03 ) in the presence of the com-bination of PWM and SAC. In 6 subjects a specific signal was obtained with our ELISPOT assay. The presence of HLA antibodies in the respective pre-cuhure supernatants supported the specificity. Conclusion The activation of PBMC with the combination of PWM and SAC, and the application of HLA-specifie ELIS-POT assay can succeed in the induction and detection of human B cells producing HLA antibodies.
