人血清中非Gal异种抗体检测方法的探讨

目的  探讨人血清中抗非半乳糖（Gal）异种抗原抗体的最佳检测条件。方法  选用α-1, 3半乳糖基转移酶基因敲除（GTKO）五指山小型猪的外周血单核细胞（PBMC) 作为靶细胞，与健康人血清混合，在不同血清浓度（4.8%、16.7%、100%）和孵育时间（0.5、1.0、2.0、3.0、6.0 h）条件下，利用流式细胞仪检测GTKO猪的PBMC上结合IgM和IgG的水平。结果  在16.7%血清浓度下，孵育时间从0.5 h延长至3.0 h，能显著提高非Gal IgM结合PBMC的水平（P < 0.01），而IgG水平的提高则差异无统计学意义（P > 0.05）。提高血清浓度也可提高非Gal IgM的结合水平，采用100%的血清浓度孵育3 h，IgM结合PBMC水平最高且有统计学意义（P < 0.01）。采用100%的血清孵育6 h，IgG结合水平升高有统计学意义（P < 0.05）。延长孵育时间和提高血清浓度不会影响PBMC的活力。结论  检测人血清中抗非Gal异种抗原抗体的最佳条件为每1×10⁵个猪PBMC，采用100%人血清浓度孵育3 h检测IgM水平，或加100%人血清浓度孵育6 h检测IgG水平。这一条件的优化有助于筛选非Gal表达抗原低表达的供体猪。

Detection methods of non-Gal xenoantibody in human serum

Objective To investigate the optimal condition for the detection of anti-non-galactose (Gal) xenoantigen and antibody in human serum. Methods Peripheral blood mononuclear cell (PBMC) obtained from Wuzhishan miniature pig models with α-1, 3-galactosyltransferase gene knockout (GTKO) were used as target cells, mixed and incubated with healthy human serum of different concentrations (4.8%, 16.7% and 100%) for 0.5, 1.0, 2.0, 3.0 and 6.0 h, respectively. The abilities of PBMC to bind with IgM and IgG were detected by flow cytometry. Results At the serum concentration of 16.7%, the ability of non-Gal IgM to bind with PBMC was significantly enhanced from 0.5 h to 3.0 h incubation (P < 0.01), whereas no statistical significance was noted in terms of IgG (P > 0.05). Increasing serum concentration could also enhance the ability of non-Gal IgM to bind with PBMC. At the serum concentration of 100% and incubation for 3 h, the ability of IgM to bind with PBMC was the highest among all groups (P < 0.01). At the serum concentration of 100% and incubation for 6 h, the ability of IgG to bind with PBMC was significantly enhanced (P < 0.05). Prolonging incubation time and increasing serum concentration did not affect the activity of PBMCs. Conclusions The optimal condition for detection of anti-non-Gal xenoantigen and antibody is determined. A quantity of 1×105 PBMC from pig should be incubated with 100% human serum for 3 h for detection of IgM level, or incubated with 100% human serum for 6 h for measurement of IgG level. This optimized condition contributes to screening the donor pigs which lowly express non-Gal antigen.