Layton G T, Smithyman A M
Int Arch Allergy Appl Immunol. 1983;72(2):158-63. doi: 10.1159/000234859.
Using optimised conditions in terms of cell concentration (2 X 10(6)/ml), duration of culture (7 days) and pH (7.0-7.1) and the highly sensitive enzyme-linked immunosorbent assay for antibody quantitation, we assessed the effects of lipopolysaccharide (LPS) and concanavalin A (Con A) on the levels of both total immunoglobulins and TNP-specific antibodies and compared the proportion of IgA anti-TNP to total IgA synthesised following the in vivo priming of mice, 5 days prior to culture, with trinitrophenylated sheep red blood cells (TNP-SRBC). LPS stimulation resulted in an increase in total IgA (ninefold), IgM (sixfold) and IgG (eightfold) synthesis over unstimulated cultures and similar increases were seen in IgA anti-TNP (eightfold), IgM anti-TNP (fivefold) and IgG anti-TNP (twelvefold). Con A stimulation resulted in a slight increase in total IgA (threefold), IgG (twofold) but a decrease in IgM (twofold) but did not appear to affect TNP-specific antibody levels. In unstimulated cultures, IgA anti-TNP comprised 7.8% of the total IgA synthesised. In LPS and Con A stimulated cultures the proportion was 6.6% and 3.2%, respectively. Inclusion of 10% fetal calf serum in the culture medium resulted in a substantial increase in the synthesis of IgG, IgM and IgA in unstimulated cultures but increased IgA levels only in LPS stimulated cultures. Our results emphasise the importance of optimising culture conditions when studying in vitro antibody synthesis and demonstrate that Peyer's patches respond quite substantially to parenterally administered antigen and that these responses can subsequently be monitored in vitro.
我们采用优化的细胞浓度（2×10⁶/ml）、培养时间（7天）和pH值（7.0 - 7.1）条件，以及用于抗体定量的高灵敏度酶联免疫吸附测定法，评估了脂多糖（LPS）和伴刀豆球蛋白A（Con A）对总免疫球蛋白和三硝基苯特异性抗体水平的影响，并比较了在培养前5天用三硝基苯化绵羊红细胞（TNP - SRBC）对小鼠进行体内致敏后，合成的IgA抗TNP与总IgA的比例。与未刺激的培养物相比，LPS刺激导致总IgA（增加九倍）、IgM（增加六倍）和IgG（增加八倍）合成增加，IgA抗TNP（增加八倍）、IgM抗TNP（增加五倍）和IgG抗TNP（增加十二倍）也有类似增加。Con A刺激导致总IgA（增加三倍）、IgG（增加两倍）略有增加，但IgM（减少两倍），且似乎不影响TNP特异性抗体水平。在未刺激的培养物中，IgA抗TNP占合成的总IgA的7.8%。在LPS和Con A刺激的培养物中，该比例分别为6.6%和3.2%。在培养基中加入10%胎牛血清导致未刺激培养物中IgG、IgM和IgA的合成大幅增加，但仅在LPS刺激的培养物中IgA水平增加。我们的结果强调了在研究体外抗体合成时优化培养条件的重要性，并证明派伊尔结对外源性给予的抗原有相当大的反应，且这些反应随后可在体外进行监测。
