Indraccolo S, Zamarchi R, Veronese M L, Mazza M R, Mion M, Veronesi A, Panozzo M, Colombatti M, Barelli A, Rocchetto P
Institute of Oncology, Interuniversity Center for Research on Cancer, University of Padua, Italy.
J Immunol Methods. 1993 Jan 4;157(1-2):105-15. doi: 10.1016/0022-1759(93)90076-j.
Optimal conditions for in vitro anti-human immunodeficiency virus type 1 (HIV-1) antibody (Ab) synthesis and detection were re-appraised. Western blot (WB) and radioimmunoassay (RIA) could detect about 1 and 10 ng, respectively, of HIV-1-specific Ab (HIV-Ab), while the sensitivity of an enzyme-linked immunosorbent assay (ELISA) was much lower. Optimal HIV-Ab recovery was obtained by culturing 2.5 x 10(6) peripheral blood mononuclear cells (PBMC)/ml from seropositive subjects for 16 days in the absence of mitogens; at higher cell concentrations, background levels were unacceptably high. The background of non-de novo synthesized HIV-Ab was due to insufficient PBMC washing and/or cytophilic immunoglobulin (Ig); a particular washing procedure, as well as 24 h peripheral blood mononuclear cells (PBMC) pre-culture, might help in limiting this phenomenon. However, results should be compared with those obtained in cultures containing puromycin especially in infants, where a higher CD16 antigen expression in lymphocytes is likely responsible for increased amounts of cytophilic Ig released in culture supernatants, compared to adults.
重新评估了体外抗1型人类免疫缺陷病毒（HIV-1）抗体（Ab）合成与检测的最佳条件。蛋白质印迹法（WB）和放射免疫测定法（RIA）分别可检测到约1 ng和10 ng的HIV-1特异性抗体（HIV-Ab），而酶联免疫吸附测定法（ELISA）的灵敏度则低得多。通过在无丝裂原的情况下将来自血清阳性受试者的2.5×10⁶个外周血单核细胞（PBMC）/ml培养16天，可获得最佳的HIV-Ab回收率；在更高的细胞浓度下，背景水平高得令人无法接受。非从头合成的HIV-Ab的背景是由于PBMC洗涤不充分和/或嗜细胞免疫球蛋白（Ig）；一种特定的洗涤程序以及24小时外周血单核细胞（PBMC）预培养可能有助于限制这种现象。然而，尤其是在婴儿中，结果应与在含有嘌呤霉素的培养物中获得的结果进行比较，与成人相比，婴儿淋巴细胞中较高的CD16抗原表达可能是培养上清液中释放的嗜细胞Ig量增加的原因。
