Yoshida C F, Mercadante L A, Gaspar A M, Maia M M, Berro O J, de Almeida E C, Schatzmayr H G
Mem Inst Oswaldo Cruz. 1984 Jul-Sep;79(3):329-36. doi: 10.1590/s0074-02761984000300006.
By the method of affinity chromatography a partially purified antigen was obtained after passing the plasma of an asymptomatic carrier of HBsAg through a column of Sepharose 4B linked to angi-HBs. This antigen was inoculated in rabbits using a schedule of 1,0 mg in the first dose and 4 other doses of 0,5 mg with intervals of approximately 15 days. Observing that blood samples collected after the 5th inoculation showed no change in antibody levels, the animals were bled on the 62th day and these immune sera were standardized with the following tests for the detection of HBsAg: Reverse passive hemagglutination (R-PHA) - using specific gamma globulin that was obtained from rabbit sera by affinity chromatography and reaching an optimal concentration of 10 micrograms/ml to sensitise SRBC at 5% fixed in glutaraldehyde. Counter immuno electrophoresis (CIEP) - using the rabbit immune sera diluted to 1/20 as a reagent for the detection of HBsAg. The immune sera was also used to conjugate new Sepharose 4B for affinity chromatography and was found having a linking capacity of approximately 0,5 to 1,0 mg of HBsAg per ml of Sepharose after complete saturation.
通过亲和层析法,将一名无症状乙肝表面抗原携带者的血浆通过与血管性乙肝表面抗原相连的琼脂糖4B柱后,获得了部分纯化的抗原。将该抗原接种于兔子,首剂为1.0毫克,另外4剂为0.5毫克,间隔约15天。观察到第5次接种后采集的血样抗体水平无变化,在第62天对动物进行采血,并用以下检测乙肝表面抗原的试验对这些免疫血清进行标准化:反向被动血凝试验(R-PHA)——使用通过亲和层析从兔血清中获得的特异性γ球蛋白,达到最佳浓度10微克/毫升,以致敏5%戊二醛固定的绵羊红细胞。对流免疫电泳(CIEP)——使用稀释至1/20的兔免疫血清作为检测乙肝表面抗原的试剂。该免疫血清还用于偶联新的琼脂糖4B进行亲和层析,发现完全饱和后每毫升琼脂糖结合乙肝表面抗原的能力约为0.5至1.0毫克。