FACTORS AFFECTING THE POTENCY OF HBIG

在上一份年度报告中，我们研究了骨料的影响， 片段对B型肝炎免疫球蛋白（HBIG）效力的影响。三 商业HBIG批次和FDA标准（参考编号2)由两个 方法，即，AUSAB方法[用于测量二价完整IgG和 片段（Fab '）2]和放射免疫沉淀（RIP）方法（以测量 二价抗体以及单价Fab/Fc和Fab 片段）。我们的研究结果表明，聚合物分数少 有效的单体和二聚体组分具有同等活性，并且富含Fab/Fc 级分可以抑制AUSAB。从那以后，我们分离并纯化了 通过以下方法从一个高度片段化的HBIG批次中分离单体及其片段： 凝胶过滤和蛋白G层析。然而，Fab/Fc仅 由于我们无法将其与IgG完全分离，因此部分纯化 单体的当用AUSAB测定时，Fab/Fc或Fab抑制了 参比品效价# 2单体，而Fc即使存在于 高浓度。此外，从高分子中分离的单体 片段化批次的效力低于来自HBIG参考编号的单体2的 含有聚集体但没有碎片。 当受到IgG亚类 分析中，发现前一种单体含有大量IgG 2， 小IgG 1，而参比编号2单体中含有大量的IgG 1。子类 分馏参考编号2通过蛋白A琼脂糖凝胶进行 pH梯度洗脱的层析。虽然该方法不能 将IgG 4与IgG 2或IgG 1有效分离，富集的IgG 1 部分具有最高的抗-HBs比活性，而IgG 2或 IgG 3组分活性较低。因此，碎片化，抑制 片段和IgG 1缺失是影响HBIG效价的因素。工作 仍在进行中。

In the previous annual report we studied the effects of aggregates and fragments on the potency of Hepatitis B Immune Globulin (HBIG). Three commercial HBIG lots and an FDA standard (ref.#2) were assayed by two methods, i.e., AUSAB method [to measure the bivalent intact IgG and the fragment (Fab')2] and a radioimmuno-precipitation (RIP) method (to measure the bivalent antibodies as well as the monovalent Fab/Fc and Fab fragments). Our results indicate that the polymer fractions are less potent, monomer and dimer fractions are equally active, and Fab/Fc enriched fractions may be inhibitory to AUSAB. We have since isolated and purified monomer and its fragments from one of the highly fragmented HBIG lots by both gel filtration and protein G chromatography. Fab/Fc, however, was only partially purified since we were unable to separate it completely from IgG monomer. When assayed with AUSAB, either Fab/Fc or Fab inhibited the potency of the ref.#2 monomer while Fc did not inhibit even when present at a high concentration. Furthermore, the monomer isolated from the highly fragmented lot was less potent than the monomer from HBIG ref.#2 which contained aggregates but no fragments. When subjected to IgG subclass analysis, the former monomer was found to contain largely IgG2 and very little IgG1 while the ref.#2 monomer contained largely IgG1. Subclass fractionation for ref.#2 was carried out by Protein A Sepharose chromatography with pH gradient elution. Although the method can not separate IgG4 efficiently from either IgG2 or IgG1, the IgGl enriched fraction possessed the highest anti-HBs specific activity while IgG2 or IgG3 fractions had low activities. Hence, fragmentation, inhibition by fragments, and loss of IgG1 are factors affecting the potency of HBIG. Work is still in progress.