Mohan S, Bautista C M, Herring S J, Linkhart T A, Baylink D J
Department of Medicine, Loma Linda University, California.
Endocrinology. 1990 May;126(5):2534-42. doi: 10.1210/endo-126-5-2534.
A variety of human cells and biological fluids have been shown to produce or contain insulin-like growth factor (IGF)-specific binding proteins (BPs). The existence of these BPs in serum and conditioned medium of cell and organ cultures has complicated radioligand assays for measurement of IGFs. Various strategies have been proposed to avoid interference of BPs with these assays, including acid-ethanol precipitation of BPs and acid-gel filtration. Many of these procedures are time consuming, exhibit low recoveries, and do not completely eliminate BP artifacts. In this study we have investigated interference of inhibitory IGF-BP (In-IGF-BP) purified from bone cell-conditioned medium in an IGF-II RRA and IGF-I RIA and developed methods to neutralize In-IGF-BP artifacts in IGF assays. In the IGF-II RRA, purified In-IGF-BP competed for [125I]IGF-II binding to H-35 cells in a dose-dependent manner and, thus, increased the apparent value for IGF-II in the medium. Fifty percent inhibition of [125I]IGF-II binding to H-35 cells was seen at 12.2 and 5.7 ng/ml unlabeled IGF-II and IN-IGF-BP, respectively. In-IGF-BP also competed for [125I]IGF-I in the IGF-I RIA; however, the interference was much less in the IGF-I RIA compared to the IGF-II RRA. Fifty percent displacement of [125I]IGF-I binding was seen at 0.25 and 5 ng/ml unlabeled IGF-I and In-IGF-BP, respectively. Our approach to eliminate BP artifacts was as follows. We knew that In-IGF-BP showed comparatively equal binding affinities with both IGF-I and IGF-II, and binding of these ligands to cell receptors (IGF-II) and antibodies (IGF-I) was very specific (2% and 0.5% cross-reactivity for IGF-I and IGF-II, respectively). Therefore, in the IGF-I RIA we blocked the In-IGF-BP artifacts by adding an excess of IGF-II, and in the IGF-II RRA we blocked the In-IGF-BP artifacts by adding an excess of IGF-I. By incubating purified In-IGF-BP with different amounts of IGF-I, we found that 30-min preincubation of 5 ng In-IGF-BP with 10 ng IGF-I completely blocked BP artifacts in the IGF-II RRA. Similarly, preincubation of In-IGF-BP with IGF-II blocked BP artifacts in the IGF-I RIA.(ABSTRACT TRUNCATED AT 250 WORDS)
多种人类细胞和生物体液已被证明能产生或含有胰岛素样生长因子（IGF）特异性结合蛋白（BP）。细胞和器官培养物的血清及条件培养基中存在这些BP，使得用于测量IGF的放射配体分析变得复杂。已提出各种策略来避免BP对这些分析的干扰，包括BP的酸 - 乙醇沉淀和酸 - 凝胶过滤。许多这些方法耗时、回收率低，且不能完全消除BP假象。在本研究中，我们研究了从骨细胞条件培养基中纯化的抑制性IGF - BP（In - IGF - BP）在IGF - II放射受体分析（RRA）和IGF - I放射免疫分析（RIA）中的干扰，并开发了中和IGF分析中In - IGF - BP假象的方法。在IGF - II RRA中，纯化的In - IGF - BP以剂量依赖方式竞争[125I]IGF - II与H - 35细胞的结合，从而增加了培养基中IGF - II的表观值。未标记的IGF - II和IN - IGF - BP分别在12.2和5.7 ng/ml时可使[125I]IGF - II与H - 35细胞的结合受到50%抑制。In - IGF - BP在IGF - I RIA中也竞争[125I]IGF - I；然而，与IGF - II RRA相比，其在IGF - I RIA中的干扰要小得多。未标记的IGF - I和In - IGF - BP分别在0.25和5 ng/ml时可使[125I]IGF - I的结合发生50%的置换。我们消除BP假象的方法如下。我们知道In - IGF - BP与IGF - I和IGF - II的结合亲和力相对相等，并且这些配体与细胞受体（IGF - II）和抗体（IGF - I）的结合非常特异（IGF - I和IGF - II的交叉反应性分别为2%和0.5%）。因此，在IGF - I RIA中，我们通过加入过量的IGF - II来阻断In - IGF - BP假象，在IGF - II RRA中，我们通过加入过量的IGF - I来阻断In - IGF - BP假象。通过将纯化的In - IGF - BP与不同量的IGF - I孵育，我们发现5 ng In - IGF - BP与10 ng IGF - I预孵育30分钟可完全阻断IGF - II RRA中的BP假象。同样，In - IGF - BP与IGF - II预孵育可阻断IGF - I RIA中的BP假象。（摘要截短于250字）
