Idiotypic analysis of monoclonal anti-fluorescyl antibodies: Localization and characterization of idiotypic determinants

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (K_a values ranging from 5 × 10⁶ to 10¹⁰ M⁻) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (γ, κ) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15–20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained γl heavy (H) chains and 4-6-10 contained both γl and γ2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which denned binding mechanism diversity. The location of determinants recognized by antiidiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotye by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (˜25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the V_H- and V_L-chain interaction must exist for the expression of these idiotypes.     

A potential radioimmunoassay system for detection of Hanganutziu-Deicher type heterophile antigen(s) and antibodies in tissues and fluids

A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The 125I-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. The antigen-antibody complex was precipitated with normal human serum as the carrier protein, followed by the addition of rabbit anti-human IgG F(ab')2 serum. With this method, different H-D antigen-active molecules were compared for heterophile H-D antigen potency with reasonable sensitivity detecting about 0.3 ng of cold glycoprotein. 8 different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attempt to correlate expression of H-D antigen on tissues with elevation of H-D antibodies. The results showed that all patients' tissues expressed the antigen(s) but only 3 of them had abnormal levels of H-D antibodies. This could have been due to excess antigens in circulation or immune complexes.     

Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies

A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.     

Cholecystokinin-like immunoreactivity in the dorsal horn of the spinal cord of the rat: a light and electron microscopic study

Cholecystokinin-like immunoreactivity was investigated with an indirect immunoperoxidase technique in the whole spinal cord with the light microscope and in the dorsal horn with the electron microscope. Intraparenchymal injections of colchicine were performed to allow the detection of cholecystokinin-like immunoreactive cell bodies. Rats treated at birth with capsaicin were also studied at the light microscope. Numerous cholecystokinin-like immunoreactive fibres and varicosities were found in the two superficial layers of the dorsal horn and in the intermedio-medial nucleus; cholecystokinin-like immunoreactive cell bodies were also present in these two regions. After neonatal capsaicin treatment, the number of cholecystokinin-like immunoreactive fibres and varicosities was strongly reduced in the dorsal horn. At the electron microscope level, cholecystokinin-like immunoreactivity was localized in numerous neurites often filled with vesicles (axon terminals and dendrites containing vesicles) and in few cell bodies and dendrites. The immunoreaction was found mainly associated with ribosomes, granular reticulum, neurotubules and vesicles. Large granular vesicles were filled with the reaction product whereas small and medium-sized vesicles showed a varying degree of immunoprecipitate around their membrane. In addition dense "granules" of precipitate were observed in numerous presynaptic neurites. Cholecystokinin-like immunoreactive axons were of small calibre and mostly unmyelinated. Cholecystokinin-like immunoreactive axon terminals made asymmetric synaptic contacts with generally unlabelled dendrites or dendritic spines. A single labelled nerve terminal could contact several different dendrites in structures resembling glomeruli. Few axo-somatic synapses but a relatively high number of axo-axonic contacts were seen. About half of these axo-axonic contacts involved pre- and postsynaptic profiles. Both light and electron microscopic observations led us to the conclusion that some of the cholecystokinin-like immunoreactive fibres of the dorsal horn originate in the spinal ganglia via capsaicin-sensitive C afferents; and some from intrinsic neurons, particularly islet cells. Other fibres may come from supraspinal centres, other local neurons or capsaicin-insensitive afferents from the spinal ganglia. The results are discussed with regard to data in the literature, particularly those concerned with the specificity of the cholecystokinin antibodies; it is hypothesized that several types of cholecystokinin-like immunoreactive peptides may be present in the dorsal horn, depending on their origin (supraspinal, intrinsic or peripheral).(ABSTRACT TRUNCATED AT 400 WORDS)     

Ecdysteroids in adults of the nematode, Dirofilaria immitis

Adult males and females of the dog heartworm, Dirofilaria immitis, were extracted separately and, following separation of the free and conjugated ecdysteroid fractions, the conjugates were hydrolysed enzymically. Both the ecdysteroids released by hydrolysis of the conjugates and the free hormones were further purified and analysed by a combination of radioimmunoassay, thin-layer chromatography and high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by gas-liquid chromatography/mass spectrometry (selected ion monitoring). Both males and females contained free and conjugated ecdysteroids. Evidence was obtained for the presence of ecdysone, 20-hydroxyecdysone, 20,26-dihydroxyecdysone and possibly ponasterone A. The possible parallel between ecdysteroid endocrinology in nematodes and insects is discussed.     

Characterization of monoclonal antibodies against prostaglandin E2: Fine specificity and neutralization of biological effects

The specificity and heterogeneity of the immune response of BALB/c mice immunized with prostaglandin E₂ (PGE₂) coupled to thyroglobulin was studied. All the animals (n = 50) responded to PGB₂, a transformation product of PGE₂. However, following repeated injections most of the animals (n = 30) were also able to respond to PGE₂. Cellular hybridizations were performed and five anti-PGE₂ monoclonal antibodies were isolated and analysed. They are mainly directed against the ring and the ω-chain of PGE₂ but their specificity toward the α-chain is more limited. The association constants are greater than to 1 × 10⁹M^?1. The monoclonal antibody 8E.57.71 (K_a = 1.3 × 10¹⁰M^?1) is particularly convenient for sensitive radioimmunoassays (detection limit 25pg/ml, when iodinated tracer is used). Anti-PGE₂ monoclonal antibodies were found to neutralize the specific binding of [³H]PGE₂ to rat brain hypothalamic receptors and to inhibit the PGE₂ induction of rat fundus muscular contraction.     

1000名健康小儿血清骨钙素含量的测定及其分析

目的 通过对不同年龄段健康儿童血清骨钙素（ＢＧＰ）含量的测定,评价正常儿童的骨骼２地血清ＢＧＰ的含量,为代谢发现有病的诊断、治疗提供依据。方法用放射免疫的方法检测１０００名１￣１４岁的健康儿童血中ＢＧＰ的含理。结果 １￣１４岁小儿血清ＢＧＰ的一在１０．６００－２４．８６６ｎｇ／ｍｌ之间,随着年龄增长,含量增加,青春期达高峰,１￣３组女孩血清ＢＧＰ一明显高于男孩。４￣７岁组男、女血清ＢＧＰ含量无差别     

毛细支气管炎患儿血浆CGRP、VIP RIA的意义

目的：通过愉洲毛细支气管炎患儿急性期和恢复期血浆中降钙素基因相关肽（CCRP）、血管活性肠肽（VIP）的含量,探讨毛细支气管炎患儿血浆CGRP、VIP放射免疫分析（RIA）的意义。方法：本文采用放射免疫分析测定了31例毛细支气管炎患儿急性期和恢复期血浆CGRP、VIP的浓度,以35例正常婴幼儿血浆CGRP、VIP的浓度为对照。结果：毛细支气管炎患儿急性期血浆CGRP浓度明显高于恢复期及正常对照组婴幼儿（P〈0．05）;恢复期CGRP浓度有所下降,但仍高于正常对照组（P〈0．05）,三组间有显著差异（F=82．50,P〈0．01）;毛细支气管炎患儿急性期血浆VIP浓度明显低于恢复期及正常对照组婴幼儿（P〈0．05）;恢复期V1P浓度有所升高,但仍低于正常对照组（P〈0．05）;三组之间有显著性差异（F=300．20,P〈0．01）。结论：CGRP、VIP定量分析结果表明毛细支气管炎患儿血浆CGRP、VIP含量与病程紧密相关。CGRP、VIPRIA对于小儿毛细支气管炎发病机制的研究以及了解其发病病程和指导治疗有着重要的意义。     

肿瘤标志物放射免疫分析进展

本文从免疫分析总体发展的过程中展示了肿瘤标志物分析的进展.从经典的竞争性RIA到以IRMA开始的非竞争性免疫分析和多分析物微点阵的出现,其中非竞争分析的成功是一个历史性转折.肿瘤标志物的诞生恰遇这一变革,大都直接采用了先进的抗体标记、双位点、夹心式非竞争性分析技术.根据位点占据的理论,竞争性分析本质上属于间接测定,非竞争性分析属于直接测定;直接量度比间接推导的结果更为精确.因此,直接测量的非竞争性方式是达到分析结果高精确、高灵敏以及其他优越性的最关键因素.在同样条件下非竞争分析的灵敏度可比竞争性分析高一百倍以上.在非竞争分析中非特异性结合很容易达到1%以下,在最佳条件下甚至可以达到0.01%,此时的灵敏度可再上升两个数量级.非竞争分析的优越性同样体现在微点阵分析中.微点阵分析是免疫分析中新的分支,尚在发展之中,肿瘤标志物检测及一般临床常规诊断对它尚无明显需求,它的主要潜力将在大规模筛选实验之中.     

血清β-HCG和P联合检测在早期异位妊娠诊断中的价值

为探讨联合检测血清β-人绒毛膜促性腺激素β-HCG和孕酮（P）在早期异位妊娠诊断及保守治疗中的价值,采用放射免疫分析法测定324例早期异位妊娠患者血清β-HCG和P水平,并与146名早期正常妊娠者作对照,比较保守治疗前后血清β-HCG和P水平的变化,观察治疗成功组患者血清β-HCG和P水平下降至正常范围内所需要的时间。结果显示,异位妊娠组血清β-HCG和P水平低于宫内妊娠组,差异有统计学意义（P〈0.05）;保守治疗成功组和失败组血清β-HCG下降幅度的差异无统计学意义（P〉0.05）,但P下降幅度的差异有统计学意义（P〈0.05）;保守治疗成功组和失败组对比,血清P比β-HCG更快下降至正常水平。因此,联合检测血清β-HCG和P可辅助确诊早期异位妊娠,为患者赢得治疗时机的同时对预后监测也具有临床意义。动态血清P测定可作为一种优于血清β-HCG的预测异位妊娠保守治疗效果的检测手段。