Development of a sensitive solid-phase radioimmunoassay technique for quantification of class-specific Escherichia coli antibodies

A specific and sensitive, quantitative solid-phase radioimmunoassay (RIA) has been developed for the detection of Escherichia coli antibodies in serum and secretions. Preparation of affinity-purified anti-E. coli standards allows accurate quantification of G, M and A classes of antibody down to 10 ng/ml using only 20 microliters of sample. This technique has considerable advantages over indirect haemagglutination in sensitivity and accuracy of immunoglobulin class detection. RIA also compares favourably with ELISA in sensitivity and sample size required. Affinity-purified standards may also be used to quantify the ELISA test.     

Rapid screening of monoclonal antibodies: new ''microstick'' radioimmunoassay

A semi-automation of fluid phase double antibody radioimmunoassay has been developed. The immune precipitate that was formed in 96-well microtitration plates was harvested and washed on microfibre filters using a Titertek cell harvester. A disc transfer system originally designed for use with the harvester was used as a quick and easy method of transferring the filter discs containing immune precipitate into vials for counting. The results of radioimmunoassay using the microtitration plate-filtration and conventional tube-centrifugation method are essentially identical. The microtitration plate-filtration radioimmunoassay has the following advantages over the conventional tube-centrifugation method: (1) there is no centrifugation required; (2) handling of microtitration plate is easier than the tubes in racks; and (3) it requires much less time to perform the assay.     

Further comparative studies on chemical properties of carcinoembryonic antigen in tumor tissues and closely related antigens in adult feces and meconium

The chemical structure of carcinoembryonic antigen (CEA) and two closely related antigens, normal fecal antigen-2 (NFA-2) in normal adult feces and nonspecific cross-reacting antigen-2 (NCA-2) in the meconium, were further analyzed comparatively. The NH2-terminal amino acid sequence of NCA-2 was newly determined to position 18 and found to be identical to that so far determined for CEA- and NFA-2. After proteolytic digestion with chymotrypsin or protease V8, the digests of these antigens showed two groups of fragments upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One consisted of the sharply banded fragments which were identical in all antigens and stained only with Coomassie brilliant blue (CBB) (five bands in the range 2500-10,000 daltons for chymotrypsin and 11 bands in the range 8000-35,000 daltons for protease V8, respectively), and the other consisted of the dispersed fragments which had variable mol. wts in the range 10,000-100,000 and were stainable with both CBB and periodic acid-Schiff reagent. Elution profiles of CEA, NFA-2, and NCA-2 from lectin columns, especially from concanavalin A-Sepharose columns, suggested some differences in oligosaccharide chains between them. These results indicate that the fundamental chemical structure of these antigens seems to be very similar to one another and is divided into two parts; an homologous portion(s) which is common to all three antigens and contains no sialylated sugar components, and a heterogeneous portion(s) which is variable among these antigens and contains sialylated sugar components.     

A sensitive enzyme-linked immunosorbent assay for the quantitation of antigen-specific murine immunoglobulin E

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of 2,4-dinitrophenyl (DNP)-specific murine immunoglobulin (Ig) E is described. The assay uses beta-galactosidase, which is conjugated to goat anti-rabbit gamma-globulin (GARG) via a method using a mild heterobifunctional cross-linking reagent, m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). The assay has a sensitivity for detection of about 200 pg/ml of anti-DNP IgE. Analyses of murine serum samples using this ELISA correlate well with those obtained using the passive cutaneous anaphylaxis (PCA) reaction in rats. With the use of automated 96-well reader, data acquisition is rapid and, therefore, this ELISA is ideal for analyses of large numbers of samples. The assay can be easily modified for the measurement of other Ig classes and of IgE of other antigen specificities.     

癌胚抗原检测方法学评价及其在胃癌诊断中的意义

为比较临床常用CEA检测方法的特异性和敏感性,探讨CEA在胃癌诊断中的意义,用ELISA、时间分辨荧光免疫法(TRFIA)、RIA、化学发光免疫法(CLIA)检测36例病理确诊胃癌患者血清CEA水平,同时检测20名健康体检者血清CEA水平.结果显示,四种方法的特异性均为100%,ELISA敏感性最低,为19.4%,CLIA的敏感性最高,为44.4%.结论是ELISA检测CEA的敏感性有待提高,CEA检测不能作为胃癌诊断的指标,可以作为部分胃癌患者疗效观察的指标.     

Mapping of antigenic determinants within peptides of a variant surface glycoprotein of Trypanosoma brucei

The location of antigenic determinants in the primary amino acid sequence of the variant surface glycoprotein of Trypanosoma brucei MITat 1.6 was investigated using monoclonal antibodies in conjunction with the known cyanogen bromide and tryptic cleavage patterns of this antigen. The cyanogen bromide digestion fragments of the antigen were purified and used to raise polyclonal antisera, which were specific for the appropriate cyanogen bromide fragment and partial digestion products, as well as recognising the intact variant surface glycoprotein. Competition radioimmunoassays were carried out between these antisera and nine monoclonal antibodies specific for MITat 1.6 variant surface glycoprotein, which have previously been characterised and shown to recognise five antigenic determinants of which only one is exposed on the surface of the living trypanosome. The binding of the monoclonal antibodies to the major tryptic peptide of MITat 1.6 variant surface glycoprotein was investigated by immunoblotting and by competition radioimmunoassay, and revealed that the five antigenic determinants recognised by the nine monoclonal antibodies are all located in the N-terminal two thirds of the MITat 1.6 variant surface glycoprotein molecule. Three of the determinants are located in an immunodominant region apparently formed by the folding together of two of the cyanogen bromide peptides. The other two determinants appear to be more conformationally labile; one of these is the determinant which is exposed on the surface of the living trypanosome, which is located in the N-terminal one third of the molecule.     

Limitations in the use of the enzyme-linked immunosorbent assay (ELISA) for identification and quantification of thermogenin

The use of immunological assays, ELISA and RIA, for the identification and quantification of thermogenin (the brown adipose tissue-specific, GDP-binding, 32 kDa uncoupling protein) raises doubts regarding the exclusive occurrence of thermogenin in brown adipose tissue. Weak reactions between mitochondria from rat liver, rat skeletal and heart muscle and hamster white adipose and thermogenin antibodies have been observed (Cannon et al., 1982; Lean et al., 1983; Hansen et al., 1984). In order to study whether these reactions were due to thermogenin in tissues other than brown adipose tissue (BAT) or due to non-specific binding of thermogenin antibodies, a protein from rat liver mitochondria and a protein from tubifex mitochondria were isolated by the same procedure as thermogenin. The 2 proteins had almost the same molecular weight as thermogenin and reacted with thermogenin antibodies in ELISA and dot-blotting, but did not bind GDP and had an amino acid composition different from that of thermogenin. It is concluded that the weak reactions seen between thermogenin antibodies and mitochondria from different tissues other than BAT are due to non-specific binding, and that antibody cross-reactivity alone is unsuitable for the identification of thermogenin.     

电针镇痛对大鼠中枢cAMP和cGMP含量的影响

目的 研究针刺镇痛与中枢环核苷酸含量变化之间存在的可能性关系。方法 运用放射免疫法（RIA）测定大鼠电针前后,全脑和不同脑区cAMP和cGMP含量的变化。结果 电针30min提高大鼠痛阈的同时,使全脑cAMP含量显著降低（P＜0．05）;间脑cAMP含量降低,端脑cGMP含量降低,而脑干则显著升高（P＜0．05）。直线相关处理和回归分析表明,大鼠脑干cGMP含量与针刺镇痛效应之间存在正相关关系。结论 针刺镇痛与中枢环核苷酸水平之间存在密切关系,提示中枢环核苷酸含量的变化可能是实现针刺镇痛的重要环节之一。     

高原低氧环境下大鼠中脑区内ＧＨＲＩＨ含量的研究

本工作选用健康Ｗistar大白鼠,由平原（海拔５m,上海）,直接引入高原低氧环境（２２６０m和３４６０m）,用放射免疫分析法９ＲＩＡ）分析了大鼠进入高原低氧环境后,在急性低氧反应期和慢性低氧习服期,中枢中脑区内生长抑素（ＧＨＲＩＨ）的含量变化。结果表明,大鼠在急性低氧应激反应期,与平原对照组相比,ＧＨＲＩＨ降低非常显著（Ｐ＜０.０１）,在３０天慢性低氧习服期,１－３d升高显著（Ｐ＜０.０１）,３－７－１５d持续性降低（Ｐ＜０.０１）,１５－３０d趋于回升（Ｐ＜０.０５）。中脑区内ＧＨＲＩＨ的这种动态变化可能在低氧性生理反应的调节中有重要的生物意义。     

不同海拔藏族人血浆AVP含量的比较研究

目的了解世居高原男女性在不同海拔高度其血浆中AVP含量的情况。方法在不同海拔高度测试者用放射免疫分析法进行AVP测定。结果男、女性随海拔高度的增高,AVP含量均有显著性差异。结论含量的变化可能与低氧性环境有关。