Radioimmunoassay for the detection of IgG antibodies to herpes simplex virus and its use as a prognostic indicator of HSV excretion in transplant recipients

We report the development of a solid-phase radioimmunoassay for the detection of IgG antibodies to herpes simplex virus (HSV), using a mouse monoclonal antibody specific for the Fc portion of human IgG as the radiolabelled detecting antibody. The binding ratio to test antigen at a single serum dilution, 1:100, correlated significantly with the endpoint titre by radioimmunoassay and with neutralising antibody titre. When compared to neutralisation the radioimmunoassay had a sensitivity of 100% and a specificity of 93%. We believe that the anomalous results are not false positives but represent an increased sensitivity of the radioimmunoassay. We found, by either radioimmunoassay or neutralisation, that high levels of antibody prior to transplantation were associated with a significantly increased risk of HSV excretion post-transplantation in both renal and bone marrow transplant recipients. Thus the radioimmunoassay is a sensitive, specific, and rapid test that can be used as a prognostic indicator of HSV excretion in transplant recipients.     

Rubella immunity by four different techniques: results of challenge studies

When 42 sera with low or inconsistent levels of haemagglutination-inhibiting (HAI) antibodies were tested by single radial haemolysis (SRH), radioimmunoassay (RIA), and enzyme-linked immunoassay (ELISA), RIA was shown to be the most reliable test for detecting low levels of antibody. SRH, however, was found to be an acceptable alternative screening test for rubella antibodies and was more reliable than HAI. Although SRH plates prepared in our own laboratory failed to detect antibodies in six sera, in five of the six, antibodies were only at a low level (RIA titre 1:20- 1:80). OriVir plates (Orion Diagnostica, Finland) failed to detect low levels of antibody in only three sera. There were six (14.3%) sera which were false positives in the HAI test. These women were shown to be seronegative by radioimmunoassay and, when three of these six volunteers were vaccinated, they developed a typical primary immune response which resembled that developed by 43 seronegative women following vaccination. Fifteen of the young women with consistently low HAI litres and one woman who was seronegative by HAI but seropositive by RIA and ELISA were subsequently vaccinated. Six (37.5%) of these women showed no significant rise in titre by any of the tests employed, while ten had a significant rise in titre, detected by at least one test, with a low level of rubella-specific IgM detectable in one.     

A micro enzyme-linked immunosorbent assay for insulin antibodies in serum

A solid-phase micro enzyme-linked immunosorbent assay for the measurement of insulin antibodies in serum is described and its performance compared with that of an established radiobinding assay. Interassay precision in the ELISA was 10% or less at widely spaced points on the dilution curves for human, porcine and bovine insulins. Specificity was demonstrated by substituting purified human gamma-globulin for the test serum and glucagon for the insulin. The influence on ELISA of endogenous insulin in the test serum was examined by measuring antibody binding before and after extraction of the insulin. The correlation between results from extracted and unextracted sera was 0.96 and the fit ideal: y = 1.00x + 0.38%. The correlation between the results of measuring insulin antibody in 256 diabetic sera by the 2 assays was r = 0.74, P less than 0.001 (human insulin) and r = 0.71, P less than 0.001 (porcine insulin). ELISA is cheap and simple to perform. We believe it may prove to be a practical alternative to radioassay in both the routine detection and investigative research of insulin antibodies.     

Monoclonal antibodies to human alpha-foetoprotein: analysis of the behaviour of three different antibodies

Three mouse monoclonal antibodies directed to different epitopes on human alpha-foetoprotein have been produced. Two are of IgG1 subclass and the third is IgA. The polyethylene glycol-dependent immunoprecipitation system, designed for conventional antisera, had to be adapted before reproducible results could be obtained with the reagents. In this adapted system, as well as in a radioimmunoassay using solid-phase second antibody, a mixture of the 3 monoclonal antibodies exhibits cooperativity. However, the sensitivity of the radioimmunoassay with pooled monoclonals is not good as that of conventional antiserum. Low affinity monoclonal antibodies have been used for immunopurification of the antigen, whilst the high affinity one is useful for antigen quantitation in a labelled antibody-dependent system which requires absolute antibody specificity.     

Enzyme-linked immunosorbent assay for quantitation of toxoplasma antibodies in human sera.

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of toxoplasma antibodies using a single serum dilution (1:800) in conjunction with a standard curve Antigen was prepared from Toxoplasma gondii cultivated in human cell cultures. A nearly linear relationship was found between the logarithms of the absorbance values of 120 human sera at a dilution of 1/800 and the titres as determined by an end point dilution ELISA. The reproducibility of the single dilution ELISA was excellent; the coefficients of variation for within-day and day-to-day tests were less than 15%. A close correlation was found between the results obtained with ELISA, indirect immunofluorescence (IF), and complement fixation (CF). The titres in ELISA were 20 to 40 times higher than in IF and 200 to 1000 times higher than in CF.     

Enzyme-linked immunosorbent assays for sperm antibody detection and antigenic analysis

Enzyme-linked immunosorbent assays for estimation of antibodies against human sperm and for determination of antigenic reactivity of spermatozoal proteins were established. Sperm immobilized on PHA-coated microtiter plates or solubilized spermatozoal antigens adsorbed on poly(L)-lysine coated microtiter plates were used as the solid phase. Assay of sperm antibodies was performed by incubation of the test samples with the solid phase followed by incubation with anti-Ig conjugated to peroxidase. Sigmoidal antibody dilution curves were obtained with rabbit and mouse anti-sperm sera. The ELISA was effectively used to screen production of anti-sperm antibodies by mouse myeloma x splenocyte hybridomas. The sensitivity of this ELISA for sperm antibodies was more than 1000-fold greater than the classical tray sperm immobilization test, and was comparable in sensitivity to a radioimmunoassay using 125I-labeled protein A as the tracer. Sperm immobilized on PHA-coated plates exhibited significantly greater antigenic reactivity in both the ELISA and RIA compared with methanol fixed sperm. In a competitive inhibition ELISA, linear Logit-log dose-response curves were obtained with detergent solubilized spermatozoal antigens. The assay was used to monitor the purification of the solubilized spermatozoal antigens by chromatofocussing; a more than 60-fold increase of antigenic potency of purified sperm antigen compared with unfractionated sperm extract was evident in the competitive ELISA.     

Detection of an antigen (AN6520), possibly related to non-A, non-B hepatitis, by monoclonal antibodies. I

The antibody to AN6520 antigen, which was isolated from the liver of a patient with non-A, non-B hepatitis (NANBH), has been detected frequently in convalescent sera from patients with NANBH by the passive hemagglutination (PHA) test. In a further study, we established hybridoma cells secreting antibodies against AN6520 antigen and obtained ascitic fluids with PHA titers ranging from 1:10(5) to 1:10(7). In immunodiffusion with AN6520 antigen, all monoclonal antibodies were found to form an identical precipitin line. These lines were also identical to those formed by rabbit antiserum against AN6520 antigen and by convalescent sera from patients with NANBH. With one of the monoclonal antibodies, 1-F12, solid-phase radioimmunoassay (SP-RIA) for detecting AN6520 antigen was developed as well as blocking RIA for anti-AN6520 antibody detection. The antigen assay was 50 times more sensitive than the reverse passive hemagglutination (R-PHA) test, with a sensitivity threshold of the 1 ng/ml of antigen solution; the antibody assay was 10 times more sensitive than PHA. The results with this blocking RIA were mostly in agreement with the data obtained by PHA. Furthermore, the antigen in human sera, which had never been detected by R-PHA test, could be detected by SP-RIA.     

Development of an enzyme immunoassay for metsulfuron‐methyl

The development of an indirect competitive ELISA for the highly effective sulfonylurea herbicide metsulfuron‐methyl (MSM) is described. As hapten, only the methylester phenyl‐sulfonamide of MSM with an additional succinic acid spacer was used. It was coupled to carrier proteins by the activated ester method. Antibodies raised in rabbits were screened for sensitivity and specificity for MSM. Using an antiserum dilution of 1:180 000 and a coating antigen (hapten‐BSA) concentration of 0.5 ng ml^?1 the IC₅₀ values achieved for the target analyte were in the 1.4 μg I^?1 range, with the limit of detection being 40 ng l^?1. The anti‐MSM antibodies cross‐reacted mainly with sulfometuron (43%), cinosulfuron (16%) and triasulfuron (10%) and, to a much lower extent, with a few other sulfonylureas. The influence of potential matrix interferences such as organic solvents, humic acids and surfactants was investigated. Gas chromatography (GC) was used as a comparison test to validate the ELISA with fortified water samples. The correlation between data from GC and ELISA analyses was 0.995 (n = 9) with a slope of 1.03.     

TrFIA乙肝病毒PreS1抗原的应用研究

目的:乙型肝炎病毒PreS1抗原时间分辨荧光免疫分析(TrFIA)的应用.方法:收集120例HBVDNA拷贝数＞103的乙型肝炎患者血清标本,同时应用TrFIA和酶联免疫吸附法(ELISA)对样本中PreS1抗原进行检测.选择荧光值较高的一例PreS1抗原阳性血清标本,倍比稀释至1:16384倍,用TrFIA试剂与ELISA定性试剂盒同时进行PreS1抗原的检测.结果:有9例标本ELISA结果为阴性,而用TrFIA可检测到PreS1抗原,χ2=7.1,P＜0.05,TrFIA方法灵敏度高于ELISA方法.一例PreS1抗原阳性血清标本ELISA在1:2048倍时结果为阴性,而TrFIA在1:8192倍时仍可检出PreS1抗原.TrFIA方法高、中、低三个浓度批内CV分别为3.57%、3.71%、6.74%;批间CV分别为3.54%、4.16%、8.52%均优于ELISA方法.50份正常体检者血清标本(乙肝标志物均阴性)用TrFIA试剂进行PreS1抗原的检测,结果均阴性,特异性100%.结论:TrFIA与ELISA相比,精密度和灵敏度有较大提高.     

Expression of antibody activity measured by ELISA. Anti-ssDNA antibody activity characterized by the shape of the dose-response curve

To demonstrate the presence or absence of antibodies, results derived from a single serum dilution in an ELISA are sufficient. However, qualitative differences in antibodies are reflected by the shape of dose-response curves. A method based on approximating the absorbance value by a polynomial p(x) = a1x + a2x2, where 1/x is the dilution factor, was used to characterize the dose-response curves in an ELISA for anti=ssDNA antibodies. The parameters used are E = a1 and A = -a2(1)/a2. It can be argues that E gives an estimate of the effective amount of antibodies in the serum and that A is essentially a function of the reaction constants between antibody and antigen.     

Specifics on the refinement and application of two serological assays for the detection of antibodies to HHV-8.

BACKGROUND: Serologic assays for the detection of antibodies to human herpesvirus type 8 (HHV-8) are important for epidemiological studies and to further investigate the proposed pathogenesis of the virus in cancer. Although a variety of assays are available, a lack of optimization and standardization makes their usefulness uncertain, and may be responsible for the controversy concerning the prevalence of infection. OBJECTIVES: To refine an indirect immunofluorescent assay (IFA) for the detection of latent antibodies and a recombinant ORF 65 ELISA for the detection of lytic antibodies in order to increase their ability to differentiate individuals at higher and lower risk for HHV-8 infection. STUDY DESIGN: Sera from Kaposi's sarcoma (KS) patients and blood donors (BDs) were used to modify assay parameters in an attempt to better discriminate between the two populations. Modifications included methods of substrate fixation, incubation times, sample dilution, and antigen/conjugate concentrations. RESULTS: Optimal modifications to the latent IFA included acetone fixation of substrate, and dilution of sera to 1:64 which enhanced detection of HHV-8 antibodies from 68 to 92% in the KS population. Similarly, successful refinement of the ORF 65 ELISA to increase the signal-to-noise ratio included the use of 88 ng of ORF 65 antigen per well and serum dilutions of 1:50. Optical density-to-cut-off ratios directly correlated with titers, thereby introducing a strategy to predict antibody concentrations. The ORF 65 ELISA and the latent IFA were both able to discriminate between the two populations but with different efficiencies. CONCLUSIONS: Although neither the latent IFA nor the ORF 65 ELISA produced perfect test indices, improvement in their performances was noted following the optimization strategies. The ELISA produced better detection of antibodies to the virus than the IFA and permitted prediction of sample titers, thus improving cost and time effectiveness.     

An ELISA method for the detection of autoantibodies to adrenal cortex

An enzyme-linked immunosorbent assay (ELISA) is described for autoantibodies to adrenal cortex. Microsomes were prepared from fresh human adrenal glands, and microtitre ELISA plates were incubated at 4 degrees C overnight with 25 micrograms antigen/ml, the optimal concentration for the system. Optimal dilution of patient's serum was 1/500. Peroxidase-labelled anti-human IgG and IgM sera were used in separate tests and o-phenylenediamine and H2O2 served as substrate. Intra-assay variance of optical density units was 4.5%, and inter-assay variance was negligible when antigen preparations from 2 different adrenal glands were compared. All sera positive or negative at first test gave the same qualitative result in a second. Non-organ-specific binding of sera containing mitochondrial or ribosomal antibodies was eliminated by a blocking ELISA system where the antigen-coated plates were incubated with test sera, and in a second step, peroxidase-labelled IgG from an adrenal antibody-positive control serum was added. In this test, optimal antigen concentration for coating of plates was 6.25 micrograms/ml and optimal serum dilution 1/50. The ELISA proved more sensitive and reproducible than indirect immunofluorescence. Adrenal antibodies detected by ELISA were usually of IgG class alone and only 1 of the 30 positives also contained IgM specificity. 30 out of 38 sera (79%) from patients with 'idiopathic' Addison's disease were positive whereas immunofluorescence revealed only 23 (61%) at first testing and another 4 positives when sera were tested on different adrenal glands. The ELISA described is useful for both scientific work and clinical diagnosis of autoimmune adrenalitis.     

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.     

Comparison of sensitivities of ELISA and radioimmunoassay for detection of class-specific antibody in mouse serum

The limits of detection of IgG1 antibody in a standard mouse antiserum by a single antiglobulin enzyme-linked immunosorbent assay (ELISA) and 2 amplified systems, a double antiglobulin ELISA and a double antiglobulin radioimmunoassay (RIA), were compared in microtitration plates with the same antigen preparation and antisera. Compared with the single antiglobulin ELISA, both amplified assays demonstrated a 64-fold increase in sensitivity for the detection of antibody at high dilutions of standard antiserum. It is concluded that the amplified ELISA offers a safer assay than the amplified RIA and of equal sensitivity for comparable consumption of antisera.     

促肾上腺皮质激素释放因子放射免疫测定法的建立

采用文献[1]的方法提取猪下丘脑促肾上腺皮质激素释放因子(CRF),免疫家兔获得CRF抗血清,与~(125)I标记的纯品CRF建立起特异的CRF放免检测。~(125)I-CRF投入量15000cpm/0.1ml,CRF抗血清1∶1600时,结合力(Bo/T)20%,检出标准CRF最小量为25～50Pg/ml;抗原竞争抑制实验表明与相关肽无交叉反应。应用上述方法检测了人、大鼠、小鼠外周血CRF含量,分别是18±14Pg/ml,36±28pg/ml,120±40Pg/ml;大、小鼠脑组织CRF含量以下丘脑最高。上述结果与国外文献一致,提示该CRF·RIA法可用于血浆及器官CRF含量检测。     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.     

Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis

Pelargonium zonate spot virus (PZSV) is identified recently in tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is a necessity. The coat protein (CP) gene of a California isolate of PZSV was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC). The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3 mg refolded protein per 200 mL of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum was able to detect the recombinant protein, the PZSV coat protein and PZSV infected plant samples. The antiserum was successfully used in indirect-ELISA at dilutions of up to 1:16,000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.     

Enzyme-linked immunosorbent assay to monitor colorectal carcinoma patients treated with a monoclonal antibody (17-1A)

An enzyme-linked immunosorbent assay (ELISA) was compared to a radioimmunoassay (RIA) for the detection and quantification of mouse monoclonal antibody MoAb 17-1A and for measurement of the host response (i.e. anti-mouse immunoglobulin in sera from patients receiving immunotherapy with MoAb 17-1A. Comparable sensitivity and reproducibility were noted with RIA and ELISA but ELISA was more rapid to perform than RIA. Thus quantitative ELISA compared favorably with the RIA for MoAb detection.     

抗牛碱性成纤维细胞生长因子单克隆抗体的制备及初步鉴定

目的：制备抗牛碱性成纤维细胞生长因子(bFGF)单克隆抗体(mAh)，并鉴定其亚类，建立ELISA检测bFGF含量的方法。方法：应用基因重组的牛bFGF免疫BALB／c小鼠，通过细胞融合，建立分泌抗bFGF mAh的杂交瘤细胞株。应用免疫沉淀技术鉴定抗bFGF mAh的亚类；应用基因重组的牛bFGF免疫青紫蓝兔，制备抗bFGF的多抗血清；将抗bFGF mAb及兔抗血清用Protein A亲和层析纯化后，建立检测bFGF含量的ELISA方法。结果：共获得3株稳定分泌抗bFGF mAb的杂交瘤细胞株；它们所分泌的mAb均为IgG1；采用夹心ELISA法检测bFGF的敏感性达ng水平。结论：抗bFGF mAb(IgG1)和多克隆抗体制备为临床应用及相关研究提供了必要的试剂。     

A user's guide to the indirect solid-phase radioimmunoassay for the detection of cytomegalovirus-specific IgM antibodies

In this review full laboratory details are given of a solid-phase indirect radioimmunoassay for the detection of specific IgM antibodies against cytomegalovirus. Practical advice is given on readily available commercial sources of reagents, a simple iodination procedure, the rapid dilution of sera under test and calculation of the results with a computer program available from the authors. Problems encountered with the assay are also detailed such as interference by rheumatoid factor, deterioration of the radiolabel and high background binding found with some sera. If these problems are avoided by the methods given in the text then the radioimmunoassay has been shown to give results of 100% specificity with 89% sensitivity for detecting congenital infection and 92% sensitivity for identifying primary cytomegalovirus infection in pregnant women.