Evaluation of Passive Hemagglutination, Solid-Phase Radioimmunoassay, and Immunoelectroosmophoresis for the Detection of Hepatitis B Antigen

Sensitivity and specificity of passive hemagglutination (RCA), solid phase radioimmunoassay (RIA), and immunoelectroosmophoresis (IEOP) were compared under experimental and clinical conditions. In dilution experiments with sera containing hepatitis B antigen (HB Ag) of known subtypes, the sensitivity for an ad subtype serum was RIA (1), RCA (1/2), IEOP (1/256) and for an ay subtype serum RCA (1), RIA (1/8), IEOP (1/128). An evaluation of the National Institutes of Health, Division of Biologics Standards test panel number 2 demonstrated HB Ag in 34 of 60 samples by RIA, in 33 by RCA, and in 25 by IEOP. HB Ag was detected in 57.5% of 200 outpatients with a tentative diagnosis of hepatitis by RIA, in 54% by RCA, and in 42.5% by IEOP. In 1,661 volunteer blood donors, 13 (0.78%) were "positive" for HB Ag by RIA, 11 (0.66%) by RCA, and 3 (0.18%) by IEOP. However, absorption experiments indicated that at least six of the above RIA positive and five of the RCA positive sera exhibited nonspecific positive reactions.     

A microtiter solid-phase radioimmunoassay for hepatitis A antigen and antibody.

A microtiter solid phase radioimmunoassy for hepatitis A antigen (HA Ag) and antibody (anti-HA) was developed. The test was more sensitive than immune adherence hemagglutination for detecting HA Ag and almost as sensitive for detecting anti-HA. The specificity and sensitivity of reagents were examined and optimum conditions for the test were determined. Radioimmunoassay, immune adherence hemagglutination, and immune electron microscopy were compared for detecting anti-HA. A serologic response to HA Ag was detected in paired sera from patients with type A hepatitis but not from patients with type B or non-A, non-B hepatitis by all three techniques.     

Evaluation of Assay Methods for Hepatitis-Associated Antigen

Assay methods for hepatitis-associated antigen (HAA) were evaluated for sensitivity, or reproducibility, or both in a series of three trials in which both research and service-oriented laboratories participated. Agar-gel diffusion (AGD) methods were found to be the least sensitive and reproducible of the commonly employed assay methods. Complement fixation (CF) tests were consistently more sensitive than either AGD or counterelectrophoresis (CEP) methods for detection of HAA. With judicious choice of the antibody reagent, sensitivity of CEP techniques was equivalent to CF methods of HAA detection. None of the three major assay methods (AGD, CEP, or CF) compared in this study were capable of consistently detecting HAA when it was present in relatively low concentrations in human serum.     

Comparison of Solid-Phase Radioimmunoassays for Baculoviruses

The sensitivity and cross-reaction of four solid-phase radioimmunoassays (RIA) for Trichoplusia ni nuclear polyhedrosis virus containing singly enveloped virions were investigated. The detection limits of each assay were as follows: Indirect RIA, 5 ng of dissolved polyhedron antigen; direct RIA, 50 ng; indirect sandwich RIA, 200 ng; and direct sandwich RIA, 300 ng. The indirect and indirect sandwich RIAs showed considerable cross-reaction with other baculovirus antigens, but the direct and direct sandwich RIAs showed cross-reaction with only one closely related baculovirus. When microtiter plates used for the solid phase were pretreated with bovine serum albumin, nonspecific binding of labeled antibodies was reduced to a minimum. Antibodies prepared by an immunoadsorption procedure showed greater specific binding than antibodies prepared by ammonium sulfate precipitation of the immunoglobulin fraction. Highly contaminated antigen could not be detected by the indirect RIA, but the direct sandwich RIA was unaffected by antigen contamination. Antigen making up 0.0025% (wt/wt) of a sample of bird droppings could be detected by the direct sandwich RIA.     

Solid Phase Radioimmunoassay for Identification of Herpesvirus hominis Types 1 and 2 from Clinical Materials

A solid phase radioimmunoassay (RIA) was developed for typing Herpesvirus hominis (HVH) strains isolated from clinical materials, and it also proved to be applicable to the direct detection and typing of HVH antigen in human and animal brain tissue. The procedure utilized virus-infected human fetal diploid cells or brain tissue smears in the bottom of 1-dram glass vials, antigen was detected through the use of intermediate HVH antisera produced in rabbits or hamsters and cross-absorbed with the HVH heterotype, and (125)I-labeled anti-species (rabbit or hamster) globulins produced in goats were used for detection of immune complexes. The cross-absorbed HVH antisera could be used at high dilutions in the RIA test, and they reacted with marked type-specificity in the RIA system. Specificity of the test was also improved by determining and using optimal concentrations of intermediate sera and of (125)I-labeled anti-species globulins. Results of typing HVH isolates by the RIA procedure agreed in all instances with those obtained by direct fluorescent antibody staining with cross-absorbed conjugates. The RIA procedure was effective and more sensitive than direct fluorescent antibody for demonstrating and typing HVH antigen directly in smears of infected human brain tissue.     

Detection of IgG antibody to measles virus by radioimmunoassay technique

A highly sensitive procedure of solid-phase radioimmunoassay (RIA) was developed for the detection of measles IgG antibody. HeLa cells persistently infected with measles virus were used as a solid-phase antigen. This technique was applied to the detection of measles IgG antibody in patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis. Normal subjects having experienced natural measles or measles vaccination and patients with various neurological diseases of non-virus nature were also examined as control groups. Measles antibody was detected at high titers in both the sera and cerebrospinal fluid of SSPE patients. Moreover, RIA/HI ratios of SSPE patients were significantly higher than those of normal subjects, suggesting the presence in the formers of antibodies to nucleocapsids at high titers as well as to viral envelopes. On the other hand, no significant difference was found in both RIA and HI titers between the sera of multiple sclerosis and those of various neurological diseases.     

Interactions of Standard Antibody with Australia Antigens in Au Ag-Ab Radioimmunoassay

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.     

Microtiter Solid-Phase Radioimmunoassay for Hepatitis B Antigen

A micro-solid-phase radioimmunoassay (micro-SPRIA) for hepatitis B antigen (HB Ag) was developed for use with microtiter serological equipment. Radiolabeled immunoglobulin G was prepared from human and animal sera containing hepatitis B antibody (HB Ab); it was not necessary to isolate specific HB Ab by immunochemical means. A micro-SPRIA prepared with guinea pig reagents was approximately as sensitive as the AusRIA radioimmunoassay, but, like the AusRIA test, yielded false positive results. A micro-SPRIA prepared with human reagents was slightly less sensitive but did not yield false positive results. These micro-SPRIA tests offer several advantages, including conservation of reagents, adaptability to other antigen-antibody systems, ease of performance (especially when testing large numbers of specimens), and economy.     

Hepatitis B Core Antigen: Immunology and Electron Microscopy

TWO DISTINCT VIRAL ANTIGENS ARE ASSOCIATED WITH THE HEPATITIS B VIRUS: the hepatitis B surface antigen (HB(s)Ag, Australia antigen) and the hepatitis B core antigen (HB(c)Ag). HB(s)Ag, purified from the serum of asymptomatic human HB(s)Ag carriers, and HB(c)Ag, purified from the liver of a chimpanzee acutely infected with hepatitis B virus, were examined by serological and immune electron microscopic methods. Antisera raised against HB(s)Ag reacted with the outer, surface component of the Dane particle and with the 20-nm spherical and tubular particles present in HB(s)Ag-positive serum, but not with the internal component of the Dane particle or with purified HB(c)Ag particles. Antisera raised against purified HB(c)Ag particles reacted with the internal component of the Dane particle and with HB(c)Ag, but not with the surface of the Dane particle or with the 20-nm spherical and tubular particles associated with HB(s)Ag. Purified HB(c)Ag particles, 27 nm in diameter, demonstrated distinct subunits. The infectious form of hepatitis B virus appears to be represented by the 42-nm Dane particle composed of a 27-nm nucleocapsid core component (HB(c)Ag) surrounded by an antigenically and morphologically distinct lipoprotein surface component (HB(s)Ag).     

一种新型H5N1禽流感病毒血凝素抗原快速检测试剂的建立

利用5株广谱特异性抗H5亚型血凝素单克隆抗体和酶联免疫渗滤技术成功地建立了一种适于现场检测H5亚型禽流感病毒血凝素蛋白的抗原快速检测试剂H5-HA(Ag)Dot-ELISA。该试剂对41株代表当前亚洲地区流行的各种遗传变异亚系H5N1禽流感病毒检测均为阳性,对多数毒株的分析灵敏度优于0.1个血凝滴度(HA titer),其中部分优于0.01个血凝滴度;比较该试剂与早期开发的同类ELISA试剂,发现前者对后者未能检出的H5N1新变异株检测均为阳性;利用该试剂和商品化Directigen Flu A(BD)试剂检测两株H5N1病毒株,提示前者灵敏度高于后者;该试剂对一株H5N1病毒的检测灵敏度与标准RT-PCR相当;该试剂对24株非H5亚型病毒检测均为阴性,显示出良好特异性。以上结果提示,此研究建立的H5N1病毒抗原快速检测试剂在H5禽流感现场检测上具有较好的应用前景。     

Time-resolved fluorescence immunoassay of thyroxine in serum: immobilized antigen approach

With T(4)-bovine IgG as a solid-phase antigen, we have developed a direct competitive-type immunoassay for serum total thyroxine (TT(4)), which depends on the competitive distribution of europium-labeled anti-T(4) monoclonal antibody between solid-phase-bound T(4) and the T(4) in the sample or standard. The captured fraction of the tracer was measured after a dissociation-enhancement step. Four different T(4) protein conjugates were synthesized, of which T(4)-bovine IgG was selected as the most favorable for the preparation of solid-phase antigen. The sensitivity was 3.5 ng/ml with a sample volume of 20 microl. T(4) values obtained by this procedure agreed well with those obtained by RIA (r = 0.967, n = 38) and EG&G Wallac TRFIA (r = 0.926, n = 64). All other quality criteria was also fulfilled with respect to precision, accuracy, and dynamic range.     

Comparative study of three commerical preparations for the detection of Australia antigen]

2,944 sera from blood donors (420 of them being new donors) were tested in four techniques: Counterelectrophoresis (CEP) and three commercial tests: Ausria II, Auscell and Hepanosticon. Over 10 HBs Ag detected by RIA, 8 were evidenced by the Auscell test and 6 by the Hepanosticon Test. False positive results (approximately 3%) represent a disadvantage to the reverse hemagglutination method; this rate increased considerably in a group of 386 patients sera also tested. The authors conclude that the results obtained with the reverse hemagglutination technique are nearing those of RIA.     

Radioimmunological analysis in studying the persistence of the plague antigen in the body of rodents and ectoparasites in natural foci in Siberia

To study the persistence of Y. pestis capsular antigen, or fraction 1 (F1), in the body of less important plague carriers in the Mountain Altai and Transbaikal natural foci, as well as in experimentally infected ticks, the liquid-phase competitive radioimmunoassay (RIA) was used for the first time. In this study RIA showed, due to its sensitivity, doubtless advantages over traditional methods, such as the passive hemagglutination (PHA) test and the antibody neutralization (AN) test, and made it possible to detect F1 in picogram amounts. RIA revealed that F1 persisted in Siberian long-tailed gophers for up 14 months after the infection of the animals in diffusion chambers and for 7 months after their infection by subcutaneous injection. Experiments on Daurian pikas confirmed that, in comparison with the PHA and AN tests, RIA ensured fourfold effectiveness in the detection of antigen F1. The study of infected mites revealed that antigen F1 could be retained in them for more than a year and detected by RIA techniques in 10% of cases. The data obtained in this investigation indicate that the persistence of microorganisms should be studied mainly with the use of new-generation tests, and RIA, being one of the most sensitive techniques, deserves wide approval and introduction into the practical work of institutions intended for plague control.     

Prevalence and significance of hepatitis B surface antigen in a general hospital.

Over a 6-month period 2025 patients admitted to New Mount Sinai Hospital, Toronto were screened for hepatitis B surface antigen (HBsAg) by counter-immunoelectrophoresis (CIEP) and radioimmunoassay (RIA). CIEP detected 12 HBsAg-positive patients and RIA 16. RIA is therefore the more sensitive test for HBsAg. Of the 16 patients 2 had liver disease previously diagnosed, 3 had malignant disease and 11 were asymptomatic carriers. Of the 11 carriers all were born in countries where the carrier rate is known to be high. Routine screening of hospital patients on admission is of no value in detecting unsuspected liver disease but is of value in detecting asymptomatic carriers, which is of importance for the patient and his family. Routine screening tests for HBsAg in Canadian hospitals that treat many patients born in countries with a known high HBsAg prevalence is recommended. Routine screening is also recommended in all hospitals in Mediterranean and Asian countries.     

Subspecificity determination of HBsAg by radioimmunoassay

A radioimmunoassay method for subtyping hepatitus B surface antigen (HBsAg) was developed. Polystyrene beads pretreated with glutaraldehyde and coated with monospecific anti-d or anti-y antibodies were used. The treatment of beads with glutaraldehyde significantly increased the sensitivity of the test. Subtyping by radioimmunoassay was found to be more sensitive than by the immunodiffusion method. Fifty-nine percent of the samples tested could be subtyped by RIA and only 32% by immunodiffusion. The technique has the added advantage of utilising a commercially iodinated anti-HBs.     

Novel potentiometry immunoassay with amplified sensitivity for diphtheria antigen based on Nafion, colloidal Ag and polyvinyl butyral as matrixes

A novel potentiometry immunoassay with amplified sensitivity has been developed for the detection of diphtheria antigen (Diph) via immobilizing diphtheria antibody (anti-Diph) on a platinum electrode based on Nafion, colloidal Ag (Ag), and polyvinyl butyral (PVB) as matrixes in this study. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log Diph concentrations, high sensitivity (51.4 mV/decade), wide linear range from 8 to 800 ng ml(-1) with a detection limit of 1.5 ng ml(-1), rapid potentiometric response (<3 min) and long-term stability (>6 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting diphtheria antigen in the clinical diagnosis.     

Comparative evaluation of methods for detecting HBsAg in sera

The results of the analysis of 1209 serum samples, made with a view to detecting those containing HBsAg, are presented. This analysis was made by the radioimmunoassay (RIA) on a polyethylene film, by the standard RIA technique with the use of a diagnostic kit obtained from Abbott Laboratories (USA) and by the passive hemagglutination (PHA) test. The RIA film technique was found to have the sensitivity of about 2 ng/ml HBsAg, which is similar to the sensitivity of the kit from Abbott Laboratories and exceeds the sensitivity of the PHA test approximately 50-fold. The percentage of detected HBsAg-positive sera, yielded by analysis with the use of the RIA film technique and the standard RIA technique, is the same. The RIA technique make it possible to detect more positive sera than the PHA test by about 2.5%.     

Relative Sensitivity of Gel Diffusion, Complement Fixation, and Immunoelectroosmophoresis Tests for Detection of Hepatitis-Associated Antigen and Antibody

The immunoelectroosmophoresis (IEOP) test was compared with gel diffusion and complement fixation (CF) tests for sensitivity in detecting hepatitis-associated antigen (HAA) in the sera of hepatitis patients, for titration of HAA, and for detection of antibody to HAA. The IEOP test was found to be slightly more sensitive than either gel diffusion or CF tests for detection of antigen in the patients' sera. Titers of HAA demonstrated by IEOP were higher than those seen in gel diffusion tests but lower than CF titers. The gel diffusion test with an "enhancement" pattern was found to be more reliable than the other two procedures for detection of low levels of anti-HAA, due to the greater inhibitory effect of an antigen excess in the IEOP system and the possible masking of low levels of antibody by anticomplementary activity in the CF test system. Staining of immunoprecipitates in the IEOP test contributed little to the sensitivity of the test for detection of HAA.     

Development of a direct microplate enzyme immunoassay for the determination of pregnanediol-3 alpha-glucuronide in urine

A sensitive direct enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was developed. The assay system involves the use of an antiserum against pregnanediol-3 alpha-glucuronide and an enzyme-labelled antigen chemically prepared by linking beta-D-galactosidase to 20 alpha-hydroxy-5 beta-pregnane 3(O-carboxymethyl)oxime. Free from antibody-bound antigen was separated by a solid-phase double antibody method, using a microplate coupled with goat anti-rabbit gamma-globulin. This solid-phase enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was validated in terms of specificity, accuracy and sensitivity. When urine samples were assayed for pregnanediol-3 alpha-glucuronide, the results obtained by the solid phase enzyme immunoassay and conventional radioimmunoassay methods agreed well (n = 30, r = 0.922). This assay system has an advantage over radioimmunoassay, because it does not require the use of radioisotopes. The procedure of this method is very simple, since it does not require purification steps of the biological samples.     

生长激素和催乳素放射免疫测定法的建立与应用

目的：建立测定大鼠垂体和血浆中生长激素（GH）和催乳素（PRL）含量的高特异性、高灵敏度的双抗放射免疫测定（RIA）法;研究急性低氧对垂体激素GH和PRL的作用。方法：用氯胺－T法进行抗原放射性碘标记;采用平衡饱和加样程序的双抗RIA法测定。结果：用该方法测定急性低氧（0．5h）时血浆和垂体GH和PRL含量,7km低氧,垂体GH含量明显升高（P＜0．05）,血浆则相反;7km低氧,明显降低垂体和血浆PRL含量（P＜0．01）;而5km低氧对GH和PRL的作用与对照组比无统计学差异。结论：本双抗RIA法具有高特异性、高灵敏度及简便易行等特性;用该法测定提示急性低氧可抑制大鼠GH和PRL的分泌。