An atomic force microscopy study in the detection of hepatitis B surface antigen by enzyme-linked immunosorbent assay.

Atomic force microscopy (AFM) was employed to study certain enzyme immunoassay steps for the detection of hepatitis B surface antigen (HBsAg). Physical adsorption of monoclonal antibodies (mAb), blocking of surface active sites free of antibodies by neutral proteins, and capture of HBsAg particles by sensitized surfaces were visualized successively in microplate wells of standard immunological plates from various manufacturers. The previously undescribed details such as "etching holes" up to 20 nm in depth were observed on the surface of plates some companies. The quantitative relationships between the optical density (OD) values obtained by enzyme immunoassay (EIA) and the number of antigen-antibody complexes in AFM were calculated.

Therapeutic AIDS vaccines.

Therapeutic HIV vaccines represent promising strategy as an adjunct or alternative to current antiretroviral treatment options for HIV. Unlike prophylactic AIDS vaccines designed to prevent HIV infection, therapeutic vaccines are given to already infected individuals to help fight the disease by modulating their immune response. The first immunotherapeutic trial in AIDS patients was conducted in 1983. Since then several dozen conventional therapeutic vaccine trials have been carried out. Unfortunately, the results have consistently shown that while HIV-specific immune responses were evident as a result of vaccination, the clinical improvement has been seldom observed. The instances of the apparent clinical benefit were invariably associated with unconventional vaccines that acted in accord with the principles of alloimmunization and/or autologous vaccination. All such vaccines were derived from the blood of HIV carriers or a cell culture and thus they inherently contained allo- or self-antigens unrelated to HIV. This intriguing observation raises the issue whether this clinically successful approach has been unduly neglected. The current strategy biased toward vaccines, which have shown little evidence of clinical efficacy, needs to be diversified and supplemented with research on alternative vaccine approaches geared toward immune tolerance induction.

[Preparation and selection of monoclonal antibodies for detecting hepatitis B surface antigen in blood sera]. / Poluchenie i otbor monoklonal'nykh antitel dlia vyiavleniia poverkhnostnogo antigena virusa gepatita B v syvorotkakh krovi.

HV monoclonal antibodies (MAb) were produced in order to improve the quality of HBsAg detection and their specific characteristics were compared with those of other MAbs. MAbs were characterized by asymmetric interactions with the antigen when used as first or second antibodies. The reactivity of a panel of HV and X MAb to ad and ay subtypes was studied by enzyme immunoassay. Mutual blocking (epitope mapping) of MAb helped select antibody couples for the creation of highly effective test system for the diagnosis of the major HBsAg subtypes. The sensitivity and specificity of MAbs were evaluated on reference and control panels of HBsAg sera and on serum specimens from a random sampling of 300 blood donors. The sensitivity of the most specific MAb pairs was 0.1 ng/ml for HBsAg subtype ay and 0.25 ng/ml for subtype ad. The specificity of attested MAb was 98.5% in incubation with stirring and 97% in static incubation. The optimal combinations of attested MAbs were used in the manufacture of Recomnathep B test system in the sandwich format.

[Construction of reference panel of immune serum against hepatitis C virus with standard level of IgG antibodies]. / Konstruirovanie referens-panelei syvorotok k virusu gepatita C s normirovannym urovnem IgG-antitel.

A panel of anti-HCV sera (lot 03HC) was prepared from human sera obtained at blood transfusion centers and infectious hospitals. Donor sera and high-titer sera from patients infected with HCV were used. For positive samples, specific sera reactive with the core and/or NS proteins of HCV 1b and 2 were selected. Positive sera were standardized by the concentrations of IgG with a pool of negative sera containing no HBsAg and antibodies to HIV, HCV, and syphilis. The sera for the panel were selected and titered in screening and specific tests. The anti-HCV panel includes negative and positive sera with low and high titers. The panel sera are stabilized and can be stored for a short time at room temperature. The anti-HCV panel of sera, lot 02HC, was certified at L. A. Tarasevich Institute for Standardization and Control as anti-HCV reference panel intended for sensitivity, specificity, and stability control of diagnostic systems for detection of antibodies to HCV in Russia.

[Development of sera reference panels for quality control of ELISA diagnostic kits in Russia]. / Razrabotka standartnykh panelei syvorotok dlia kontrolia kachestva immunofermenthykh test-sistem v Rossii.

In the past 5 years, the investigators of the "VECTOR" SRB VB and the L.A. Tarasevich State Institute of Standardization and Control of Medical Biological Preparations have jointly designed sera reference panels containing anti-HIV-1 IgG, anti-HCV IgG, and anti-HAV IgM which have been approved as national standard panels. The panels are intended for use in controlling the specificity and stability of the most widely used ELISA diagnostic kits and immunoblot test systems during production, control, and application stages. Some problems of development and production of these panels, including the representation of different sera in the panels and the selection of specific IgG concentrations in the different sera in the panel are described. The authors also attract attention to the stabilization of the specific characteristics of panel sera during storage and transportation.

[Design of reference panels of serum for testing the quality of HIV antibody tests in practical laboratories]. / Konstruirovanie otsenochnykh panelei syvorotok dlia testirovaniia kachestva provedeniia analizov na antitela k VICh v prakticheskikh laboratoriiakh.

The authors define the scientific basis for development of panels of reference sera intended for effective control of the quality of enzyme immunoassay of HIV antibody screening. Special attention was paid to developing the technology of preparing standards highly stable serum samples with a preset concentration of anti-HIV antibodies. The resultant panel of reference sera was tried in screening for anti-HIV antibodies by enzyme immunoassay at seven diagnostic laboratories. Mathematical analysis of the results permits the detection of the minimal errors in studies with the use of serum panel of practical laboratories.

[Constructing the reference panel of sera with standard level of IgG-antibodies]. / Konstruirivanie standartnykh panelei syvorotok c normirovannym urovnem IgG-antitel.

Fundamentals of designing reference panels of sera for effective control of commercial test systems and immunoblotting, intended for detecting antiviral antibodies, have been developed. Reference low-titer panels of anti-IgG antibodies to HIV-a and hepatitis C virus have been designed. A reference panel contains diluted reactive sera with a standard level of IgG antibodies and native sera with undetectable level of antibodies to the major viral antigens from risk group subjects. The reactive sera of a panel contain the whole spectrum of antibodies to all principal viral antigens.

[The principles of creating standard serum panels with a low concentration of HIV-1 antibodies for the quality control of commercial immunoenzyme test systems]. / Printsipy sozdaniia standartnykh panelei syvorotok s nizkoi kontsentratsiei antitel k VICh-1 dlia kontrolia kachestva kommercheskikh immunofermentnykh test-sistem.

The main approaches are defined to the production of sera with the required concentration of antibodies to HIV by the dilution method and to formation on their basis of serum panels according to theoretical distribution of optic density (OD) values. It was shown to be principally possible to prepare panels of positive sera with low concentration of HIV-specific antibodies in a dry stable form, and their practical importance in the evaluation of the sensitivity of enzyme immunoassays was demonstrated. The evaluation of the quality of commercial test systems is based on the position and shape of the histogram of OD values distribution for panel sera for the controlled test systems.

[The comparative characteristics of the peroxidase conjugates used in Soviet immunoenzyme test systems for detecting HIV antibodies]. / Sravnitel'naia kharakteristika peroksidaznykh kon''iugatov, primeneniaemykh v otechestvennykh immunofermentnykh test-sistemakh dlia vyiavleniia antitel k VICh.

Five lots of peroxidase conjugates were studied in enzyme immunoassay systems "Recombinant-HIV" and "Peptoscreen-2" for detection of antibody to HIV. The conjugates differed from each other by the source of generation and methods of preparation. The conjugates were studied in biochemical tests and ELISA. When panels of sera from HIV-infected children and adults were employed, the advantages of using the anti-immunoglobulin conjugate over protein A-peroxidase conjugate were demonstrated, as the former increased the sensitivity of the test systems to HIV antibody detection.