Sensitivity Of Antibody Detection Research Articles

日本脳炎ウイルス持続感染C6/36細胞を用いた血清学的迅速診断法について

The usefulness of persistently infected C6/36 (C6/J-121) cells with Japanese encephalitis virus (JEV) for a rapid serodiagnostic test was examined with the sera of men, swine and laboratory animals by indirect immunofluorescent antibody (IFA) technique. Detection of specific antibodies was completed within 3 to 5 hours. Nonspecific fluorescence frequently observed in other IFA tests was few. The sensitivity of antibody detection and the serological specificity of IFA (IgG) were similar to those of hemagglutination-inhibition (HI) test. For the detection of IgM antibodies in sera of JE-patients and naturally JEV infected swine, this method was found to be simpler and more sensitive and rapid than the conventional HI test which required 2-ME treatment of the sera.

Screening of monoclonal antibodies using antigens labeled with acetylcholinesterase: Application to the peripheral proteins of photosystem 1

An original immunoenzymatic screening method, based on the use of antigens labeled with the stable enzyme acetylcholinesterase (AChE, EC 3.1.1.7), is described. The high turnover of this enzyme results in a very sensitive detection of antibodies. In this method, monoclonal antibodies from the supernatants of hybridoma cultures are immobilized on a solid phase coated with anti-mouse immunoglobulins and react simultaneously with the appropriate antigen labeled with biotin molecules. In a second step, biotinylated acetylcholinesterase is in turn associated to the system via avidin interactions and subsequently detected by a colorimetric assay. The method appears more sensitive and easier to use than either the corresponding radioimmunological test using a 125I-iodinated antigen or the same type of enzymatic immunoassay performed with biotinylated horseradish peroxidase instead of biotinylated AChE. The combined use of microtiter plates, solid-phase separation, and colorimetric detection allows a high level of automation of the method which makes it very efficient to process a large number of samples. This technique has been successfully applied to the screening of monoclonal antibodies directed against peripheral proteins of the photosystem 1 (PS1) membrane complex in photosynthesis. A complete set of antibodies recognizing these PS1 components was selected. The same technique was also tested in competition immunoassays and appears to be a very precise and useful tool for quantifying PS1 polypeptides in different biological extracts, including sodium dodecyl sulfate-denatured membranes. This can be of special interest for studying the biogenesis of membrane complexes.

Human retroviral env and gag polypeptides: serologic assays to measure infection.

Humoral antiviral responses to human retrovirus infections identify persistently infected individuals and can be used to characterize virus-host interactions. Antibodies to native viral polypeptides have been reliably measured, although quantitation of env antibodies is difficult due to a lack of purified antigens. To quantitate antibodies to env antigens, bacterially expressed cloned env polypeptides from the transmembrane regions of human T lymphotropic virus types I and III were applied to nitrocellulose filters in an immunodot assay. A combination of the sensitivity of the Western blot procedure and the specificity of peptides from defined viral sequences was used to detect 49/49 HTLV-III/LAV-infected individuals previously defined as seropositive by radioimmunoprecipitation sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these HTLV-III/LAV envelope seropositive people, 22% lacked antibody to p24 in a radioimmunoassay. In contrast, the sensitivity of antibody detection to HTLV-I env antigens and p24 were comparable. Antibodies to HTLV-I and HTLV-III/LAV env transmembrane peptides were not cross-reactive. Levels of antibody to env antigens of both HTLV-I and HTLV-III/LAV persisted without change for at least 26 mo, suggesting that most infections represent stable virus-host interactions. The use of bacterially expressed env peptides offers a rapid serologic approach for distinguishing human retroviral infections and can be used to define immune responses to specific regions of the viral genome.

A procedure to increase the sensitivity of solid-phase immunoassays

A strategy to increase the sensitivity of antibody detection is described. In this method, streptavidin is used as a cross-bridge between a biotin-conjugated second antibody and a DNA plasmid simultaneously labelled with biotin and 32P. With this simple approach antibodies can be detected with a significantly higher sensitivity as compared to the standard procedures using an 125I-labelled second antibody.

Stepwise amplified immunoperoxidase (PAP) staining. I. Cellular morphology in relation to membrane markers.

A novel procedure for the assay of monoclonal antibodies is described. The technique is based on a combination of three principles. Unlabeled (sheep) antiserum to mouse immunoglobulin (Ig) and complexes of peroxidase with mouse monoclonal antiperoxidase (monoclonal PAP complexes) are used as reagents in a variant of the unlabeled antibody enzyme (PAP) method, described by Sternberger. The amount of peroxidase eventually bound to a monoclonal antibody can be varied over a wide range by repetition of incubation cycles with anti-mouse Ig and monoclonal PAP complexes. During the assay, incubations and wash steps are performed by immersion of whole slides. The influence of repetitive incubation cycles with anti-mouse Ig and monoclonal PAP complexes on background staining and detection of monoclonal antibodies at low concentrations was quantitated in a model system. At a given primary antibody concentration, a linear relationship was found between peroxidase activity and the number of incubation cycles. Application of the technique to the detection of monoclonal antibodies bound to cell-surface antigens is described. Peripheral blood cells were labeled in suspension with monoclonal antibodies. Cytocentrifuge preparations of labeled cells were prepared, and such preparations were fixed before stepwise-amplified PAP staining. Cells showed intense specific staining. Morphological detail of stained and unstained cells is preserved, allowing morphological analysis of labeled cells and rapid analysis of monoclonal antibody specificity. Because the reagents used in the assay can be produced in large quantities with uniform quality, the technique can be readily automated. This, together with the possibility to increase the sensitivity of antibody detection in a controlled, stepwise fashion to levels that cannot be reached with "single-step" techniques, may further expand the applications of monoclonal antibodies.

The use of monoclonal antibodies for sensitive detection of adducts of polycyclic aromatic hydrocarbons to DNA

The use of monoclonal antibodies for sensitive detection of adducts of polycyclic aromatic hydrocarbons to DNA

The binding of staphylococcal protein A by the sera of different animal species.

The capacity of purified immunoglobulin or serum to bind (125I)-labeled staphylococcal protein A (SPA) was measured by means of an immunofiltration assay that facilitated the examination of large numbers of sera and required only a minute quantity of each. Sera from 80 species, including humans, laboratory animals, domestic animals, and a variety of African mammals were examined. A wide interspecies variation in the SPA-binding capacity of serum immunoglobulins was confirmed. Only small variations were observed among individuals within the same species with one notable exception. A greater than 10,000-fold variation in SPA-binding capacity was observed among sera from nine goats. Interspecies differences in serum SPA-binding capacity correlated well with taxonomic differences, and the serum SPA-binding capacity correlated well with taxonomic differences, and the serum SPA-binding capacities of African mammals corresponded closely to those of their more common relatives. The sensitivity of antibody detection by indirect (125I)SPA immunoassay was shown to be determined mostly by the SPA-binding capacity of the serum examined. The titer of antibody to influenza A/Texas/1/77(H3N2) virus in sera from several different species, when measured by (125I)SPA immunofiltration assay, was directly proportional to the SPA-binding capacity of the serum and was not proportional to the hemagglutination inhibition titer. An (125I)SPA immunofiltration assay for antibody to Lassa virus in the serum of Mastomys natalensis (which binds SPA only one-thousandth as well as human serum) was at least as sensitive as the standard fluorescent antibody assay.

Viroimmunoassays: Detection of virus-antigen/ antibody complexes with an immunoadsorbent

A sensitive assay for antibodies, antigens and haptens was developed with the use of a virus as an indicator of the formation of the antigen/antibody complex. For our model studies, dinitrophenyl-ΦX174 phage (DNP-ΦX174) was used as the hapten coupled bacteriophage. Direct neutralization of DNP-ΦX174 by anti-DNP antibody can be detected at antibody levels greater than 200 pg. In contrast, 2 pg (1·5 × 10 −5 pmole) of antibody can be measured by simply removing DNP-ΦX174, which has reacted with anti-DNP, from uncomplexed ΦX174. As described in this communication, this can be accomplished rapidly and conveniently by using antibodies coupled to a solid support. The assay can also be used to detect DNP antigenic determinants. In this way, 0·01 pg of DNP-bovine serum albumin was detectable. The assay is specific and can be completed within several hours. The viroimmunoassay may have general application for the sensitive detection of antibodies or antigenic determinants.

Response of children to Neisseria meningitidis polysaccharide vaccines.

Children aged two to six years were inoculated with various amounts of group C Neisseria meningitidis polysaccharide vaccine. Doses of 5, 25, and 50 tg were less satisfactory in eliciting antibody than larger doses of 100 or 250 [tg. Antibody response was directly related to age. Children in three of the vaccinated groups were inoculated again eight months later with 50 ctg of both group A and group C vaccines. Booster responses occurred in all groups, and those who received the highest doses originally still had the greatest amount of antibody to group C N. meningitidis. All responded to group A vaccine. The hemagglutination method did not offer sufficient sensitivity in detection of antibody, but the radioimmunoassay procedure was satisfactory. However, even with this sensitive technique, the response in children was of lower magnitude than that in adults given smaller doses of vaccine.

A Mathematical and Experimental Study of the Kinetics of Neutralization of Bacteriophage øX 174 by Antibodies

Summary The kinetics of the in vitro neutralization of φX 174 by guinea pig antibody has been studied under dilute conditions. A mathematical model comprising a system of n differential equations has been constructed to represent this reaction. To account for the behavior of reaction samples in a bioassay, the model includes an infectivity function for the phage-antibody complexes. Experimental results show that dissociation can be neglected in the in vitro reaction. The model explains discrepancies such as departure from first-order kinetics that appear when a neutralization reaction is studied by “direct plating” and “decision tube” methods. Proceeding logically from the model, a new antibody assay, “complex inactivation (CI)” is described. Results from this assay support the model and suggest a more sensitive antibody detection system. Using the CI assay, phage neutralization experiments showed that over 99% of φX exhibit homogeneity in their association activity