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Results for “Immunosorbent Techniques”

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At least 19 recordsLinked to original sources
PubMed1979

A solid-phase immunosorbent technique for the rapid detection of rubella IgM by haemagglutination inhibition.

The rubella haemagglutination inhibition (HAI) test has been modified for the detection of rubella-specific IgM. The rubella HAI test is performed in microtitre plate wells in which IgM from patients' sera has been selectively retained by anti-human IgM bound to the polystyrene surface. The test requires only anti-human IgM-coated microtitre plates, in addition to the standard rubella HAI reagents. The results obtained in this test, a solid-phase immunosorbent technique (SPIT), are in good agreement with results obtained by the sucrose density gradient centrifugation technique (DGCT). Advantages include the essentially unrestricted number of sera which can be tested daily, the availability of results within 24 h and the lack of interference by rheumatoid factor and by rubella-specific IgG.

Antibodies, Viral↗
PubMed1979

Solid phase immunosorbent technique (SPIT) for the demonstration of specific IGM antibodies by hemagglutination inhibition and passive hemagglutination.

The adsorption of protein to glass and plastic has been well documented. It has also been demonstrated that the absorbed antigen or antibodies retain their activity in as much as they are able to react with a specific antibody respective specific antigen(1). Furthermore the adsorbed antignene or antibody complex may also react with a later added antigen. Anti IgM is adsorbed to the solid phase and will retain IgM from the patient's serum. If the patient's serum contains specific IgM this will react with the specific antignene. If the antigen is a hemagglutinin it will combine with the specific IgM and therefore added erythrocytes will not hemagglutinate. If the antigen is coated on erythrocytes the coated erythrocytes will combine with the specific IgM and hemagglutination will take place. The reaction is schematically demonstrated in Fig. 1.

Erythrocytes↗
PubMed1979

Detection and quantification of foot and mouth disease virus by enzyme labelled immunosorbent assay techniques.

Enzyme labelled immunosorbent assays (ELISA) have been developed to detect and quantify foot and mouth disease (FMD) virus using flexible plastic microtitre plates. The methods were successful for the specific detection of FMD virus and were 50 to 100 times more sensitive than the complement fixation test. The application of the ELISA techniques to FMD virus typing and subtyping, and to the assay of antigen concentration during manufacture of vaccines is discussed.

Aphthovirus↗
PubMed1978

Enzyme-labelled immunosorbent assay techniques in foot-and-mouth disease virus research.

The indirect ELISA technique has been developed successfully to measure antibodies to foot-and-mouth disease virus (FMDV) in cattle sera. Preliminary studies using a standard serum assay show that reproducible results are obtained. The method should prove useful for the examination of antibody titres in sera from large numbers of cattle or other animals.

Animals↗
PubMed1978

IgE in experimental schistosomiasis. II. Quantitative determination of specific IgE antibodies against S. mansoni: a follow-up study of two strains of infected rats. Correlation with protective immunity.

Parasite specific IgE antibodies in rats infected with Schistosoma manoni were measured by passive cutaneous anaphylaxis (PCA) reactions and by the technique of immuno-adsorption. Two strains, one a low IgE producer (Fischer rats) and the other a high IgE producer (Hooded-Lister rats) were studied. In Fischer rats, a time course study of the occurrence of IgE antibodies and resistance to reinfection was made. Parasite specific IgE levels measured by immuno-adsorpiton were much lower than total IgE levels and a similar percentage of specific IgE (about 8%) was in the two strains.IgE antibodies were maximum at day 30 and day 60 after infection; however, a third peak at day 90 was observed only in Fischer rats. Some discrepancies between results obtained by PCA and immunosorbent techniques have been observed, which could be explained by differences in the affinity of IgE antibodies during infection or by the presence of total IgE in the PCA assay. There was a close parallelism between specific IgE antibodies levels and the course of immunity in Fischer rats. This parallelism supports the view that IgE could play a pre-eminent role in protective immunity in rat schistosomiasis.

Animals↗
PubMed1976

RAST using crude and purified anti-IgE.

The sensitivity of the RAST using anti-IgE in 125I-labelled IgG fractions of sheep antiserum was compared to that using anti-IgE purified by immunosorbent techniques in tests with three allergens (grass pollens, Aspergillus fumigatus and the detergent enzyme "Alcalase") on sera from 248 workers in a detergent factory. Both anti-IgE reagents measure the same antibody but the RAST procedure using the crude anti-IgE reagent is less sensitive than that using the immunosorbent-purified anti-IgE in its ability to detect circulating IgE in subjects with positive skin-prick tests. In general the agreement between positive RAST and positive skin test was improved when only skin tests equal to or greater than 3 mm were considered positive. With Alcalase, antigen non-specific binding by the crude anti-IgE reagent may give false positive results. Optimal conditions for the preparation of allergosorbents with this allergen are defined. Predictive equations relating the results of RAST and skin test show that the hitherto arbitrary definition of a positive RAST result is statistically valid.

Allergens↗
PubMed1978

Methods for establishing a working immunoradiometric assay for serum ferritin.

The two-site immunoradiometric assay for measurement of serum ferritin requires purified human ferritin and an avid high-titer antihuman ferritin antibody. Some of the antibody is radioiodinated following purification by immunoadsorption. All methods for preparation of these materials for the assay are described in minute detail. Performance of the assay itself and calculation of results are also described, and attention is drawn to several potential pitfalls. Adherence to these detailed descriptions will help to eliminate difficulties experienced by centers wishing to establish their own working serum ferritin assay.

Adolescent↗
PubMed1977

Detection of bursa and thymus-specific alloantigens in the chicken.

CH strain chickens selected from the F2 progeny of a cross between CH (B10/10) and B14B (B14/14) strains carried bursa (BA) and thymus (TA)-specific alloantigens of B14B parental origin. The BA marker was present on 95% of bursal cells but only on 10% of splenic and blood B lymphocytes. The TA marker was expressed by 80% of thymus cells and weakly by 45% of splenic and 70% of blood T lymphocytes. The adopted immunological and genetic protocol offers a feasible approach towards detection of differentiation antigens in major histocompatibility complex-homozygous but 'minor' alloantigen-segregating populations.

Animals↗
PubMed1978

A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue.

A highly sensitive method for the immuno-histochemical localisation of carcinoembryonic antigen (CEA) is described. This method is based on the binding of a peroxidase-antiperoxidase complex (PAP) to anti-CEA antibodies by means of an anti-gamma-globulin which reacts with both the anti-CEA and the antiperoxidase antibodies. Using the technique described here, CEA was localised in conventionally processed normal and cancerous colonic tissue. In normal as well as in neoplastic tissues, a CEA-specific staining of cell membranes and cytoplasm was demonstrated. In frozen sections of normal colonic tissue CEA was found even at high dilutions of the first antibody; this indicates the high sensitivity of the method. The applicability of the method to conventionally processed and thereby well preserved tissue specimens opens the possibility to identify CEA by light microscopy even at very low concentrations.

Animals↗
PubMed1976

Antibody quantitation using an immunoadsorbent and the unlabeled antibody enzyme method.

Measurement of specific immunoadsorbent-bound antibodies has been accomplished by the unlabeled antibody enzyme method (Sternberger et al., 1970). Sepharose-4B containing specific antigen (or ligand) is treated with diluted specific immune serum (primary serum), such as 1 ml of serum diluted 6000-20,000 fold. followed by antiserum against immunoglobulin G (IgG) of the primary serum and then by peroxidase-antiperoxidase (antigen-antibody) complex (PAP) derived from the same species as the primary serum. Radiolabeled primary antibody and anti-IgG have confirmed the stoichiometry of the reaction. The immunoabsorbent binds the antibody of interest quantitatively and to equal extent after 15 min or 48 h. The enzymatic activity of the PAP complex followed a direct linear relationship to its concentration indicating the stability of binding in the PAP complex. A direct relation between the enzymatic activity measured when both the primary antiserum and the anti-IgG are used allows for quantitation of the antibody level of the primary serum.

Animals↗
PubMed1978

Improved immunoadsorption procedure with anion-exchange bacterial cell columns.

Bacterial cell columns for immunoadsorption were prepared with Streptococcus cells and triethylaminoethyl cellulose (Cellex-T) matrix material as a model system. Good column flow properties and satisfactory retention of the cells were obtained with ratios as high as 2 ml of packed cells/3 g dry weight of cellulose. Anion-exchange fractionation of whole serum by the Cellex-T was prevented by using 0.25 M NaCl in the developing buffer. Antibodies were adsorbed directly from whole serum and recovered in high yield by desorption at pH 2.3. Pre-exposing bacterial cells to formalin and washing them with acetone was necessary to ensure that they remained on the columns. One strain of Streptococcus salivarius (SS 908) was satisfactorily retained on a column only after cells were labeled with fluorescein isothiocyanate and washed with acetone. The means by which Cellex-T retains bacterial cells appears to be a combination of electronic attraction and physical entrapment.

Chromatography, Ion Exchange↗
PubMed1978

Bovine alpha-fetoprotein. Isolation and characterization.

Bovine AFP was purified by ion exchange chromatograph on C.M. cellulose and DEAE Sephadex A-50, gel filtration and immunosorbent technique. AFP was homogeneous when studied by gel electrophoresis under non denaturing and denaturing conditions, by ultracentrifugation and by immunological methods. The following molecular data were obtained: 1. Sedimentation equilibrium indicated a molecular weight of 66,500 and sedimentation velocity gave s degrees 20, w = 4.71 S. A partial specific volume v = 0.737 ml g-1 was derived from density measurements. 2. From these data, a Stokes radius of 3.26 nm, a diffusion coefficient D20 w = 6.61 10(-7) cm2 sec-1 and a frictional ratio f/fo = 1.21 were calculated. 2. Sodium dodecylsulphate disc electrophoresis suggests a molecular weight of 67,000. 3. Gel filtration pointed to a molecular weight of 75,000. 4. Microheterogeneity of AFP was demonstrated by isoelectric focusing. The isoelectric point of the major component is 4.6. 5. The chemical composition was determined. AFP is a glycoprotein containing 7 per cent carbohydrate including 1.67 per cent hexoses, 2.38 per cent N-acetyl glucosamine and 1.8 per cent N-acetyl neuraminic acid.

Amino Acids↗
PubMed1977

Immunological measurements of conformational motility in regions of the myoglobin molecule.

The conformational motilities of three regions of the sperm whale myoglobin molecule and of an isolated peptide of myoglobin have been examined by measuring the equilibrium constant for the native equilibrium nonnative transition. The immunological approach of Furie et al. (Furie, B., Schechter, A.N., Sachs D., and Anfinsen, C.B. (1975), J. Mol. Biol.92, 497-506) was used with convenient modifications. Antibodies specific to the nonnative conformations were used in assaying for competition between the radioactively labeled peptide and native myoglobin. Labeling was by 125I iodination of the peptide or its 3-(4-hydroxyphenyl)propionyl derivative, and separation of the immune complex from the free peptide was either by ammonium sulfate precipitation or by centrifugation of the antibodies immobilized on Agarose beads. For the antigenic regions of the sequence (1-55), the measured conformational equilibrium constant was 840 +/- 200 at 22 degrees C; the value for the C-terminal region (132-153) was 280 +/- 120 at 25 degrees C, while that for the region (66-76) adjacent to the heme group was greater than 2.5 x 10(6). Measurements on the isolated peptide (132-153) indicated that 1% of the molecules adopt native-type folding in aqueous solution at 36 degrees C.

Amino Acid Sequence↗