Quantitative Precipitin Test Research Articles

Immunochemical Studies of Exocellular Polysaccharides of Tremella and Cryptococcus.

Comparative immunochemical studies of exocellular glucuronoxylomannans of several species of the genera Tremella and Cryptococcus were done with rabbit antisera prepared to two yeast-form strains (T7 and T19) of Tremella fuciformis Berk. Quantitative precipitin test showed that glucuronoxylomannans of Cryptococcus were precipitated with the antiserum to T. fuciformis T7 but not with the antiserum to T. fuciformis T19. In contrast, glucuronoxylomannans of Tremella are reactive with antiserum to T. fuciformis T19. Hapten inhibition tests and structural analyses of glucuronoxylomannans indicated that presence or absence of β-(1→2)-linked d-xylose short chain in glucuronoxylomannan may contribute to the serological difference among species of Tremella and Cryptococcus.

Immunochemical studies on blood groups. The combining site specificities of mouse monoclonal hybridoma anti-A and anti-B.

Mouse monoclonal hybridomas, five anti-blood group A, three anti-B, and one anti-AB, produced by various methods of immunization, have been characterized by quantitative precipitin tests and the fine structures of their combining sites have been mapped by oligosaccharide inhibition assays. The combining sites of antibodies of each specificity differed among themselves. Three of the five monoclonals were specific for difucosyl and two for monofucosyl A determinants. All but the anti-AB were strictly specific for blood group A or blood group B erythrocytes; all of the anti-A monoclonals gave essentially equivalent titers in hemagglutination tests with A1 and A2 erythrocytes except for a monoclonal anti-A prepared by immunization with a human gastric cancer cell line. The data provide additional evidence for the heterogeneity of the antibody response to the different antigenic determinants present on blood A and B substances and emphasize the importance of difucosyl determinants which comprise most of the determinants on the water-soluble blood group substances.

Detection of antibodies against infectious bursal disease virus: a comparison of three serological methods

Four different oil emulsion infectious bursal disease virus (IBDV) vaccines were inoculated into four-week-old specific pathogen-free chickens. At weekly intervals for five weeks, sera were obtained from the vaccinated birds and from uninoculated control birds and examined for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA), the quantitative agar gel precipitin (QAGP) test and the virus neutralisation (VN) test. There was a highly significant correlation between the mean responses to all tests; the highest correlation (0.818) was between VN and QAGP and the lowest (0.573) between QAGP and ELISA. Generally the ELISA detected positive sera earlier than the VN test which in turn was more sensitive than the QAGP test. The ELISA and QAGP test were less variable, more reproducible and easier to perform than the VN test.

Antigenic reactivities of chemically modified .BETA.-lactoglobulins with antiserum to bovine .BETA.-lactoglobulin.

Analysis of the quantitative precipitation of bovine β-lactoglobulin (β-Lg) with rabbit antiserum to β-Lg indicated that there were at least four antigenic sites on β-Lg. To study the antigenic property of bovine β-Lg, we examined the antigenic reactivity of anti β-Lg serum with β-Lg specifically modified with chemical reagents by immunodiffusion analysis, a quantitative precipitin test, and an enzyme-linked immunosorbent assay.Modification of arginine residues, tryptophan residues, or sulfhydryl groups had little effect on the antigenic reactivity. A significant decrease in the reactivity was observed when β-Lg was acetylated, succinylated, modified with diethylpyrocarbonate or coupled with glycine amide.These results suggest that 1.1 of three arginine residues, two tryptophan residues, and one sulfhydryl group are out of the antigenic sites, but there is a possibility that the amino group, histidine residue and carboxyl group may play an important role in the antigenicity of bovine β-Lg.

A general approach to standardization of the solid-phase radioimmunoassay for quantitation of class-specific antibodies

The feasibility of using an anti-human immunoglobulin/human immunoglobulin/[ 125I]anti-human immunoglobulin ‘sandwich’ in a solid-phase radioimmunoassay to produce a standard curve which could be used to quantitate antigen-specific antibody of a particular immunoglobulin class was investigated. The amount of secondary antibody (SAb) bound was determined as a function of whether the primary antibody (PAb) was bound to its specific solid-phase antigen or by a solid-phase anti-human immunoglobulin. No significant difference between the two values was observed. Quantitation of anti-tetanus toxoid antibody by this method was in good agreement with quantitative precipitin tests. Comparison of SAb binding as a function of the way the PAb is bound was extended to class-specific PAb by use of murine monoclonal antibodies to meningococcal antigens. In most cases somewhat greater binding of SAb occurred when PAb was bound to antigen, but in several cases where low avidity antibody and/or poor quality antigens were used, greater SAb binding occurred when PAb was bound by anti-mouse immunoglobulin. The results indicate that this approach may be useful as a general method for standardizing the SPRIA and other solid-phase immunoassays such as the ELISA to measure class-specific antibody.

A modified quantitative precipitin test which is rapid, sensitive and reproducible

A modification of the standard quantitative precipitin test is described. The new procedure, which utilizes polyethylene glycol (PEG) at both the 37°C and 4°C incubation stages, provides a convenient quantitative precipitin test assay which is more reliable and much faster than the standard assay. Moreover, the modified or PEG assay gives a quantitative measure of antibody concentration even when antisera of low titre are tested. The sensitivity of the capillary-tube test is also enhanced when the supernatant solutions contain PEG.

Standardization of the quantitative agar gel precipitin test for antibodies to infectious bursal disease

Variations in physical test parameters, antigen batch and concentration and test reader were examined for their effects on the apparent titre of infections bursal disease (IBD) sera in the agar gel precipitin (AGP) test. The British Standard IBD serum was included and its value for standardizing the AGP test was assessed. It was found that the physical test parameters and the reader affected test results significantly but that the use of the British Standard reduced such effects to insignificance. Antigen concentration was found to be of no significance within certain limits but for clarity high antigen concentrations were recommended.

IMMUNOCHEMICAL INVESTIGATIONS OF HUMAN PITUITARY β‐LIPOTROPIN

Antiserum to human beta-lipotropin has been raised in rabbits and characterized by gel double diffusion, quantitative precipitin, microcomplement fixation and biological neutralization tests. A sensitive radioimmunoassay for human beta-lipotropin has been developed. It was demonstrated that the antiserum shows specificity for this hormone. Human beta-melanotropin and human beta-endorphin showed only a minimal degree of cross-reaction, while ovine beta-lipotropin showed a partial but significant degree of cross-reactivity.

Analysis of Parameters Affecting the Solid Phase Radioimmunoassay Quantitation of Antibody to Meningococcal Antigens

An indirect solid phase radioimmunassay (SPRIA) performed in flexible polyvinyl microtiter plates was modified for quantitative determination of antibodies to various bacterial antigens. Parameters affecting quantitation of the assay were investigated by using meningococcal serotype antigens and rabbit antisera plate was essentially complete after 1 hr at 37 degrees C, and only 4 to 8% of the bound antigen was released during the course of the assay. About 90% of the specific primary antibody (PAb) added to the antigen-coated wells was bound after overnight incubation at room temperature, whereas, 10 to 25% of the bound PAb was released before completion of the assay. Under conditions of limiting PAb the time required for saturation binding of 125I-anti-immunoglobulin (secondary antibody = SAb) to the PAb was dependent on the concentration of SAb. At a concentration of 20 ng SAb/25 mul, maximum binding occurred in 12 to 16 hr at 22 degrees C. Uncer conditions of extreme SAb excess the cpm of 125I-SAb bound was directly proportional to the amount of PAb bound. Under these conditions the cpm of 125I-SAb bound per well can be related to the amount of PAb added per well by a time-dependent calibration coefficient K(t) which is the product of three parameters: 1) the specific activity of the 125I-SAb, 2) the ratio of PAb bound to SAb bound and, 3) the fraction of added PAb remaining bound to the plate. Experimentally determined values for these parameters were used to calculate K(t) and quantitate the specific antibody in nine rabbit antisera with the SPRIA. A close correlation (r=0.985) was found between these results and the results of quantitative precipitin tests performed by using the same antisera and antigens. Although the SPRIA results were an average of 33% lower, a more accurate value for K(t) can easily be determined by performing the SPRIA on several sera calibrated by the quantitative test. Reproducibility of the SPRIA for 10 replicate determinations was +/- 6.7%. The assay described is capable of measuring a minimum of about 0.5 mug antibody/ml of serum and appears to be applicable to many different antigens.

Immunological evidence for substrate-induced conformational alterations in human carbonic anhydrase B.

The inhibition by specific antibody of carbonic anhydrase B activity towards bicarbonate was uncompetitive (Ki 9.5 X 10(-7) M) whereas that of activity towards p-nitrophenylacetate was mixed-type (Ki 9.2 X 10(-7) M). Differences in the immunological reactivity of the enzyme in the presence of the substrates was documented by quantitative precipitin tests and by the Farr technique. In the presence of bicarbonate, nearly half of the antigenic determinants of the enzyme were altered to such an extent that antibodies directed against these determinants in the native form did not bind. This antibody fraction was separated and shown to react with the native enzyme in different molar ratios than the total antibody population. It produced only a very limited inhibition of the activity towards CO2 and p-nitrophenyl acetate. The immunological reactivity of the enzyme with both the total antibody population and the fraction that is nonreactive in the presence of bicarbonate was identical in the absence of substrates and in the presence of CO2 and p-nitrophenyl acetate. It is suggested that the native enzyme form exhibits enzymatic activity towards CO2 and p-nitrophenyl acetate, whereas in the presence of bicarbonate a "conformational adaptation" is induced.

Purification and immunochemical characterization of type e polysaccharide antigen of Streptococcus mutans.

The type-specific antigen of Streptococcus mutans strain MT703, serotype e, has been chromatographically purified and characterized. Two chromatographic fractions were obtained from saline extracts which reacted with both anti-MT703 whole-cell serum and Lancefield group E serum. The major fraction (eI) was identified as a polysaccharide composed of 37% glucose, 56% rhamnose, 5% protein, and 0.3% phosphorus, whereas the minor fraction (eII) contained 66% protein in addition to 10% glucose and 17% rhamnose. The immunological specificity of these antigens was found to be the same by immunodiffusion in agar gel. Another fraction with a negative charge (eIII) reacted with polyglycerophosphate antisera from Streptococcus mutans and Streptococcus pyogenes. For comparison, the MT703 antigen in a hot trichloroacetic acid extract (eA) and the group E antigen from a saline extract of cells of strain K129 (EI) were similarly purified by anionic ion-exchange chromatography. Although the ratio of glucose and rhamnose in eA was 1:0.9 and in eI and eII approximately 1:1.5, reactions of identity were obtained in gel diffusion against specific anti-e serum. This difference in ratio is probably a result of the extraction procedures. Both the type e and group E antisera were reactive with both eI and EI antigens. The adsorption of group E antiserum with MT703 cells removed all E antibody, whereas type e-specific antibody remained after adsorption with K129 cells. These results suggest that eI antigen possesses both e and E specificities, whereas EI possesses E only. These findings were supported by the quantitative precipitin test and immunodiffusion and/or immunoelectrophoretic patterns in agar gel. Methyl-beta-D-glucopyranoside markedly inhibited the precipitin reaction in both type e and group E sera. However, a significantly stronger inhibition by cellobiose of type e serum than of group E serum indicates that a beta-linked glucose-glucose dimer is the predominant antigenic determinant of the e specificity. The presence of both e and E specificities on a single polysaccharide molecule was demonstrated by the use of purified e antigen released from a specific e-anti-e complex. This antigen reacted with a group E-specific serum as well as a type e-specific serum. An examination of five S. mutans type e strains showed the presence of group E specificity also, whereas the I, II, and IV serotypes of group E streptococci only possessed the group E specificity.

Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia.

The cell wall peptodoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The petidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histoloyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other clostridia as well as various types of C. botulinum.

Effects on specific antibodies on the catalytic activity of L-asparaginase from Serratia marcescens and Escherichia coli.

Rabbit antisera against highly purified L-asparaginase from Serratia marcescens and from Escherichia coli showed up to 60% inhibition of the catalytic amidohydrolysis of L-asparagine when combined with the homologous enzyme. This inhibition was diminished somewhat against the heterologous enzyme. Kinetic studies in the presence of these antisera showed an increased Kmapp for both homologous and heterologous enzymes using L-asparagine as substrate. In contrast, kinetic studies employing the poor substrate, L-glutamine, showed activation attributable to specific antibodies. This was seen in lower Kmapp values and up to twofold increases in the Vmax over the normal rabbit serum controls. The high degree of cross-inhibition (approximately 80%) and the low degree of cross-reactivity in the quantitative precipitin test (approximately 34%) suggest that these two enzymes possess structural similarities located mainly in the regions of the catalytic sites.

Precipitin and mitogenic behavior of dimeric and tetrameric concanavalin A.

The results of double diffusion and quantitative precipitin tests show that in contrast to the concanavalin A tetramer, the dimer is incapable of precipitating glycogen. The results support previous work suggesting that dimers composed of two fragmented monomer units possess only one binding site for carbohydrate (McKenzie and Sawyer, J. Biol. Chem. 1973. 248: 549). On the other hand, dimer and tetramer species had similar mitogenic activity as measured by the incorporation of radioactive thymidine by mouse spleen lymphocytes and induced cap formation to similar degrees. Results are discussed in terms of the affinity and valency of the polymeric forms and their stability at the cell surface.

Interactions between ricinus agglutinin and human IgM and IgG.

Ricinus agglutinin, purified until homogeneous, precipitates serum glycoproteins with terminal nonreducing galactose residues including IgM and IgG. Almost 100% of IgM reacted with anti-IgM and ricinus agglutinin in quantitative precipitin tests. In similar tests, almost 100% of polyclonal IgG was precipitated by excess anti-IgG, whereas only about 10% reacted with ricinus agglutinin. In quantitative precipitin analyses and affinity chromatography experiments with insolubilized ricinus agglutinin and isolated monoclonal IgG1 and IgG3 proteins, only IgG3 proteins reacted with ricinus agglutinin.

Use of Radioimmunoassays to Determine the Concentration of Streptococcal Group-Specific Antibodies in Rabbit Antisera

A radioimmunoassay was developed for the quantitation of antibodies to the Group A, A-variant, and C carbohydrates in rabbit streptococcal antisera. The assay employs radiolabeled purified Group carbohydrate which has been tyrosylated to allow for incorporation of 125-I. This assay has an advantage over the intitative precipitin test because it measures non-precipitating antibody in addition to precipitating antibody. Furthermore, 7S anti-IgG in rabbit antisera give a falsely elevated value for the antibody concentration in the quantitative precipitin test. This did not occur with the radioimmunoassay. The assay described is reproducible, sensitive, and uses little antisera.

Immunogenicity of the delta-haemolysin of Staphylococcus aureus.

SUMMARY Antibody to δ-lysin of Staphylococcus aureus was produced in rabbits given injections of purified antigen. Removal of normal serum inhibitors was effected by ammonium-sulphate fractionation and ion-exchange chromato-graphy. The purified anti-δ-lysin showed a single line of precipitation with purified antigen in Ouchterlony agar gel-diffusion tests and when subjected to immunoelectrophoresis. The line of precipitation also coincided with the zone of haemolysis caused by δ-lysin when the agar gel-diffusion test was performed in blood-agarose plates. A plot of the data obtained in a quantitative precipitin test performed with purified antibody and δ-lysin showed a characteristic curve confirming their interaction; in control tests with non-immune sera in place of δ-antibody, no precipitation was evident.

A Monoclonal IgM With Antibodylike Specificity for Phospholipids in a Patient With Lymphoma

Abstract Both murine and human myeloma proteins have been shown to have antibodylike activity for serum lipoproteins. We studied a 58-yr-old woman (M.L.) with a lymphoma and a VDRL titer of 1:4096 and a nonreactive FTA-ABS. Serum electrophoresis revealed an M-component which was shown to be IgM-kappa by immunoelectrophoresis and 2-mercaptoethanol treatment of the serum reduced the VDRL titer to 1:64. Immunoelectrophoresis (IEP) of the purified IgM against sonicates of the specific components of the VDRL antigen (cardiolipin, lecithin, cholesterol) produced a precipitin line with both cardiolipin and lecithin. All IgM in the serum could be removed by absorption with cardiolipin, and elution experiments demonstrated the specificity of this reaction. A quantitative precipitin test demonstrated that at the point of equivalence, 0.009 mg of antigen (cardiolipin) reacted with 0.4 mg of IgM (antibody). Additional studies showed that purified IgM (M.L.) gave a positive reaction with nine of 15 phospholipids tested. Similar studies on sera from 50 normal individuals, ten patient’s sera without M-components giving false positive VDRL reactions, and serum from one patient with a congenital absence of IgA and IgG and increased IgM showed no evidence of such a precipitin reaction with any phospholipid tested. Thus, it appeared that the IgM-kappa monoclonal protein behaved like an antibody directed against specific phospholipid antigenic components. The role of lipid antigens in the initiation of such neoplastic and immunologic abberrations should be further investigated and monoclonal proteins screened more extensively against such antigens.

The assay of pneumococcal capsular polysaccharide by immunodiffusion

A simple gel diffusion technique was compared with the quantitative precipitin test for evaluating concentrations of pneumococcal polysaccharide. The analysis showed that results with unknown samples compared closely by the two methods.

The antigenic specificity of different states of aggregation of tobacco mosaic virus protein.

Abstract It has been shown by immunoelectrophoresis that tobacco mosaic virus (TMV) preparations usually contain TMV protein oligomers and that TMV protein preparations contain aggregated material in the form of disks. When these contaminations were minimized, it was possible to obtain antisera of narrow specificity and to prove the existence of antigenic determinants specific for the polymerized and monomeric forms of TMV protein, respectively. When rabbits were immunized with formalinized TMV free of contaminating protein oligomers, antisera were obtained that did not react with depolymerized protein. The monomer of TMV protein was found by quantitative precipitin tests to have an antigenic valence of about five. It can therefore no longer be disputed that TMV protein is a multideterminant antigen.