Quantitative Precipitin Test Research Articles

Polysaccharide of Coccidioides immitis.

Pappagianis, D. (University of California, Berkeley), E. W. Putman, and G. S. Kobayashi. Polysaccharide of Coccidioides immitis. J. Bacteriol. 82:714-723. 1961.-Soluble polysaccharide from mycelia or culture filtrates of Coccidioides immitis was found to consist mainly of mannose, but also included small quantities of galactose and another reducing sugar. Isolation of the polysaccharide by ethanol precipitation provided accompanying nitrogenous material. There was 3 to 4% nitrogen, present in amino acids, but in a nondialyzable (possibly protein or peptide) form. The average molecular weight of the complex was 31,700. Attempts to separate the nitrogenous and polysaccharide materials by chemical methods or by moving boundary electrophoresis were unsuccessful; ultracentrifugation showed a single peak. Despite these findings, there appeared to be several antigenic species present, as indicated by multiple lines of precipitation in double diffusion and quantitative precipitin tests. In the latter, polysaccharide-containing fractions gave precipitates without measurable carbohydrate, suggesting separability of nitrogenous and polysaccharide components.

Mouse Antibody

Summary It has been shown that peritoneal fluid of mice immunized intraperitoneally with Ea in Freund's adjuvants may contain as much as 2 mg of Ab N/ml. Qualitatively, mouse peritoneal fluid appears to be very similar to mouse serum. Mouse antibody behaves like rabbit antibody in the quantitative precipitin test; insoluble precipitates are formed in extreme antibody excess, and soluble complexes are formed in extreme antigen excess. The anti-Ea antibody, the only one studied, was found only in the γ-globulin portion of the fluid. Various factors affecting the precipitin reaction such as incubation temperature, length of incubation and NaCl concentration have been investigated, and conditions which permit satisfactory and reproducible results have been established.

The immuno-enzymology of muscle proteins. I. General features of myosin and 5′-adenylic acid deaminase

Antisera produced in rabbits against electrophoretically homogeneous chicken skeletal muscle myosin A showed relative homogeneity by immunochemical criteria in only two animals. Sera from eleven other similarly injected animals showed varying degrees of heterogeneity in quantitative precipitin tests and Ouchterlony tests. Some of the antibodies in these latter antisera were directed against the small amounts of 5′-adenylic acid deaminase (AMP-deaminase) normally present in the myosin preparations. Myosin adenosinetriphosphatase (ATPase) activity is not inhibited on combination and precipitation by its specific antibody. Its K m remains unchanged. AMP-deaminase, on the other hand, is completely inhibited. AMP-deaminase can be separated from myosin preparations by means of the antibody prepared against partially purified AMP-deaminase. The antibodies directed against myosin and those directed against AMP-deaminase are species and organ specific. This specificity is at variance with the results obtained with fluorescein-labeled antimyosin. The differences are discussed.

Die Immunologie der Lebermikrosomen

Microsomal suspensions prepared to consist predominantly of fragments of the endoplasmic reticulum are sufficiently stable at neutral pH for use in the Heidelberger Kendall quantitative precipitin test. This test characterizes the microsomes as antigens with several determining groups. The antibody binding capacity of an average microsomal vesicle, 100 mμ in diameter, varies from 20 to 200 antibody molecules according to different antisera and different microsomal preparations. The presence of tissue specific antigens in the microsomal fraction is unequivocally demonstrable by the quantitative precipitin test. Tissue specific and non tissue specific microsomes can to a certain extend be precipitated independently.

Preparation and Immunologic Properties of Trifidin A, an Antigen from Giant Ragweed Pollen

Summary A material, Trifidin A, has been isolated from giant ragweed pollen. It is chromatographically and electrophoretically homogenous. Quantitative precipitin, Ouchterlony and Oudin tests have been used to establish that Trifidin A is a single antigen. It is shown that Trifidin A has skin reactive properties and is active physiologically on sensitive individuals.

Proteins of green leaves VIII. The distribution of fraction I protein in the plant kingdom as detected by precipitin and ultracentrifugal analyses

1. 1. Rabbit antisera against Fraction I protein from Nicotiana debneyi were prepared. 2. 2. Quantitative precipitin tests with the γ-globulin fraction of the pooled antisera showed no overlap between the regions of antigen and antibody excess, which was taken as evidence for the serological homogeneity of the reaction. 3. 3. A series of tobacco-cytoplasm samples from which Fraction I had been partly removed by means of ultracentrifugation in a separation cell was prepared. A positive correlation was shown to exist between the removal of Fraction I and the change in the antigen-dilution end-points of the samples. 4. 4. Cytoplasmic preparations from plants represnnting the major divisions of the plant kingdom were tested for the presence of Fraction I protein by means of analytical ultracentrifugation and precipitin analyses. Agreement between the two methods was good. 5. 5. The presence of Fraction I was shown in representative plants of the major divisions of the plant kingdom.

Über die Rezeptorsubstanz für den Phagen T 5

Quantitative precipitin tests indicate the absence of serological differences between the receptor substance for the bacteriophage T5 and its inactive analog from B/1,5. The implications of this result are discussed. The serological system employed here has several advantages allowing an analysis of successive events taking place in a precipitin reaction.

Studies on the O Antigen of Salmonella Typhosa

Summary Immunological studies have demonstrated that when a somatic antigen of the “Boivin” type is freed of protein, the resultant lipopolysaccharide exhibits no loss in antigenic activity. Indeed antigenic activity in mice, rabbits, and man is increased considerably. This product approached immunological homogeneity in the quantitative precipitin test more closely than the “Boivin” type antigen, as evidenced by an improved supernatant analysis and the formation of soluble antigen-antibody complexes in the region of far antigen excess. The essentially complete agreement of agglutinin nitrogen values with total precipitable N values for horse and rabbit antisera, together with evidence that serological specificity was entirely comparable to that of intact typhoid bacilli, all provide evidence that the immunologically active form of the somatic O antigen in S. typhosa is the lipopolysaccharide.

Studies on Vi Antigen

Summary Vi antigens derived from three V form enteric species by a uniform isolation procedure were examined in a variety of immunological tests. The antigens derived from E. coli and P. ballerup were significantly more active than the Vi antigen obtained from S. typhosa in hemagglutination, quantitative complement fixation, and quantitative precipitin tests. Antigenic activity measured in terms of stimulation of antibody production in rabbits and active immunization of mice against challenge with S. typhosa revealed that coli Vi antigen was the most potent, followed by Vi antigen from ballerup, while the Vi antigen isolated from S. typhosa was the least effective. These differences were established most clearly in the mouse protection tests. The relationship of these biological activities to specific viscosity of the antigen preparations and the possibility of this being a causal relationship was considered. Attention was drawn to certain similarities between the biological properties of the Vi antigens and the pneumococcal polysaccharides.

A Preliminary Report on the Purification of Foot-and-Mouth Disease Virus by Serological Methods

Summary Repeated injections of a fraction from normal bovine tongue epithelium into rabbits elicited the production of precipitins. It was observed that most of the infectivity of purified virus preparations was also precipitated by the antiserum to normal bovine tissue. Quantitative precipitin tests indicated that at least 90% of the most highly purified virus preparation tested consisted of material serologically indistinguishable from normal host tissue. Virus produced in guinea pigs did not react with the antiserum. Although mixtures of normal bovine tissue antigen and guinea pig virus gave comparable serological precipitates, most of the infectivity of the guinea pig virus remained in the supernatant serum. The results suggest that foot-and-mouth disease virus may possess certain serological characteristics of the host in which it was produced.

Precipitation and Inhibition of Carboxypeptidase by Specific Antisera

SUMMARY 1. Crystalline bovine carboxypeptidase was used as an antigen in ob- taining rabbit antisera which contained precipitating antibodies. Studies showed that the system contained only a single antibody and a single antigen as judged by quantitative precipitin tests and by observations with the agar diffusion method. 2. The antibodies interacted slowly with the enzyme to produce a non- competitive inhibition since the extent of inhibition was not influenced by substrate concentration. Moreover, competitive inhibitors of carboxy- peptidase of low molecular weight had no influence on the antigen-antibody interaction. 3. At high temperatures (25-39.5’), the inhibition is described by the equation for an interaction of 1 molecule of antibody for each active site of enzyme. At 6.4”, the data are described by an equation where the enzymatic site reacts with 2 molecules of antibody.

Studies on Vi Antigen

Summary A study was made of the immunological properties of the Vi antigen obtained from Escherichia coli 5396/38 by a NaCl-ethanol fractionation procedure. The purified Vi antigen could be absorbed on collodion particles or on erythrocytes which then became specifically agglutinable by Vi antisera. It possessed excellent serological activity and specificity in these tests and also in the quantitative complement-fixation and quantitative precipitin tests. In the range of antibody excess the Vi antigen precipitated in a uniform manner the Vi antibody in coli, ballerup, typhoid and purified Vi-antisera. Supernatant fluid analysis in the quantitative precipitin test with E. coli antiserum indicated that the purified antigen approached but did not quite meet this criterion of immunological homogeneity. The antigen was markedly effective in the protection of mice against challenge with virulent strains of Salmonella typhosa. It was antigenic for mice, rabbits and man, evoking the production of antibody reactive with both Vi antigen and with Vi strains of S. typhosa. The antibody produced in rabbits possessed mouse protective potency and in vitro activity essentially comparable to the Vi antibody obtained by immunization with Vi strains of S. typhosa, although certain differences in these activities were pointed out. The antigen was as effective as V form S. typhosa in the removal of mouse protective potency from various Vi antisera.

Studies on Immunization Against Plague

Summary A water soluble carbohydrate-protein immunizing antigen, Fraction IA isolated from Pasteurella pestis, was studied for its electrophoretic and serological homogeneity. Determination of the 50% immunizing dose of Fraction IA in mice showed that Fraction IA was a potent immunizing agent against infection with P. pestis. Twelve to 22 micrograms of the antigen were sufficient to protect 50% of the mice against 100 MLD of a highly virulent strain of the organism. The antigen preparations were electrophoretically inhomogeneous. Quantitative precipitin reactions showed that although the Fraction IA preparations were homogeneous on the basis of the analysis of the supernates, a serologically active minor component could be detected by absorption studies. However, the portion of the curves of the reaction for the zones of antibody excess and equivalence are believed to be the results essentially of the precipitation of Fraction IA and its antibody. The lack of complete inhibition of precipitation in the extreme antigen excess zone in the Fraction IA system was probably due, in part, to the serological activity of the minor component. However, removal of this component resulted in an increased but still incomplete inhibition of precipitation. This suggests that the carbohydrate moiety may be serologically active and may act in a manner similar to that of the pneumococcus polysaccharide. It is believed that the quantitative precipitin test, using a standard serum, can be used to assay preparations of P. pestis antigen by determining the amount of antigen required to bring the system to equivalence.

The Use of Chicken Antiserum in the Immunochemical Studies of Edestin

Summary The immunochemical relationships of edestin and its homologous chicken antiserum using 8% NaCl concentration throughout the system are reported. No significant difference was found between the amount of immune precipitate obtained with chicken anti-rabbit globulin serum incubated at either refrigerator or room temperature. On freezing and thawing chicken antiserum, a non-specific precipitate is obtained in which some antibody is lost. Ten per cent NaCl concentration appears to be slightly better than either 8% or 12% NaCl to carry out the quantitative precipitin test with the edestin-anti-edestin chicken serum system. The significance of the immunochemical relationships obtained with the edestin-anti-edestin system are discussed.

THE PROTEINS IN UNHEATED CULTURE FILTRATES OF HUMAN TUBERCLE BACILLI

1. Only two serologically different proteins were found in the unheated culture filtrates of both virulent and slightly virulent tubercle bacilli. One of them was the protein which had a sedimentation constant of 3.4 S, and the other was in filtrate fractions with a constant of 2 S. 2. That these proteins were distinct was demonstrated by three methods: quantitative precipitin and precipitin absorption tests with rabbit antisera, skin tests in guinea pigs actively sensitized with the culture filtrate fractions, and skin tests in passively sensitized guinea pigs. 3. A third antigen of unknown nature was found by means of the precipitin tests, but only in certain fractions from the virulent culture filtrate. 4. The protein with the constant of 3.4 S could not be demonstrated serologically in an O.T. made from the same culture filtrate as the unheated preparation from the virulent organism.

PRECIPITIN REACTIONS OF HIGHLY PURIFIED INFLUENZA VIRUSES AND RELATED MATERIALS

Antisera to purified PR8 virus, to purified protein from normal allantoic fluid, and to purified normal mouse lung particles were obtained from hyper-immunized rabbits and used in quantitative precipitin tests employing various purified preparations of influenza virus and related materials as antigens. The results of those tests indicated that the most highly purified preparations of PR8 or of Lee influenza virus obtained from infectious allantoic fluid contain an antigen characteristic of normal allantoic fluid and likewise that highly purified mouse lung PR8 virus contains an antigen characteristic of normal mouse lungs. Since the infectivity of virus preparations which were ultracentrifugally and electrochemically homogeneous was precipitated by the appropriate antisera to normal antigens, it was concluded that the normal antigens constitute a part of the 100 mmicro particles with which influenza virus activity is at present deemed to be associated. It was estimated from quantitative precipitin data that the most highly purified preparations of PR8 and of Lee influenza viruses obtained from infectious allantoic fluid contain at least about 20 and 30 per cent, respectively, of an antigenic structure characteristic of the sedimentable protein of normal allantoic fluid.

Fractionation of a Type 1 Strain of Group “A” Hemolytic Streptococci

Summary Proteic fractions were isolated from a type 1 strain of hemolytic streptococcus by grinding and extracting undried cells in the cold. One of the most active fractions was sub-fractionated by means of sodium chloride. The serological activity of the fractions was determined by quantitative precipitin-tests. Tryptic digestion of streptococcal proteins indicated the serological activity to be in large part due to difficultly digestible or possibly non-proteic components which appear to be major antigens or haptens of the cell. The presence of C-substance in protein and protein-digest fractions was regularly encountered and its importance in the reactivity of fractions toward antisera emphasized.