Precipitin System Research Articles

An immunologic precipitin system between soluble nucleoprotein and serum antibody in systemic lupus erythematosus.

Double diffusion in agarose was employed for the characterization of a soluble nucleoprotein antigen that gave precipitin reactions with sera from patients with systemic lupus erythematosus. The soluble antigen that was isolated from calf thymus nuclei was demonstrated by enzyme degradation and ultracentrifugation studies, and by immunologic analysis, to be a complex of deoxyribonucleic acid and a moiety susceptible to proteolytic digestion. Antibody to soluble nucleoprotein did not react with the DNA portion of the complex released after proteolytic digestion or with purified calf thymus DNA. Immunologic reaction of identity was obtained between the soluble nucleoprotein antigen and synthesized DNA-histone complex, suggesting that the protein moiety of soluble nucleoprotein might in part be composed of histone. Antibody to soluble nucleoprotein was present in 51% of systemic lupus erythematosus sera examined and was more common than antibody to deoxyribonucleic acid.

Immunoelectrophoretic analysis of serum proteins in agar and in agarose.

SummarySeveral antigen-antibody precipitin systems were compared by immuno-electrophoretic analysis using agar and agarose as the supporting medium. Electroendosmosis was reduced in agarose, mobility of components was more rapid, lateral separation of alpha and beta globulin components was increased and better definition of individual precipitin arcs in this medium was observed. Some precipitin reactions which failed to develop in agar gave clear reactions in agarose. The reverse was never observed. There were some individual antigen-antibody reactions in which there appeared to be little change in either the shape or position of the precipitin arcs on either medium, but increased clarity was always observed on the agarose medium. The advantages of agarose for I.E.A. when increased sensitivity is required, as in quantitating antigens or in testing for homogeneity of purified proteins, should be considered.

A DIRECT IMMUNOLOGIC ASSAY OF HUMAN SERA FOR BENCE JONES PROTEINS (L-CHAINS).

Abstract Rabbit antiserum to the L-polypeptide chains of pooled human normal γ 2 -globulin has specificity for antigenic sites which are relatively inaccessible in intact γ-globulin as well as for exposed determinants of the L-chain. Absorption of such antiserum with normal γ 2 -globulin yields a reagent which detects Bence Jones proteins in the presence of other serum globulins. This absorbed antiserum is apparently specific for the core determinants of the two major BJ groups rather than specific for any antigenic subgroup. By means of a double diffusion precipitin system in agar, such antiserum can detect a concentration of 0.02 mg. per milliliter of Bence Jones protein of both major antigenic groups. Absorption of this antiserum with individual BJ proteins provides a group-specific reagent. Application of this antiserum in a study of normal human sera and normal casual urines fails to detect the small amounts of BJ proteins normally present. Fifteen of 17 sera from patients with multiple myeloma and macroglobulinemia reveal detectable amounts of BJ proteins with elements of both groups present in several cases. A similarly high incidence of Bence Jones proteinuria is recorded.

A Turbidimetric Analysis of a Hemocyanin-Antihemocyanin Precipitin Reaction

relative concentrations of the antigens and antibodies which are present. The rate at which the precipitate forms is likewise dependent upon proportions of the principal reagents, and also upon other factors such as avidity of the antibodies, the size of the antigenic molecule, and temperature. Combinations between antigens and antibodies occur in a matter of seconds, even at 0?C (1,2). Precipitation, the visible stage of the serological reaction, also may occur in a matter of seconds or, depending upon the conditions involved, may require from several hours to several days. Boyd (3) studied the times of flocculation of mixtures of various dilutions of the hemocyanin of Limulus and various dilutions of antiserum. Other workers (4, 5, 6) have made analyses of the amount of composition of the precipitates of reactions which have been permitted to go to completion. Martin (17) made an extensive study of the development of turbidities in precipitin reactions obtained with Pneumococcus Type I SSS. His analysis included the entire range of combining proportions of antigen and antibody. His observations were made at room temperatures and at times ranging from 10 seconds after the mixing of reagents to 5 minutes of reaction-time. Lanni (7) made turbidimetric and microscopic analyses of the precipitates formed in ovalbumin and pneumococcus polysacharride precipitin systems, individually, and after they had been mixed together. He graphically recorded the amount and rates of precipitation in selected dilutions of his reagents; there were curves for the regions of antigen-excess, optimal-proportions, and antibody-excess. His recording-times began at 20 seconds following the mixing of reagents and continued, at frequent intervals, up to 30 minutes of reaction-time. This paper presents a detailed, turbidimetric analysis of a hemocyanin-antihemocyanin system for reaction-times ranging between 20 seconds and 7 days at room temperatures. The rate of precipitation and amoun' of precipitate are recorded for the entire range of antigen

Detection of certain serologic imbalances in human sera by agar columns.

Abstract : The detection of normal and abnormal human sera by quantitative analyses of agar column diffusion patterns has been confirmed and amplified. Baseline precipitin patterns from 29 sera, assumed to be normal, were compared with diffusion spectra of 131 sera from hospitalized subjects. The same fast- moving faint zone, previously observed and believed to be associated with an increased concentration of mucoprotein, was evident. Diffusion data on this precipitin system, together with the data associated with albumin and globulins, were analyzed in order to detect certain serologic imbalances. These analyses showed serologic imbalances in hospitalized subjects in 36 percent more cases than was shown by the usual clinical C-reactive protein test. The results paralleled the physician's estimate of the stress produced by the particular disease state or condition.

The Agar Diffusion Precipitin Technique: A Comparison of the Simple and Double Diffusion Methods

Summary The simple and double diffusion methods were studied experimentally by means of specially designed plates. The 2 methods were compared with respect to their sensitivity, reaction range, reaction time, resolving power and susceptibility to concentration and temperature artifacts. The following observations were made: The potential sensitivity of the double diffusion method is somewhat greater than that of the simple diffusion method.The reaction range of the simple diffusion method is unlimited in the antigen excess zone but is restricted to only a narrow segment of the antibody excess zone. The reaction range of the double diffusion method is unlimited in both directions.The effective reaction time, i.e., the time interval during which a precipitate band persists in the column, is the same for both methods at intermediate or high concentrations of antigen and antibody, but at relatively low concentrations of the reactants the effective reaction time of the simple diffusion method is shorter than that of the double diffusion method.The resolving power of the double diffusion method for mixed precipitin systems is considerably better than that of the simple diffusion method.The sensitivity of the simple diffusion method to concentration and temperature changes is much greater than the sensitivity of the double diffusion method. Under average laboratory conditions temperature artifacts in double diffusion columns or plates have never been observed, but special precautions must be taken to prevent such artifacts in simple diffusion columns.

Interpretation of the Ouchterlony Precipitin Test

Summary Under ordinary test conditions specific precipitates in the agar plate do not interfere with the free diffusion of unrelated antigens and antibodies. Identical antigens always give rise to fusion of their specific precipitates. Unrelated antigens may produce complete intersection, partial intersection or fusion of their specific precipitates, depending on the relative concentration of the two precipitin systems. Comparison of several heated, formalized and urea-treated proteins with untreated controls resulted in complete fusion of their specific precipitates. Further studies are desirable in order to elucidate the precipitate patterns yielded by cross-reacting immune systems.

Precipitin Production in Chickens

Summary The effects on the chicken precipitin system of a Hofmeister series of sodium salts were investigated. With any particular ionic species there were differences in the amounts of specific precipitation on varying the ionic strength, and at any one ionic strength there were differences in varying the ionic species. Certain characteristic patterns emerged. At low ionic strength (up to 0.26) precipitation was larger in the iodide medium than in any of the other ionic media. On increasing the ionic strength above 0.26 precipitation in the iodide medium decreased. This inhibition of the reaction was even more striking in the thiocyanate medium. These results suggested that a certain amount of anion binding by the antibody-antigen reactants increased precipitation but that greater amounts of binding decreased precipitation. With anions such as chloride and sulfate precipitation increased with each increase of ionic strength. In fact the maximal precipitation for the chicken system in the various sodium salts occurred at high ionic strengths (about 1.5) of chloride and sulfate. These results in turn suggested that a salting out of soluble antibody-antigen aggregates is an important condition for obtaining complete precipitation with the chicken system. Additional data which also demonstrated the operation of a salting out effect was obtained by reacting the chicken precipitin system in potassium phosphate buffers of varied concentration.

Antigens in Scarlatinal Erythrogenic Toxin Demonstrable by the Oudin Technic

Summary Dick toxin contains a mixture of antigens for which specific antibodies exist in the sera of horses immunized for the production of scarlatinal antitoxin. At least eight distinct precipitin systems are represented in a mixture of commercial antitoxin and standard erythrogenic toxin employed in this laboratory. Several methods of identifying individual precipitin systems, based on the Oudin technic, have been examined: Specific adsorption of one antibody from the serum gives the most unequivocal results. Replacement of the antigen layer on agar in which zones have been established is a reliable method, but produces some unexplained effects. Agreement between the slopes of lines representing the relation between concentration of antigen and rate of migration should be used only as supportive evidence when dealing with ill-defined preparations. At least one antigen present in Dick toxin, antigen A, is derived from the heart-infusion broth used in toxin production. The erythrogenic toxin proper is represented by the fourth zone (D) in tubes containing our standard toxin and antitoxin.