Non-Specific Complement Fixation Research Articles

Adherence inhibition assay: a specific serological test for detection of antibodies to Mycoplasma pneumoniae.

Antibodies directed against the adherence-mediating protein of Mycoplasma pneumoniae were measured by an adherence inhibition assay. Pretreatment with antibody-containing sera reduced the attachment of sheep erythrocytes to Mycoplasma pneumoniae layers grown in flat-bottom microtiter plates. The degree of attachment of erythrocytes was estimated by lysis with distilled water and measurement of absorbance in a microtiter reader. Sera tested included 126 sera from patients with suspected Mycoplasma pneumoniae respiratory infection and 60 sera from patients with serologically confirmed respiratory infection of other origin. Examination of human sera by both complement fixation using glycolipid as antigen, and the adherence inhibition assay indicated a high degree of specificity of the latter. Furthermore, titer increases parallel to complement fixation titers were found in six paired sera, and testing of separated IgM showed high reactivity in this immunoglobulin fraction. The adherence inhibition assay is a reproducible method which is relatively easy to perform. It may be of importance especially when a non-specific complement fixation reaction due to cross-reaction is suspected.

Significance of Thromboplastic Activity of Antigens Used in Complement-Fixation Tests

ConclusionsThese results give further evidence of the correlation between clotting and complement activity and indicate that antigens possessing thromboplastic activity may give nonspecific complement fixation.

A NATURAL ANTIBODY THAT REACTS IN VITRO WITH A SEDIMENTABLE CONSTITUENT OF NORMAL TISSUE CELLS

Continued serological investigations of the sedimentable constituents of normal and neoplastic tissues have shown that the blood serum of normal rabbits will fix complement in mixture with saline extracts of normal rabbit tissues. The phenomenon has proved referable, not to anticomplementary effects of serum or antigen nor to so called non-specific complement fixation, but to a naturally occurring serum principle, hitherto unrecognized, which reacts specifically in vitro with a sedimentable constituent of normal tissue cells. The principle exists in the blood of nearly all adult rabbits but is absent from that of rabbits less than 1 month old. It can be salted out from serum with ammonium sulfate and is destroyed when heated at 65 degrees C. for 20 to 30 minutes. Its titer was found to run parallel in general with that of two natural antibodies also present in normal rabbit's blood (natural Wassermann reagin, natural anti-sheep hemolysin); but absorption tests showed it to be distinct from these. Because of its properties, the serum principle has been termed the natural tissue antibody. The substance with which the natural tissue antibody reacts is regularly present in saline extracts of many normal tissues,-those of rabbits and of other species as well. Kidney and liver tissues always yield it in abundance, while spleen, brain, and testicle provide somewhat less; heart and voluntary muscle extracts contain relatively little, and non-nucleated erythrocytes and skin are practically devoid of it. The results of affinity and absorption tests indicate that it is nearly or quite the same from whatever tissue or species derived. It is readily sedimentable in the high-speed centrifuge, little or none remaining in the supernatant liquid of potent suspensions spun at 25,000 R.P.M. (45,400 g) for 1 hour. It either does not come away into alcohol or is inactivated thereby, is readily destroyed by heat (56-70 degrees C. for 30 minutes), and diminishes notably in antigenic potency upon standing overnight in saline suspension or when the tissues containing it are kept in glycerol. Its properties suggest that it may be a protein. The implications of the findings are discussed in relation to the formation of the natural antibody and its place amongst serological phenomena, to so called "non-specific" fixation of complement and other serological complexities, and with particular reference to the character of the sedimentable constituents of normal and neoplastic tissue cells.

The Kahn Reaction with Serum of Different Animals

While making routine Kahn tests for the diagnosis of syphilis, I had occasion from time to time to examine serum from different animals, including cattle, sheep, hogs and horses. It was amazing to note that many of the serums give positive reactions. Nonspecific complementfixation reactions with serum from various animals have been reported by many workers.1 The Wassermann test especially has been subjected to extensive study. I am not familiar, however, with any report of

On the Mechanism of True and of Non-Specific Complement Fixation Reactions

The complement fixation test for tuberculosis gives a non-specific or falsely positive reaction if a balanced physiological saline, akin to Tyrode's solution, replaces the plain 0.9% NaCl used in the test. The balanced saline which gives the strongest non-specific reaction contains 0.888% NaCl, 0.009% CaCl2, and 0.003% MgCl2; both Ca and Mg salts are required, and it is only over a very limited range of concentration of these salts that the non-specific reaction occurs. Petroff's1 whole bacillus antigen is more effective than any other examined in producing this non-specific reaction, while “fat-free ”tuberculo-antigens do not give this non-specific effect. In the absence of antigen, i. e., in the anticomplementary control tubes, balanced saline assists hemolysis, so that one-third of the normal unit of complement may produce complete hemolysis.The mechanism of this falsely positive reaction is as follows:1. The surface properties of the (Petroff) antigen particles are modified when incubated with complem...

ON THE MECHANISM OF COMPLEMENT FIXATION

As previously described, a falsely positive or non-specific complement fixation reaction can be produced with certain tuberculo-antigens; the mechanism of the reaction is described in the present article. Examination of the antigen in the non-specific reaction shows that it can assume the same complement fixing power, cohesiveness and oil-wettability as antigen sensitized by immune serum. This indicates that fixation of complement is not a specific immune reaction, but only a secondary effect due to the surface properties of suitably modified antigens.

A Study of the Thermolabil and Thermostabil Antilysins (Anticomplementary Substances) of Human Serum

Summary and Conclusions Anticomplementary substances (antilysins) in human sera may be divided into two kinds: (a) those removed by heating at 56°C. (thermolabil antilysin) and (b) those not removed by heating (thermostabil antilysin). Human sera do not develop antilysins as a result of heating, as may occur with rabbit, dog and mule sera. Steril sera develop thermolabil but not thermostabil antilysins. Steril sera kept at 37°C. may develop these thermolabil antilysins in from three to seven days; at room and lower temperatures (0 to 2°C.), longer periods are required. Fresh sera rapidly dried in filter paper do not develop anticomplementary properties. Human sera containing various bacteria and particularly staphylococci rapidly develop thermolabil and thermostabil antilysins. Staphylococci alone rather than their products elaborated during cultivation in broth and serum, produce the antilytic effects of contaminated serum. Large amounts of hemoglobin in salt solution and serum, steril and contaminated, exert anticomplementary activities before and after heating. Traces of hemoglobin are usually free of these antilytic effects. Steril and contaminated sera containing antilysins gradually become alkaline in reaction; neutralization of this alkali with hydrochloric acid does not remove the antilytic activities of the sera. Definite changes in the hydrogen ion concentration of steril and contaminated human serum before and after the development of antilysins, could not be determined with colorimetric methods. Steril human sera containing thermolabil antilysin showed a slight increase of total protein and especially of the globulin fraction, as determined by the refractometric method of Robertson; contaminated sera containing thermolabil and thermostabil antilysins showed a marked increase of total protein and especially of the albumin fraction, probably due in part to the presence of bacteria. Anticomplementary sera passed through a Kitasato filter which removes the antilysins, showed a reduction in total protein and especially of the globulin fraction. Heating anticomplementary sera at 56°C. for thirty minutes, which reduced the content of antilysins, had no appreciable influence upon the protein constituents. The removal of ether soluble lipoids from anticomplementary human sera did not remove the thermolabil and thermostabil antilysins. The antilysins of human sera are closely allied with the protein constituents and especially the globulin fraction. Absorption of human sera with barium sulphate tends to remove a portion of thermolabil and thermostabil antilysins; kaolin, bone ash and charcoal also remove antilysins, but to a lesser degree. Absorption of human sera with washed erythrocytes does not remove thermolabil or thermostabil antilysins as may occur with the thermostabil antilysins of dog serum; erythrocytes exposed to the antilysins of human serum do not acquire an increased resistance to serum hemolysis. Filtration of diluted human sera through new, chemically clean and steril Katasato filters removes all thermolabil and thermostabil antilysin; likewise the filtration of diluted heated dog serum removes the substances responsible for non-specific complement fixation reactions. Filtration of the sera of luetic persons has practically no influence upon the antibody concerned in the Wassermann reaction. Filtration of freshly prepared bacterial antigens removes the antilysins; filtrations of antigens for the Wassermann test as alcoholic extracts of beef heart reinforced with cholesterin diluted 1:20 with saline solution, does not remove the antilysin but completely removes the antigenic substance. I beg to express my appreciation of the kindness of Professor Kolmer for outlining the experiments and technic of this investigation and for his supervision of the work; also to Dr. Charles Weiss, for aid in conducting the hydrogen ion determinations, and to Dr. Hatai and Dr. Toyama for aid in the refractometric work.

Studies in Non-Specific Complement Fixation. I. Non-Specific Complement Fixation by Normal Rabbit Serum

The object of this communication is to direct attention to the observation that in complement-fixation tests when normal rabbit serum is used, according to the usual technic, in an active condition or after heating at 55 C. for half an hour for the purpose of inactivation, nonspecific complement absorption or fixation may occur, not only in the presence of lipoidal antigens, but with various bacterial antigens as well. This is a matter of considerable importance in view of the fact that rabbits are commonly employed for experimental studies in syphilis, and likewise for the preparation of immune amboceptors for the study and differentiation of bacterial species and proteins in general. This possibility of non-specific complement fixation is to be remembered and reckoned with in complement-fixation work with rabbit serum, especially in experimental syphilis, as otherwise erroneous conclusions may be drawn, if based alone upon complementfixation tests.

Studies in Non-Specific Complement Fixation. II. Non-Specific Complement Fixation by Normal Dog Serum

Studies in Non-Specific Complement Fixation. II. Non-Specific Complement Fixation by Normal Dog Serum

Studies in Non-Specific Complement Fixation. III. The Influence of Splenectomy and Anesthetics on the Non-Specific Complement Fixation Sometimes Shown by Normal Rabbit and Dog Sera

This investigation was undertaken in the hope of obtaining some understanding of the part played by the spleen in hemolysis and of the mechanism by which the red cells develop an increased resistance to hemolytic agents after splenectomy. Antischkow1 has shown that the feeding of rabbits with cholesterin leads to an increased deposition of anisotropic fats in the spleen. Eppinger2 and King3 have found that dog's blood after splenectomy shows an increase in total fats and cholesterin and a decrease in unsaturated fatty acids. These observations naturally suggest that if the spleen has such an important influence on fat and lipoid metabolism, this mechanism may in some way be concerned in the changes which the spleen effects in the red cells. That the change in the red cells which is responsible for their increased resistance to hemolytic agents after splenectomy is a characteristic of the cells themselves and not a change in the serum, is indicated by the work of Karsner and Pearce,4 tho it must be admitted in view of King's results that it may be an antihemolytic power of the serum, dependent upon an increase in cholesterin content. As stated in the first paper5 of this series, several investigators have noted the occurrence in presumably normal rabbits of positive complement fixation with lipoidal antigens. The reason for this is not known, but we have evidence at hand that lipoidal substances

Studies in Non-Specific Complement Fixation. V. The Effect of Heat on Normal Rabbit and Dog Sera in Relation to Antilytic and Non-Specific Complement-Fixation Reactions

Having considered in previous articles1 the property of normal rabbit and dog sera of yielding non-specific complement-fixation with various lipoidal and bacterial antigens, and the influence of anesthetics and splenectomy2 upon this property, and the relation of serum lipoids to the process,3 in the present article we shall study further the nature and mechanism of the process, and the influence of certain factors upon it, and we shall consider methods for lessening or abolishing its effects in complement-fixation tests with these sera for specific immune bodies.

Studies in Non-Specific Complement Fixation. IV. The Relation of Serum Lipoids and Proteins to Non-Specific Complement Fixation with Normal Rabbit and Dog Sera

Since ether and chloroform during anesthesia have been found to influence the substances in normal rabbit and dog sera responsible for absorbing complement in a non-specific manner,1 probably through some influence on the serum lipoids, we have continued this investigation to determine more definitely the relation of these lipoids to the process by extractions of serum with lipoid solvents, as ether and chloroform, and by feeding and immunizing experiments with various lipoids.