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Plaque assays for derepressed autoimmunocompetent B lymphoid cells with specificities for two murine erythrocyte surface autoantigens have been developed. Using purified anti-HB and anti-X anti-erytlirocyte autoantibodies, fluid-phase complement fixation reactions were performed to determine the influence of pH and ionic strength on the efficiency of autoantibody-mediated cytolysis of murine erythrocytes. Anti-HB autoantibody exhibited optimal cytolysis of bromelin-treated erythrocytes at pH 7.0, ionic strength 0.15; haemolysis of intact erythrocytes by anti-X autoantibody was observed over a broad range with maximum sensitivity and specificity at pH 6.6, ionic strength 0.14. NZB spleen ceils secreting anti-HB autoantibody were detected in modified direct haemolysis-in-gel assays, were neutralized by soluble HB autoantigen, and appeared to represent derepressed B lymphoid cells of anti-HB type. Anti-X plaque-forming cells, not demonstrable using conventional assays, were detected under conditions corresponding to the pH and ionic strength optima determined for complement-dependent cytolysis using purified autoantibody. NZB spleen cells secreting anti-X autoantibody were detected in indirect assays employing monolayers of erythrocytes, were neutralized by soluble X autoantigen, and appeared to represent derepressed B lymphoid cells of anti-X type. The distribution of observed plaque diameters in both HB and X assays suggested that more than 95% of all cells secreting autoantibody were detected. [ABSTRACT FROM AUTHOR]

An autoantibody reacting with microsomal membranes has been characterized by a distinctive immunofluorescence pattern on proximal renal tubules and hepatocytes. The microsomal nature of the antigen was demonstrated by absorption and quantitative complement fixation studies. These results showed the antibodies to be quite distinct from the mitochondrial antibodies found in primary biliary cirrhosis. Microsomal antibodies have so far been detected in sixteen cases, of whom twelve had liver disorders. These antibodies, although rare, may provide a serological marker for a small proportion of active chronic hepatitis cases differing in several respects from other recognized subgroups in this disease. [ABSTRACT FROM AUTHOR]

Antigenic determinants of Australia antigen (AuAg) may be determined by haemagglutination inhibition with ease and high sensitivity. The reaction depends on the inhibitory effect of the test antigen-positive sample on the agglutination by specific antibody of glutaraldehyde-fixed sheep erythrocytes which have been tanned and coated with AuAg of different specificities. The standard monospecific antibody reactants are directed against one or other of the common antigens, a and Re, or against the subtypic determinants, d,y,w and r. Because it is more sensitive than conventional immunodiffusion or electrosyneresis, and more convenient than radioimmunoassay, haemagglutination inhibition is most suited to large-scale determinations of AuAg subtypes in samples of relatively low antigenic activity. [ABSTRACT FROM AUTHOR]

Thirty-five weak A samples including fourteen A3, eight Ax, seven Aend, three Am and three Am were studied in order to determine their A antigen site density, using an IgG anti-A labelled with 125I. The values obtained ranged between 30,000 A antigen sites for A3 individuals, and 700 sites for the Ae1 red cells. The hierarchy of values observed made it possible to establish a quantitative relationship between the red cell agglutinability of these phenotypes measured under standard conditions, and their antigen site density. [ABSTRACT FROM AUTHOR]

The effects of Ca++ on primary and secondary immune responses to SRBC in vitro was investigated using the Marbrook technique. During the primary immune response three periods could be distinguished: first, a Ca++-independent lag period (0-24 hours after antigenic stimulation); second, a period with an absolute requirement for Ca++ (24-36 hours after antigenic stimulation), which is related to a proliferative phase of antigenically stimulated cells; and third, a period (later than 48 hours and up to 72 hours after antigenic stimulation), which is inhibited by Ca++ and which can be enhanced by removing Ca++ from the medium. This third period is related to the differentiation step(s) leading to antibody-forming cells. During the secondary immune response only a partial inhibition of immune response was observed after removing Ca++ from the medium at the time of antigenic stimulation. Addition of Ca++ to EGTA-containing culture medium at any time relative to the initiation of the secondary immune response enhanced the response, but, in contrast to its effects on a primary immune response, never completely restored it. Removal of Ca++ later than 6 hours after initiation of the response resulted in a decreased inhibition of the immune response and in an increased switch from 19S to 7S antibody-forming cells. This differentiation step was enhanced by removing Ca++ from the medium and was inhibited by Ca++ added to the medium. The results suggest that Ca++ controls the mechanisms involved in the antibody formation by an antagonistic action on cell proliferation and cell differentiation. [ABSTRACT FROM AUTHOR]

Ig levels were determined by radial immunodiffusion in uncomplicated cases of acute hepatitis with or without Australia antigenaemia. Initial sera from Australia antigen negative cases showed a striking elevation in IgM levels when compared to Australia antigen positive cases (6.5 versus 1.9 mg/ml). None of twenty-four Australia antigen positive cases exceeded 3 mg/ml IgM, and only 3/58 Australia antigen negative cases exhibited values below 3 mg/ml. initial sera from Australia antigen positive and Australia antigen negative subjects did not differ in concentration of IgG, IgA, or IgD. Serial determinations of IgG revealed a transient fall in patients with Australia antigen positive hepatitis, and a rise in Australia antigen negative cases. Asymptomatic, Australia antigen positive, Guaymi Indian subjects were compared to matched Australia antigen negative controls from the same indigenous group and no differences in the concentration of IgG, IgM, IgA or IgD were found, although elevations of IgG and IgM were common in both groups. No evidence of abnormal proteins was found when sera were tested by cellulose acetate electrophoresis or by immunoelectrophoresis versus immunoglobulin-specific anti- sera. Ultracentrifugal analysis failed to detect `7S' IgM. [ABSTRACT FROM AUTHOR]

Methods for isolation and purification of precipitating and non-precipitating guinea-pig antibodies are described. The physicochemical properties of γ1 and γ2 non-precipitating antibodies are similar to γ1 and γ2 precipitating ones. Biological properties are also similar excepting the reverse Arthus reaction, which is positive with the precipitating and negative with the non-precipitating antibodies. Bivalence of these antibodies was experimentally demonstrated. Precipitating antibodies Ko do not differ greatly from those obtained with the corresponding non-precipitating ones. The incapacity to precipitates with the antigen may be a consequence of a steric impediment. [ABSTRACT FROM AUTHOR]

A system is described with which an antibody response to soluble chicken ovalbumin can be obtained in dissociated spleen cell cultures from primed mice. This was assayed by determining specific anti-ovalbumin plaque-forming cells (PFC). Only IgG-producing cells (indirect PFC) were seen. The mice received alum-precipitated ovalbumin i.p. and cultures were set up either late in the primary splenic response (23–27 days after injection) or near the peak of the response (10–14 days). With both groups secondary responses were seen in the presence of antigen on the fourth day of culture. In the second group PFC were present in the cultures at a high level initially. The number of PFC de- creased over the first 2 days, the rate of decrease being greater in the presence of antigen. When mice were primed with alum-precipitated ovalbumin together with B. pertussis as adjuvant, responses could not now be obtained in culture. With mice taken late in the primary response (23–27 days), a considerable number of PFC were present in the cultures initially. The number decreased throughout the 4-day period and the decline was accelerated by antigen. With mice taken near the peak of the response (10–14 days) no PFC were seen in culture, even on the first day, in the presence or absence of antigen. The first phenomenon, suppression by antigen, is considered to be of general significance since it may also be seen in cultures from mice receiving no B. pertussis as described. It is suggested that the second phenomenon is due to adverse culture conditions resulting from the white cell mobilization initiated by B. pertussis; after 23–27 days conditions have recovered to a point where suppression but not the secondary response can occur. The suppressive effect of antigen could be related to antigenic competition or tolerance. [ABSTRACT FROM AUTHOR]

The effect of some non-ionic polymers on immunoprecipitin ring formation (in the assay of IgE in serum by simple radial immunodiffusion (SRD)) was investigated. Polymers were either added to the antigen well after the antigen and/or they were included together with the antibodies in the gel. A considerable enhancement of ring width was found when the low molecular weight polyethylene glycol, Carbowax 200, was added to the antigen well following antigen addition. On the other hand, incorporation of Carbowax 200 or other nonionic polymers into the gel before antigen delivery caused a decrease in precipitin ring width. The presence of Carbowax 6000 in the gel improved the visualization of the precipitate although its presence slightly diminished the ring width of the precipitate. [ABSTRACT FROM AUTHOR]

Immunological tolerance was induced by the exposure of mouse spleen cells to mixtures of polymerized flagellin and antibody in vitro for 6 hours, at 37°, prior to thorough washing and in vitro challenge with optimally immunogenic doses of polymerized flagellin. The induction of antibody-mediated tolerance depended critically on the ratio of antigen and antibody to which the cells were initially exposed. Low zone tolerance could be induced with subimmunogenic concentrations of monomeric flagellin, in amounts as low as 2 pg/ml (about 10-14 molar) and the appropriate concentration of antibody (a titre of 10-3). In contrast tolerance could not be induced with subimmunogenic concentrations of polymeric flagellin in the presence of a wide range of antibody doses. There was a remarkable similarity between the tolerant states obtained in vitro with tolerogenic doses of polymeric flagellin, and with immunogenic and subimmunogenic concentrations of flagellin in the presence of antibody, suggesting that the cellular basis of the tolerant state remains the same. An antigen-focusing hypothesis has been proposed to integrate the mechanisms of high zone and antibody-mediated tolerance at the cellular level. [ABSTRACT FROM AUTHOR]

Formation of aggregates in mixtures of antigen and antibody was followed by measurement of the intensity of light scattered at 90° and of the ratio of the intensities of light scattered at 45° and 135° under varied environmental conditions—concentration, antigen/antibody ratio, temperature, concentration of neutral salts, hydrogen ion and organic solutes. We propose that the aggregation of antigen-antibody complexes is accelerated by change in the structure of antibody molecules that occurs on combination with antigen. The effect of solutes on the rate of aggregation may be due to reduction of the association constant of the combination of antigen and antibody or to inhibition of the change in structure of antibody on combination. Acceleration of the early stage of aggregation by increase of temperature is attributed to increased rate of change in the structure of antibody. [ABSTRACT FROM AUTHOR]

Quantification of the parameters in an antigen-antibody fluorescent reaction in which it was possible to vary the amount of the antigen, was carried out in a model system. Formalinized erythrocytes were coated with different amounts of 131I-labelled human IgM. The cells were then exposed to fluorescein-conjugated antiserum to human IgM. The fluorescence intensity of individual cells was measured with a microspectrofluorimeter. As the cells were coated with varying amounts of protein it was possible to investigate the relationship between the quantity of protein antigen and the intensity of fluorescence. [ABSTRACT FROM AUTHOR]

Rabbit C3 has been identified in serum by agarose gel electrophoresis and by immunoelectrophoresis, and it was shown that calcium ions slow its mobility. It was purified by methods previously used for human C3. Characteristic conversion products similar to those in other species were produced after various in vitro treatments. Reproducible changes occurred during sub-anaphylactic antigenic chal- lenges in the rabbit which were reflected in total serum complement activity. Anaphylactic death was associated with profound changes in haemolytic complement and C3. These findings provide a basis for further study in the rabbit during experimental immunological disease. [ABSTRACT FROM AUTHOR]

Antigen-antibody precipitates, prepared either in solution in antibody excess or by cutting precipitin lines from immunoelectrophoretic plates have been shown to bind specifically to the homologous antigen in tissue sections. This finding has been used as the basis of an immunofluorescent technique in which antigen-antibody precipitates are applied to a section and then stained with a fluorescein-conjugated antiglobulin reagent. The technique allows the localization of antigens in tissue even where they are not characterized or purified and where monospecific antisera are not available. [ABSTRACT FROM AUTHOR]

Comparison of iso-precipitating antisera produced in chickens in the laboratories of the three authors has shown that three distinct systems of isoantigens of the blood scrum of chickens exit. Two of these systems are of allotypic antigens present respectively on the IgG and IgM immunoglobulin molecules. They are identified by antibodies raised in response to immunization with bacterial agglutinates. The third system is identified by both ‘natural’ and isoimmune antibodies. The serum component carrying the antigens A, B and O of this system is unknown. The system is described as GA-NS. [ABSTRACT FROM AUTHOR]

Precipitin lines were observed in double diffusion tests between mouse gut tissue homogenates and serum from the same animal. Other tissues failed to produce this result. The line-forming components of the tissue are lipoidal while serum reactants are free of lipid. Purified γ-globulin, IgG, does not react with tissue constituents. An IgM component can be identified as one reactant. Lipo- polysaccharides extracted from E. coli 014, Forssman antigen and Kidd-Friede- wald antigen could not be demonstrated as reactants. It appears that certain tissue components which may be lipoproteins, and lipid free serum components, one of which is IgM, react in a way which resembles immunological interactions but which arc believed not to be so. [ABSTRACT FROM AUTHOR]

Summary. The capacities of BSA and DNP-protein conjugates to evoke reagin formation in rabbits were compared. Reagins to DNP generally appeared earlier and disappeared more rapidly from the circulation than did anti-BSA reagins. Initial formation of reagins proceeded with a logarithmic phase indicating a doubling time of 7-8 hours. Booster antigen injections resulted in some cases in a reagin response after a shorter latent phase than that observed after primary immunization. A secondary reagin response was more readily evoked in rabbits with low titres of agglutinating antibodies than in those with high titres. Anti-DNP reagins were demonstrable in a higher percentage of the injected rabbits than were anti- BSA reagins. The two types of reagins were equally sensitive to heat and 2-mercaptoethanol. A positive correlation between serum levels of anti-DNP but not anti-BSA reagins and agglutinating antibodies was demonstrated. Some evidence that a low antigen dose was more efficient than a high dose in evoking reagin formation was obtained. Treatment of rabbits with 6-mercaptopurine during the 1st week following antigen injection resulted in an increased latent phase and an enhancement of the production of anti-BSA reagins and some suppression of the formation of anti-DNP reagins. [ABSTRACT FROM AUTHOR]

The immunogenic capacity of protein antigens has been compared for the free and peritoneal exudate cell (PEC)-bound forms. The response of OBA mice to BSA provides the reference system, but lysozyme, ovalbumin, HSA and modified BSA were also studied. Uptake is approximately equally efficient in vivo and in vitro. PEG-bound antigen, estimated by radioactivity is far more potent than the free form in inducing primary immunization. The following properties were also found: (i) viable PEG are required; (ii) irradiation of the cell donor 2–7 days before giving antigen inhibits immunization, but irradiation after uptake does not do so; (iii) mice are susceptible to immunization during their phase of recovery from paralysis; (iv) PEG do not retain large amounts of antigen for long; (v) the activity does not depend solely on a minor, phagocytosis- prone fraction of the antigen; (vi) allogeneic transfer of PEG reduces their immunogenic capacity; (vii) paralysed hosts are not susceptible to immunization, but PEG from paralysed donors are effective; and (viii) the enhancement of immunogenic capacity does not apply to the secondary response. The conclusion may be drawn that the retention of small quantities of antigen by macrophages plays an essential role in some, but probably not all types of immune response.

When lymphoid cells from hypersensitive rabbits are incubated with antigen, biologically active substances are formed and released which are capable of inhibiting the migration of normal non-sensitized mesenchymal cells. In the present paper some basic parameters of their production were determined. These substances were regularly obtained after 6 and 18 hours incubation, but not after 2 hours. Under more favourable cultivation conditions (lower density of lymphocyte suspension) an increased activity in the cell extracts as compared with the supernatants was observed. Another critical factor in the production of these substances is the quantity of antigen added. Ten micrograms of PPD leads to the production and liberation of a highly effective substance. A lower dose of antigen results in the liberation of a substance into the supernatant which by itself is almost inactive, but becomes more active when more antigen is added. The efficiency of the released substances was determined by serial dilution. The inhibiting activity was maintained at 1:5 and 1:20 and sometimes at 1:100 dilutions. [ABSTRACT FROM AUTHOR]

A procedure demonstrating the usefulness of combining fluorescence with agar diffusion is presented. A minor obstacle to its use is the means of permanently recording the colours that are obtained. The method described gives the conditions under which photographs can be taken. However, since the degree of fluorescence may vary from specimen to specimen exposure times, f-openings and conditions of lighting must be varied for optimum results. [ABSTRACT FROM AUTHOR]

γM cross-reacting antibodies in group O sera produce mixed agglutination with secretor buccal cells, but not with non-secretor cells. The mixed agglutination with sera which also contain immune anti-B is produced only with A buccal cells and B indicator red cells, and not with B buccal cells, and is inhibited by B secretor saliva or galactose, but not by A secretor saliva. Mixed agglutination with sera containing immune anti-A is produced only with B buccal cells and A indicator red cells, and is inhibited by A secretor saliva or 2-acetamido-2-deoxygalactose (N-acetyl-D-galactosamine), but not by B secretor saliva. If two sera, one containing immune anti-A and the other containing immune anti-B, are mixed together in equal volumes, mixed agglutination is no longer produced with either A buccal cells (and B indicator red cells) or B buccal cells (and A indicator red cells). These observations are thought to be most readily explicable by the hypothesis that the combining site on the cross-reacting antibody is smaller than on specific anti-A or anti-B, and that it reacts with that part of the antigenic determinant which is common to both A and B antigens. As a consequence of the restricted size of the combining site, it is suggested that cross-reacting antibody will have a lower affinity for A or B antigens than specific anti-A or anti-B does, and competes unsuccessfully with specific anti-A or anti-B for combination with antigen on buccal cells. [ABSTRACT FROM AUTHOR]

Clearance of T1 or T2 bacteriophage from the circulation of rabbits takes place in two phases; a rapid first phase and a slower second phase. Secondary stimulation of rabbits with a similar dose of phage to the primary dose results in a more rapid clearance of bacteriophage particles from the circulation. This increased rate of clearance can be demonstrated prior to the production of any detectable humoral antibody. The enhanced secondary clearance appears to be specific for the particle concerned, and does not occur using non-antigenic carbon particles. [ABSTRACT FROM AUTHOR]

Comparative tests were done to find the best conditions under which formalinized sheep red cells can be tanned and then coated with chromatographically purified ovalbumin. Variation in the time taken for tanning or coating, the amount of antigen used, the temperature during coating, the use of different batches of tannic acid, ovalbumin or of cells formalinized by different methods had little effect on sensitivity. to agglutination by antibody. A slight increase in sensitivity was obtained by coating at a low pH, by increasing the quantity of tannic acid employed and by heat denaturation of the antigen. [ABSTRACT FROM AUTHOR]

The studies reported here indicate that tanned red cells coated with a mixture of chromatographically purified ovalbumin and conalbumin give agglutination titres which are directly related to the amount of antibody (as meas- ured by quantitative precipitation) present against either of these antigens in mixed sera. With whole (unabsorbed) mixed sera, these coated cells record the titre of the major antibody present, whether this is anti-ovalbumin or anti-conalbumin. There was no evidence of greater sensitivity to anti-conalbumin. A trace of ovalbumin in a preparation of conalbumin used to coat cells is fully sufficient to enable them to react with any anti-ovalbumin in a serum. Twice recrystallized ovalbumin still contains some conalbumin. Sera raised against it may contain unexpectedly high levels of anti-conalbumin and this could cause confusion in tanned red cell tests. [ABSTRACT FROM AUTHOR]

The radioactively labelled third (C′3) and fourth (C′4) components of human complement were dissociated from erythrocyte membrane-complement complexes. The differential effectiveness of various reagents in causing dissociation suggests that C′3 and C′4 are bound to membrane sites by hydrophobic bonds. Density gradient ultracentrifugation of the dissociated components showed that they are bound in macromolecular form. Their electrophoretic mobility was greater than that of their native counterparts. In physical and immunochemical properties, dissociated C′3 and C′4 closely resembled the respective haemolytically inactive conversion products that, during immune haemolysis, accumulate in the fluid phase. It is concluded that in immune haemolysis C′3 and C′4 become linked to membrane sites only following modification by their respective converting enzymes. [ABSTRACT FROM AUTHOR]

Two random linear co-polymers composed exclusively of D-amino acids were found to elicit a primary response in mice; the sera were assayed by the indirect precipitin reaction. 247, p (D-Tyr, D-Glu, D-Ala) was studied intensively and compared to a similar polypeptide, 253, p (L-Tyr, L-Glu, L-Ala), of op- posite optical configuration. The primary response to the D-isomer in adjuvant is highly dose-dependent, showing a sharp maximum around 1 μg/mouse. while that to the L-isomer in adjuvant is largely dose-independent. At low doses the D-isomer is as immunogenic as the L-isomer. Re-injection of the D-isomer led to no increase in titre; re-injection of the L-isomer gave a typical secondary response. The antibodies formed to both isomers were highly stereospecific. The D-isomer was 50–1000 times as efficient at inducing paralysis as the L-isomer, and the paralysis was specific. The differences between the D- and L-isomers are thought to be explained by the much greater capacity of the D-isomer in inducing immunological paralysis. [ABSTRACT FROM AUTHOR]

The immune response in guinea-pigs to linear and multichain copolymers of two, three or four different amino acids including tyrosine, glutamic acid, alanine and lysine, was studied. Delayed-type response was favoured in the case of preparations of relatively low molecular weight, containing only tyrosine and glutamic acid in their potential antigenic specificity determinants. Delayed sensitivity to one of these preparations was passively transferred with lymphoid cells. Antibody response resulted from the immunization of animals with preparations of relatively high molecular weight and with a more heterogeneous chemical composition. The antibody formation was intensified and accelerated, when p-azobenzenearsonate conjugates of the polypeptides were used as antigens. Variations in the immunizing dose had little or no effect on the nature of the reactions. A response of exclusively delayed type could not be intensified by repeated immunizations. The presence of mycobacteria in the immunizing injection was essential for the elicitation of the response to multichain, but not to linear copolymers. [ABSTRACT FROM AUTHOR]

Antisera to specific proteins in herpes simplex infected cells were produced by immunization of rabbits with infected rabbit kidney cells. These antisera were highly virus specific and produced up to twelve lines in immunodiffusion tests against infected cell extracts. Acrylamide electrophoresis and immunoelectrophoresis revealed up to ten virus specific proteins of varying size. [ABSTRACT FROM AUTHOR]

During the GVH reaction produced in adult (C57Bl ... x CBA ...) F1 hybrid mice by injection of C57Bl cells a profound drop in serum immunoglobulin levels was observed. The fall in IgG and IgA values was not reversed by antigenic stimulation with BSA, but the IgM level rose somewhat after BSA through never to the level seen in immunized controls. The antibody response to BSA, as measured by passive haemagglutination, was grossly depressed in experimental animals. Animals undergoing the GVH reaction showed less local inflammatory response at the site of Freund's adjuvant injection than did controls. The possible importance of this immunoglobulin deficiency in the clinical presentation of the GVH syndrome is considered. [ABSTRACT FROM AUTHOR]

Immunization of HSA-tolerant animals with sulphanil-HSA induces the formation of antibodies that react with native HSA. To test whether the anti-HSA response does actually represent complete restoration of the original reactivity, the antibodies produced following immunization of tolerant animals with sulphanil-HSAS were compared to those produced by normal rabbits immunized with the native antigen. Gel-diffusion analysis of the precipitins disclosed that the patterns of the two types of antibody were not completely identical. The antibodies of the 'restored' tolerant rabbits were directed mainly towards an antigenic determinant of the native HSA, to which normal rabbits immunized with HSA did not respond. When the 'restored' rabbits were challenged with native HSA after a prolonged rest period, the antibodies formed thereafter were completely identical with anti-HSA produced by normal HSA-immunized rabbits. Thus, complete restoration of the original immunological reactivity to HSA was achieved following an intermediary stage of atypical anti-HSA elicited by the conjugated protein. Attempts to break down natural tolerance to RSA by treatment with sulphanil-RSA failed to give any evidence of an autoimmune elimination of RSA. To test whether this inability to break tolerance to RSA was due to the presence of an excess of native protein, animals that had been made tolerant to HSA were immunized with a mixture of sulphanil-HSA and native HSA. Under such conditions, the termination of acquired tolerance was inhibited. The possible relevance of these observations to the cellular basis of immunological tolerance is discussed. [ABSTRACT FROM AUTHOR]

Specific changes in the soluble antigens (exoantigen) of Trypanosoma brucei have been studied in relation to the process of antigenic variation Such antigens, prepared from variant substrains of trypanosomes, were compared immunologically by agglutination, precipitin and mouse protection tests. Direct and absorption tests demonstrated that specific antibodies were produced to the exoantigen of each variant population and that the immunogenicity of the antigenic components as infection proceeds. [ABSTRACT FROM AUTHOR]

K+, Cs+, Mg++ and Ca++ all reduce the velocity of the precipitin reaction relative to that observed in the presence of equal concentrations of Na+. The nature of the anion in the medium affects the velocity even more strongly, the velocity being greatest in the presence of F- and least in the presence of I-. The inhibitory power of anions runs in parallel with their position in the lyotropic series. The polyhydroxy compounds sucrose, glucose, fructose, mannitol meso-inositol and glycerol also reduce the velocity of the precipitin reaction at concentrations of 0.3 M and higher. They also decrease slightly the amount of material that is eventually precipitated. Urea inhibits in a manner similar to the polyhydroxy compounds, but simple amides such as formamide, N-methyl-formamide and N,N-dimethyl-formamide are much more powerful inhibitors, so that inhibition can be demonstrated at concentrations as low as 0.02 M. An attempt is made to explain these effects in terms of known facts about the combination and precipitation of antigens and antibodies. [ABSTRACT FROM AUTHOR]

A mathematically defined extension of Landsteiner's concept of serological specificity is given. It is a general method that compares tile relative specificity of two antisera. The resultant comparison is independent of the relative potencies of the antisera. It is valid for antisera prepared either against pure antigens or against mixtures of antigens. The method has been applied to data taken from the literature, involving haemagglutination, precipitin and enzyme reactions, and to new experimental data. Rabbit antisera were prepared against mouse erythrocytes and lymphocytes, and against three mouse ascites tumours. The potencies of these unabsorbed antisera were determined by immune cytolysis against each of these cell types. All antisera possessed moderate to strong cytolytic potencies against all mouse cells tested, but showed only low to moderate specificities. [ABSTRACT FROM AUTHOR]

The peptide chains (A and B) isolated from the 7S γ-globulin of a single donor known to be Gm(a+ b + ) and Inv(a + ), were tested for the presence of allotypic specificities. Gm factors were present only on the A chain and the Inv factor was found only on the B chain. Ten pathological B chains, of which five were antigenic type I and five type II, were studied; all were negative for Inv(a), but Inv(b) specificity appeared to be associated with B chains of both antigenic types. [ABSTRACT FROM AUTHOR]

Rabbits made immunologically unresponsive by neonatal administration of HSA, HGG or BSA were given a course of intravenous injections of the respective antigens, adsorbed on alum, after a lapse of 13-27 months since the last administration of antigen. 8/12 responded to HSA, 4/5 to HGG, 9/10 to BSA, as judged by immune elimination of antigen, but this was delayed in onset and slow compared with that in previously untreated rabbits. The antibody formed was small in quantity and usually failed to precipitate with antigen. The sedimentation coefficients of 131I-labelled antigens, in the presence of excess antibody, were measured by ultracentrifugation through a sucrose density gradient. These showed that only small complexes were formed in some of the non-precipitating antisera. In one instance the diffusion coefficient of the complex was also measured, by a technique based on diffusion through agar gel. The calculated molecular weight of the complex, 330,000 indicated the presence of only two combining sites on the antigen. Combination of the anti-HSA sera with an HSA fragment was also measured. Whereas the amount of the fragment bound by ordinary hyperimmune anti-HSA sera was about one-fifth the HSA bound, the amounts bound by the test sera were relatively much less. Some non-precipitating sera failed to bind the fragment, although they bound HSA. These findings indicate that following neonatally induced immunological unresponsiveness the capacity to respond to antigen returns piecemeal in respect of different parts of the antigenic mosaic, and that it may be severely restricted. The theoretical implications are discussed. [ABSTRACT FROM AUTHOR]

The velocity of precipitin reactions has been measured by following the increase of turbidity of a mixture of antibody and antigen in a spectrophotometer at selected wavelengths from 360 to 720 mμ. For constant amounts of antibody and antigen the measured turbidities are greater at shorter wavelengths. The reaction proceeds fastest in antibody excess and slowest in antigen excess. A lag in the development of turbidity is evident in the equivalence and antigen excess zones. The reaction is speeded up by working in smaller reaction volumes, and by hypotonic concentrations of sodium chloride. Hypertonic concentrations of sodium chloride slow the reaction and cause a lag to become apparent in the antibody excess zone. These effects on the velocity of the reaction are much more pronounced than the effects on the amount of material precipitated. 3,5-Di-iodotyrosine slows down the reaction between heavily iodinated bovine albumin and its antibodies. [ABSTRACT FROM AUTHOR]

7S and 19S components of rabbit anti-A antibody, labelled with 131 have been separated by chromatography on DEAE-cellulose. The reactions of these components with human group A1, A2 and AB red cells have been investigated. At saturation point A1 cells combine with 0.22 μg. of 7S antibody per million cells, this corresponds to a minimum of 8.3 × 105 A-antigen sites per cell, A2 cells can take up only one quarter as much antibody, and combine much less avidly with it than do A1 cells. Cells cam take up only about one-fifth as many 19S as 7S antibody molecules. 19S antibody is more avid and, on a molecular basis, is 750 times more efficient at agglutinating red cells than 7S antibody. The significance of these findings is discussed with regard to the differences between A1 and A2 antigens, and the valence and mode of attachment to the red cells of the antibodies. [ABSTRACT FROM AUTHOR]

Rabbit antisera against mouse cells have been absorbed with normal mouse serum and used to detect mouse cell-bound antigens. Mouse ascites tumor cells and their products have mainly been used as source of antigens but those described here are normal mouse components. Seven antigens have been found by agar diffusion precipitin methods and data are given to show that each is a separate molecular entity. Information is provided for the identification of each component. [ABSTRACT FROM AUTHOR]

Immunologically immature and mature Arbor Acre White Rock fowls were splenectomized and precipitin production after a single inoculation of bovine serum albumin was assayed. Fowls splenectomized at 4, 9 or 17 weeks of age were intravenously injected at 6, 12, 20 weeks; a subcutaneous or intravenous inoculation was given to 12-week-old birds splenectomized at 8 weeks. The splenectomized animals showed a delayed antibody appearance and a later day of peak titre. Only the animals which were splenectomized at 17 weeks showed a depression. These results suggest that a greater number of immunologically competent cells are present in the spleen and the non-splenic tissue of young animals only can compensate for the removal of this organ. [ABSTRACT FROM AUTHOR]

Passive cutaneous anaphylaxis in the guinea pig has been studied with rabbit antibody to a series of antigens of differing molecular weight. The results indicated that at a given antibody level the weight of antigen needed to elicit a reaction increases with its molecular weight. Previous observations have been confirmed that the amount of antigen needed to elicit a reaction at a high level of antibody is less than that required at a lower level. The results suggest that extremely small amounts of small molecular weight antigens might be sufficient to produce anaphylactic symptoms in highly sensitive individuals. [ABSTRACT FROM AUTHOR]

A quantitative immunochemical method has been developed suitable for titration of antigens and antibodies in complex systems. This method can be used for antigens migrating towards the anode in agar-gel electrophoresis only and for the corresponding antibodies in case that their mobility equals to that of gamma globulin. A number of bacterial toxins or toxoids complies with this condition. The reaction takes place in agar gel. The reacting components are transported into the reaction arena by means of electric field; identification of individual components is facilitated by the fact that each precipitating antigen-antibody system is characterized by the electrophoretic mobility of antigen. This method makes possible the quantitative determination of a number of antigenic components in crude diphtheria toxin and tetanus toxoid. In suitable conditions this method makes also possible the titration of individual determinant antigen groups or, on the contrary, their corresponding antibodies. The results achieved indicate the usefulness of this method for the study of the immunochemical structure of toxins. It is possible, by using a suitably modified method, to titrate some non-precipitating systems. [ABSTRACT FROM AUTHOR]

Particulars are given of immunization procedures carried out in rabbits in an unsuccessful attempt to produce lupus factor. Experiments on the substrate specificity and inhibition of the lupus reaction are described. From the results it is inferred that the antigenic determinants involved in the lupus reaction lie in the ‘backbone’ configuration of native nucleo-proteins, both nucleohistone and most probably nucleoprotamine. [ABSTRACT FROM AUTHOR]

Antibodies to human serum albumin differ from one antiserum to another in the degree of firmness and reversibility of combination with the antigen. These differences are reflected in different behaviours during electrophoresis of soluble antigen-antibody compounds. Molecules of each kind of antibody seem also able to combine with the antigen in more than one way. [ABSTRACT FROM AUTHOR]

Preliminary experiment on the mixed agglutination reaction suggests that this reaction will afford a useful method for identifying the species of origin of cells maintained in culture. The reaction depends on the presence of antigens characteristic of the species, common to both tissue cells and red cells. Culture cells derived from man, ox, pig and rat could be distinguished one from the other. Fibroblasts of the mouse may be differentiated from those of the rat by means of a rat anti-mouse red-cell serum or a mouse anti-rat red-cell serum. Experiments are reported on trial absorption procedures to render the sera completely species-specific in their reactions. [ABSTRACT FROM AUTHOR]

A sensitive indirect haemagglutination technique is described which depends upon attachment of the protein antigen to the erythrocyte with difluorodinitrobenzene. The attachment of human gamma globulin to human erythrocytes has been thoroughly studied using sera containing the Rose-Waaler factor as the agglutinator. It has also been shown that horse and rabbit serum proteins and egg white may be attached to cells by the reagent. The technique is simple and sensitive but an absorption step is necessary to remove a non-specific agglutinin to cells which have been treated with the reagent. [ABSTRACT FROM AUTHOR]

A state of acquired immunological tolerance to bovine gamma globulin (BGG) has been observed in CBA mice injected with 10 mg. BGG within 12 hours of birth. Tolerance was demonstrated by a lack of immune elimination of 131I labelled BGG (BGG-131I) after prior challenge with Freund's adjuvant containing BGG. The degree of tolerance was found to diminish when the time between the tolerance injection and the challenge injection with adjuvant was increased. Mice were found to be tolerant 4 months after injection of 10 mg. BGG at birth but other similarly treated mice were found to be partially immune when challenged 6 months after the tolerance injection. This diminution of tolerance could be prevented by injections of antigen into adult mice whilst they were still tolerant. Precipitin and haemagglutination tests showed that antibody to BGG was present in mice shown to be tolerant to BGG by the antigen-elimination technique. The Ouchterlony double-diffusion technique showed that the mice were tolerant to one fraction of BGG but immune to at least one other fraction. The tolerated fraction of BGG was identified by means' of starch-gel electrophoresis. [ABSTRACT FROM AUTHOR]

Human chorionic gonadotrophin (HCG) is antigenic in rabbits when injected with Freund's adjuvant. The HCG used for immunization showed the presence of five antigens in the Ouchterlony plates against the homologous antiserum. Three of these antigens were common to human serum and one of these three to normal human urine. Rabbit antiserum to HCG absorbed with human serum did not form any antigen-antibody precipitin line with either normal human serum or normal human urine. It formed two precipitin lines with HCG. One ml. of the rabbit antiserum to HCG could inhibit even 150 I.U. of HCG as tested by the Aschheim-Zondek test in mice and ovarian hyperaemia test in rats. The absorbed antiserum could inhibit hormonal activity of HCG even when the antiserum and HCG were injected simultaneously at separate sites. [ABSTRACT FROM AUTHOR]

Rabbit spleen cells, which have been treated in vitro with certain rabbit or guinea-pig antisera (e.g. against human serum albumin) and washed, are capable of specifically adsorbing radio-isotope labelled antigen. The antibody responsible for this effect appears to be distinct from the main precipitating anti-body in the serum and the term ‘cytophilic antibody’ has been suggested. The activity of the cytophilic antibody is not destroyed by heating at 56° C. for half an hour. The cytophilic antibody is not adsorbed by red cells. Spleen cells which have been treated with methanol before treatment with antiserum take up less antigen than non-methanol-treated cells tested in the same way. However, the addition of flesh normal spleen cells to the methanol-antibody-treated cells before the addition of antigen increased the uptake of the latter. [ABSTRACT FROM AUTHOR]

An in vitro assay procedure for evaluating the antibody response to glomerular basement membranes, lung basement membranes and tendon fibrils indicated a sharing of identical or similar antigenic components. At least 70 per cent of the glomerular basement membrane-binding antibodies are absorbable by tendon fibrils, and eluates from such tendon preparations are not nephrotoxic. Within the species employed (man, rabbit and dog) for glomerular basement membrane sources, there appears to be no species specificity as regards the presence of the nephrotoxic component. Immunization of sheep with either canine or rabbit glomerular basement membrane leads to the elaboration of a haemagglutinin for the respective species. [ABSTRACT FROM AUTHOR]

The distribution of antigen and antibody in radial double diffusion systems was studied by means of materials labelled with 131I or 14C. These studies showed that, once a precipitate begins to form, the assumption that antigen and antibody obey the laws of free diffusion is invalid. They also showed that, in the systems used, no antigen and very little antibody diffused past the zone of visible precipitation. It was found that accurate estimates of the diffusion constants of antigens could be obtained by allowing antigen and antibody to diffuse from troughs set at right angles and by measuring the angle of the precipitin line. Examples of the use of this method, and a theoretical treatment are given. An alternative method for estimating the size of antigens is to use the ‘molecular sieve’ properties of gelatin gels, which are sharply graded with the concentration of the gel. [ABSTRACT FROM AUTHOR]