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Soluble extracts of rat lymphoid cells were characterized in order to determine the physical relationship between the major histocompatibility (Ag-B) antigen and species or xenoantigen. Antigenic determinants recognized by rabbit anti-rat antiserum were separated from those recognized by alloantiserum by polyacrylamide gel electrophoresis (PAGE) and selective immunoabsorption. Thus, this species antigen and major H antigen are present as separate molecules on the membrane of rat lymphoid cells. [ABSTRACT FROM AUTHOR]

Spent culture media of all the human cell lines tested have been found to contain the antigenic activity present on the ll,000-Dalton HL-A common portion fragment of the HL-A antigen molecule that appears to be a characteristic, invariant portion of HL-A antigen molecules. From the culture medium of one of these lines, RPMI 1788, a lymphoid cell line, the substance carrying HL-A common activity was isolated, which was shown to be identical to the HL-A common portion fragment with respect to molecular size, electrophoretic mobility, isoelectric focusing patterns, and certain antigenic characteristics. By an isolation procedure involving differential ultrafiltration, gel filtration, and column electrophoresis, 8 litres of the culture medium yielded 1.5-2.0 A280 units of the substance representing 15-20 per cent of the HL-A common antigenic activity originally present. A single protein band with a Rf of 0.47 was obtained by disc electrophoresis. The molecular size was shown to be about 11,000 Daltons by gel filtration and by sodium dodecyl sulphate acrylamide gel electrophoresis. Upon isoelectric focusing two bands were obtained which corresponded exactly to those obtained with HL-A common portion fragment prepared from papain-solubilized HL-A antigen preparations by acid dissociation. The isoelectric point of the major band was 5.0. The reactions of this substance with rabbit antisera against human lymphoid cell membrane and against the substance were essentially identical to the reactions of HL-A common portion fragment with these same antisera. [ABSTRACT FROM AUTHOR]

The presence in each antiserum of different antibody components, with different binding affinities, quantitatively affects the outcome of an inhibition reaction. A detailed analysis permits the recognition of the following conditions. (a) At high antigen concentrations, the antiserum is approaching saturation; the only effect of adding unlabelled antigen is competition with the labelled antigen for the available antibody sites. (b) At intermediate antigen concentrations, only a fraction of the antibody components, those of higher affinity, are contributing to the binding reaction; in addition to competition, unlabelled antigen produces an increase in the number of reacting antibody sites. The resultant of these two counteracting effects can be predicted according to the slope of a simple linear function. (c) Lastly, at low antigen concentrations, the added antigen does not have any significant inhibitory effect. As a result of this analysis, practical indications are available for selecting antisera for radioimmunoassays on the basis of their avidity parameters, for choosing the most suitable antigen range for each antiserum, and for interpreting the shape of inhibition curves in terms of the avidity and heterogeneity of the antiserum. [ABSTRACT FROM AUTHOR]

Evidence is presented to substantiate an earlier report that macrophages are the operative cells in the initiation of lymphocyte trapping. Several procedures which increase phagocytosis in mice of a soluble antigen (BGG), including transfer of specific serum or of adherent and non-adherent immune cells, or the transfer of in vitro sensitized macrophages, enhance the trapping potential of BGG. Animals tolerant to BGG trapped effectively when challenged with aggregated BGG, or when passively immunized and challenged with soluble BGG. The trapping defect in tolerant animals is thus due to a specific defect in immune responsiveness rather than an inherent failure of the trap mechanism. The dissociation between trapping and immune responsiveness is discussed. [ABSTRACT FROM AUTHOR]

Injection of C57B1 mice with endotoxin or other bacterial antigens normally produces (i) a rise in the serum level of a factor that stimulates granulocyte and macrophage colony formation in vitro and (ii) a rise in the numbers of granulocyte and macrophage progenitors in the blood. Preinjection of the mice with polymerized flagellins of Salmonella species specifically depressed both t)'pes of response. After a single injection of fiagellin, depressed responsiveness began within 24 hr, persisted for several months and could be passively transferred to normal mice by injection of immune serum. Responsiveness was partially restored by 850 R whole body irradiation. [ABSTRACT FROM AUTHOR]

Determinations of antibody titres with the ammonium sulphate precipitation technique showed an accuracy of ± 10 per cent (P<005). Calculation of antibody avidities with Sips' distribution formula, using suitably diluted anti- sera, gave a ten times greater accuracy than either undiluted or too highly diluted antisera. The accuracy was lower with the formula of Celada, Schmidt and Strom (1969) probably due to the narrow antigen ranges employed. Comparison of avidity calculation according to Sips (1948) and Celada et al. (1969) showed a good correlation (r = 0.73-0.79; P.<001). No correlation between antibody titre and antibody avidity values in the sera (r = 0.03-0.19) was observed. [ABSTRACT FROM AUTHOR]

Previous reports have shown that liposomal model membranes are appropriate objects for investigating the molecular basis for complement-dependent membrane damage. The present study describes a different application for liposomes: their usefulness in screening sera suspected of containing antibodies against potential lipid antigens. Liposomes, actively sensitized with untreated or alkali-treated lipid A derived from Salmonella Minnesota, were found to release trapped glucose marker when incubated with a source of unheated complement and the anti-lipid A serum whose preparation is described in the preceding paper [1]. In the presence of complements, the anti-lipid A serum could also promote loss of glucose from liposomes which had been actively sensitized with various untreated and alkali-treated S or R from S. minnesota lipopolysaccharides. In contrast, several different anti-R-form sera had little effect on liposomes sensitized with either untreated or alkali-treated lipid A. These results are in agreement with observations on the immunological specificity of lipid-A antibodies using complement-dependent hemolysis of passively sensitized human erythrocytes as test system. Alkali-treated, bu not untreated, lipid A could also be employed for passive sensitization of liposomes; in this regard, lipid A behaves similarly to S and R form lipopolysaccharides. [ABSTRACT FROM AUTHOR]

From E. coli 0100:K?(B):H2 a lipopolysaccharide and an acidic polysaccharide were extracted with saline. The lipopolysaccharide was purified by repeated ultracentrifugation and the acidic polysaccharide by fractional precipitation with cetavlon. Both substances contained rhamnose, galactose, glucosamine, glycerol, and phosphate. The lipopolysaccharide was degraded in 1% acetic acid at 100° and the degraded polysaccharide was fractionated on Sephadex G-50. Thus a fraction was obtained which represents the specific polysaccharide moiety of the lipopolysaccharide. comparison of this fraction with the acidic polysaccharide (chemical analysis, periodate oxidation, alkaline hydrolysis, partial acid hydrolysis) showed that both substances have not only the same composition (rhamnose, galactose, glucosamine, glycerol and phosphate in the molar ratios of 2:1:1:1:1) but also the same structure. Both substances consist of a main chain (composed of rhamnose, galactose and glucosamine) to which glycerol is bound via a phosphodiester bridge from one of its primary hydroxyl groups to one of the rhamnose residues. Some alternatives for the structure are presented. In serological tests (passive haemagglutination, bacterial agglutination, immune precipitation and inhibition of these) it was shown that acidic polysaccharide and lipopolysaccharide have identical reactivities in anti-O and anti-OK antisera. This confirms the structural identity of the polysaccharides. It is concluded that E. coli 0100:K?(B):H2 contains no polysaccharide which represents a K antigen. The O antigen of this strain contains glycerol phosphate as (part of) a determinant group. Precipitating antibodies, directed against this group are present in anti-OK antiserum and not in anti-O antiserum. In this context the phenomenon of O inagglutinability of E. coli atrains, which carry K antigens, is discussed. Possible implications in the definition of K antigens of the B type in human enteropathogenic E. coli strains are presented. [ABSTRACT FROM AUTHOR]

An antigen that can be derived from rabbit kidney can be identified by use of suitable rabbit antisera as being non-tissue specific in addition to being an autoantigen, since it had been found to be distributed in kidney, heart, adrenal and liver. The purification of this autoantigen has been accomplished by successive use of four different methods. These were the salting out with ammonium sulphate, gel filtration on Sephadex G-200, dialysis against distilled water, and chromatography on DEAE-cellulose. The heterogeneity of the products derived from the procedure at each step was analysed by means of analytical electrophoresis and ultracentrifugation, and also by gel diffusion precipitation. The final product was a homogeneous component, according to the criteria that were used. It had a sedimentation coefficient of 4.1S, suggesting a mol. wt of approximately 60,000. This autoantigen is of similar molecular size to several others that are, in contrast, adrenal specific or kidney specific. [ABSTRACT FROM AUTHOR]

The antigenic components of adrenal extract were fractionated by chromatography on DEAE-cellulose. Using a stepwise elution procedure, it was found that a component of major interest, termed Pk3b, was eluted by a buffer of pH 6.70 and 0.09 M in phosphate-chloride. This antigenic preparation reacted with several rabbit antisera that had been prepared by isoimmunization with rabbit adrenal homogenate, in contrast to several other fractions, which reacted with only one (or two) of the antisera. Pk3b also showed a high degree of homogeneity, as shown by immunodiffusion, paper electrophoresis, and analytical ultracentrifugation. By the latter technique, an extrapolated sedimentation coefficient of 4.45S was determined. The further comparison of this antigen in immunodiffusion, along with an antiserum and the adrenal extract from the antiserum-providing animal, confirmed again that this is an autoantigen, and that it is one of five autoantigens of the adrenal gland. [ABSTRACT FROM AUTHOR]

Antigenic determinants recognizable by rabbits were found to be present on the molecular fragments (48,000 Daltons) which were obtained by papain-solubilization of the membrane fractions of cultured human lymphoid cells and which carried the HL-A determinants. Results were obtained which suggest that these antigenic determinants are present in common on these molecular fragments carrying HL-A determinants regardless of their HL-A specificity and are restricted to the molecular fragments which carry HL-A determinants. The study was made by use of radioimmune methods involving the binding of radioiodine-labelled soluble HL-A antigen preparations by anti-HL-A alloanti- sera and by rabbit antisera raised against the membrane fractions of cultured human lymphoid cells. [ABSTRACT FROM AUTHOR]

Three different types of antigenic determinants were demonstrated in soluble collagen with the aid of rat, rabbit and chicken antisera to native collagen. Helical antigenic determinants which require an intact triple-helical structure of the molecule are mainly recognized by rat antisera. Renaturation of the serologically inactive unfolded polypeptide chains (denatured collagen) is accompanied by a significant recovery of serological activity. Central antigenic determinants which are probably located in the same regions of amino acid sequence are less accessible in the native antigen and become exposed upon denaturation. Equal titres for both types of determinants are found in chicken antisera. Immunization with denatured collagen, however, revealed a response restricted to the central type. Isolated antibodies specific for terminal non-helical antigenic determinants, as yet only known to occur in rabbit antisera, reacted equally well with native collagen, the unfolded polypeptide chains and with small cyanogen bromide peptides. Independence of conformation is therefore suggested for these antigenic structures. [ABSTRACT FROM AUTHOR]

A soluble histocompatibility antigen was extracted from normal rat lymphoid cells by exposure to an hypertonic KCl solution, The antigen was assayed by inhibition of monospecific cytotoxic (CT) alloantiserum and shown to have the specificity AgB 4 or Rt H1.1, the major locus private antigen of the dark agouti strain. Yields were ∼2000 CT inhibitory units (ID50) per 109 cells. Gel filtration of the extract over Sephadex G-200 resulted in an approximate four-fold purification with a final concentration of 530 ID50 units per mg protein. The major portion of the antigen appeared at the front of the inner bed volume but a minor included peak at about 65,000 mol wt. was also seen. Yields and specificity were greatly increased by (a) not washing the cell suspension before extraction (b) removing excess salt from the extract by dialysis rather than gel filtration (c) removing insoluble nucleic acids from the dialysed extract by ultracentrifugation. [ABSTRACT FROM AUTHOR]

Ram SS (supernatant fraction of spermatozoa ground with Baker's fluid) and SP (seminal plasma) were analysed by double diffusion and immunoelectrophoresis in agar using six different rabbit antisera. Immunoelectrophoretic patterns of SS from ten rams show frequencies of band subpatterns which indicate polymorphism of spermatozoan antigens. Also, bands from different rams with the same diffusion characteristics but different electrophoretic mobility and others with different antigenic determinants confirm protein polymorphism. SS and SP of three rams from Blackface, Dorset and Merino breeds were examined by agar diffusion and immunoelectrophoresis at pH 5 and 8.2. Five to six spermatozoan antigens were distinguished from 4-5 SP antigens. When similar antigens from two rams were further tested, it was possible to show some difference in electrophoretic mobility or antigenic determinant. [ABSTRACT FROM AUTHOR]

Cross-reactions between immunoglobulins from twelve mammalian species were observed by immunoelectrophoresis with antisera prepared in rabbits against the purified IgG of each species. The Fc fragments of the IgG from man, pig, goat, sheep and cow, were compared by examination of the cross-reactions before and after absorption of the antisera with the various IgG. Eight antigenic determinants could be detected; some of them were species-specific, others were shared by IgG from two or more species. A close resemblance was found between goat, sheep and cow IgG. [ABSTRACT FROM AUTHOR]

Complexes of Haemophilus influenzae cells with and without intact capsular material were prepared with type specific antisera. Thin sections of positively stained specimens were prepared and examined in the electron microscope. In the absence of capsular material the antibodies were observed bound close to one another in the immediate area of the bacterial cell wall. When capsular antigen was present the antibodies were farther separated from one another in the complex and appeared to be attached at random sites along the capsular polymers with occasional individual particles being observed. This positive staining approach appears to be a useful method to observe the molecular structure of precipitated immune complexes. [ABSTRACT FROM AUTHOR]

A radioimmunoassay for the quantitative determination of the immobilization antigens of Paramoecium is described. The use of this technique in the identification of closely related antigens is discussed in terms of antigen 'profiles', derived from precipitin tests with several antisera. [ABSTRACT FROM AUTHOR]

The secretory immunoglobulin of the guinea-pig, IgA, was antigenically unique when compared with the 7Sγ1-, 7Sγ2- and γM-globulins, although it did share Fab determinants in common with the 7S&gamma2 globulin. Specific antigen binding capacity was detected in serum IgA after sequential oral and parenteral sensitization with purified protein antigens. IgA was prominent in saliva and succus entericus; in colostrum its concentration was 4–8 times that in normal serum. IgA in serum and secretory fluids sedimented at similar rates (≈12S), although colostral IgA contained an additional ≈16S population. Serum and secretory IgA appeared antigenically identical, however, serum IgA was especially sensitive to mild reductive procedures and became ≈7S after treatment. Serum IgA migrated as a β-protein on electrophoresis and was faster than IgA of colostrum. Antisera to IgA were produced by a procedure which involved simple precipitation of secretory IgA with an antiserum containing antibodies to common determinants of guinea-pig Ig. [ABSTRACT FROM AUTHOR]

Antigenic competition between the Fc and Fab fragments of rabbit IgG in mice could be abolished by passive immunization with antiserum against the dominant antigen (Fc). The induction of tolerance to Fc also eliminated antigenic competition. Anti-Fd production, generally very poor in response to rabbit lgG, was considerably enhanced by these procedures, indicating that it is also subject to antigenic competition with Fc. A model is proposed to explain antigenic competition which proposes that it is the 'co-operative antibody' produced to the dominant antigen which acts as an inhibitor to antibody production to the suppressed antigen by competing for sites —presumably on macrophage membranes—where cooperation occurs. The merits and difficulties of such an explanation are discussed. [ABSTRACT FROM AUTHOR]

The titres of complement fixing antibodies in the sera of rats injected with the soluble fraction of rat brain emulsified in Freunds' complete adjuvant (FCA) were usually below 10. In contrast, injections of the nuclear, mitochondrial or microsomal fractions of rat brain in FCA were followed by the appearances of heat stable complement fixing and haemagglutinating antibodies within a few days, the highest antibody levels being attained about 6 days after the first injection of a particulate fraction. The microsomal fraction was the most efficient particulate antigen. After 2 weeks of immunization about 60 per cent of the complement fixing activity of the antisera was due to 19S antibody and the remainder to 7S antibody. The response to injections of the nuclear and microsomal fractions of rat liver followed a similar time course but produced levels of complement fixing antibodies that were consistently lower than those engendered by the corresponding brain antigens. [ABSTRACT FROM AUTHOR]

An in vitro assay for macrophage cytophilic antibody is described, based on the measurement of the adherence 125I-labelled antigen to peritoneal exudate cells pretreated with antiserum. Using this assay, the production of cytophilic antibody to SRBC, BSA, polymerized flagellin, flagellin and a CNBr digest of flagellin was investigated in guinea-pigs, rats, rabbits and mice. Antigens injected in either Freund's complete or incomplete adjuvant were almost equally efficient at inducing cytophilic antibody production. In contrast, antigens injected in saline failed to induce detectable cytophilic antibody. The ratio of cytophilic antibody titre/passive haemagglutinating antibody titre was calculated and it was found that the ratio for rabbit >guinea-pig >mouse > rat. Furthermore, in those sera which were examined, cytophilic antibody was only detected in the 7S fractions. [ABSTRACT FROM AUTHOR]

Ox red cell antigens F, V, Ε1, J, L, R', S' and T' have been recovered in soluble membrane protein obtained by a butanol extraction method. F, V and Ε1 have been studied in more detail and are found in the sialic acid rich fraction of the membrane protein. Isoimmunization with protein containing F and V antigens produced excellent specific antisera for these antigens. Antigens lost in butanol extraction were not destroyed by sonication, and it has not been possible to regenerate activity for these lost antigens. [ABSTRACT FROM AUTHOR]

The precipitation band produced by hepatocytes from freshlydispersed adult rat liver against liver-specific antiserum in agar gel immunodiffusion plates is still exhibited after the cells have been maintained in organized culture on Millipore membranes for 10 days. [ABSTRACT FROM AUTHOR]

Rabbit antisera to mouse spleen cells and to thymocytes have been analysed by microcomplement fixation and double gel diffusion. The antisera fixed complement more efficiently with the native particulate fraction than with the soluble fraction of mouse spleen cells. Structural antigens could be solubilized partly with hypertonic salt solutions. The solubilization of structural antigens could be shown by a decrease of the antigenic activity of the particulate fraction after the extraction and a concomitant appearance of the antigenic activity in the solubilized fractions. The solubilized fractions were able to induce antibodies to structural antigens, but antibodies to cell sap antigens were also induced to a lesser extent. No gel precipitation could be observed between the tested antisera and the solubilized fractions. [ABSTRACT FROM AUTHOR]

Using radioactivity assays and radioautography, it has been confirmed that the majority of the guinea-pig anti-BSA cytophilic antibodies are found in the γ2 IgG fraction. The cytophilic activity was directly demonstrated to be present in the Fc portion of the IgG molecule. Furthermore, the Fc portion quantitatively accounts for the cytophilic activity of the whole IgG molecule. [ABSTRACT FROM AUTHOR]

The enhancing effect of dextran on the precipitability of various antigens with immune sera obtained from different stages of immunization was studied. The greatest effect was observed with antisera from early stages of immunization which in many cases gave no or weak immunoprecipitates in the absence of water soluble non-ionic polymer dextran, using the conventional double diffusion technique. [ABSTRACT FROM AUTHOR]

The preparation is described of an antiserum specific for the locally produced immunoglobulin in whey from mammary glands of ewes infused with antigen about 1 month before lambing. In immunoelectrophoresis, the antiserum failed to react or reacted weakly with serum and with colostral whey from non-infused glands of primiparous ewes but reacted strongly with a protein of β-electrophoretic mobility in the colostrum and milk from infused glands. The majority of the antibody in whey from antigen-infused glands was eluted from DEAE Sephadex by buffer of relatively high ionic strength in association with the antigenically stimulated protein. Antibody activity in these fractions was heat stable. The antigenically stimulated protein was shown to be a carbohydrate-rich immunoglobulin distinct from IgG1, IgG2 and IgM. It is concluded that it is analogous to IgA of other species. Comparison of the antigens in serum and whey reactive with the specific antiserum gave no evidence for the existence of an additional antigenic component associated with the IgA in whey. IgA was readily detected in whey from noninfused glands of older multiparous ewes, in the contents of the small intestine but not in concentrated mixed saliva. It is suggested that antigenic stimulation of mammary glands and probably some other mucous surfaces of ewes awakens an almost dormant local system of antibody production analogous to the IgA system in other species. [ABSTRACT FROM AUTHOR]

Bursal and thymic antigenic specificities have been shown in the chicken in an immune adherence assay using cross-absorbed rabbit antisera. [ABSTRACT FROM AUTHOR]

A simple test system is described, for the demonstration of antigen-antibody reactions capable of causing eosinophil granule lysis in vitro. The antigen preparations used were extracts of the nematode Amplicaecum robertsi and body fluid of Ascaris suum. Antisera were obtained from rats infested with Amplicaecum. Eosinophils were obtained from the peritoneal cavity of normal rats. Centrifugation of the cells to form a cell button was an essential step in the procedure. Lysis of eosinophils occurred with antiserum obtained from the animals between the 12th and 32nd days of infestation with Amplicaecum, and was accompanied by vacuole formation in macrophages and mast cell disruption. The reaction was most pronounced during the 3rd week. Serum from adrenalectomized infested animals caused the most marked changes in eosinophils. Serum from cortisone- treated infested animals failed to cause eosinophil changes. Attempts at purification of the antigen in Ascaris body fluid resulted in two fractions with marked activity in the test system. The same two fractions were found to form precipitin lines on agarose gel diffusion against rat antiserum. It is postulated that antigen-antibody complexes soluble in low concentration were responsible for the changes observed in the eosinophils, macrophages and mast cells. One or more labile factors in the serum were found to be necessary for eosinophil granule lysis. The evidence, though incomplete, would favour the suggestion that both labile antibody and complement were necessary. [ABSTRACT FROM AUTHOR]

A quantitative method for determining specific antibody in the different classes of immunoglobulins is described. The technique is based on the binding of all antibody present in an immune serum with antigen, and the sub- sequent precipitation of the bound immunoglobulins with a heterologous antiserum specific for the antigen employed. The concentration of each immunoglobulin class is determined by means of single radial immunodiffusion, before and after removal of its antibody-active portion. The accuracy of the measurements is essentially that of the immunodiffusion technique, and the whole analysis, applied to four immunoglobulin classes, requires only 0·06 ml of serum. The applicability of the technique has been tested using sera from mice immunized with ferritin by different routes. Antibody activity was predominant in the IgG1 and IgG2 immunoglobulin classes. However, measurable anti-ferritin activity in the IgM and IgA classes was also found in a limited number of sera. [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]

Rabbit and chicken anti-human thrombocyte sera absorbed exhaustively with human plasma, fibrinogen, erythrocytes, leucocytes and liver powder, were used for the investigation of organ specific antigens of human thrombocytes. The absorbed chicken anti-human thrombocyte serum revealed a very high agglutinating and fluorescence activity, which aided the characterization of two different types of organ-specific antigens of thrombocytes; antigens common to human and animal platelets, and antigens specific for human platelets only. The two types of antigens were found in all platelet fractions examined, the membrane, granule and soluble fractions. [ABSTRACT FROM AUTHOR]

Summary. An agar gel radial diffusion technique has been devised to examine the amount of IgG and relative amounts of As 4 and As 5 in the sera of twenty heterozygous As/4,5 rabbits immunized with Type III pneumococci. Serial absorption of the sera with the polysaccharide SSS III antigen before reaction with anti-IgG and anti-allotypic sera was used to show how much specific lgG antibody was present in each serum and the distribution of this antibody between molecules bearing one or other or neither of the allotypic determinants. The results indicate that anti-SSS III antibody generally occurred as As 4 rather than As 5 molecules although in some animals this preference was reversed. Non-allotypic (b-minus) molecules contributed significantly to the antibody response. [ABSTRACT FROM AUTHOR]

Antisera against components of guinea-pig complement were raised in rabbits by: (1) Using as antigen a suspension of killed Proteus species bacteria which had been allowed to combine with their homologous antiserum in the presence of guinea-pig complement in optimum proportions. (2) By injection of the β1C component of guinea-pig complement adsorbed to zymosan particles. The antisera raised by the two methods contained antibodies mainly against the β1C component of complement. When coupled with FJTC both antisera were found useful in detecting sites of complement fixation. The fluorescence anti-complement technique was found four times more sensitive than the indirect method for detecting antigen—antibody reactions in the presence of diminishing concentrations of antigen. It was only twice as sensitive for detecting antigen—antibody reactions in the presence of diminishing concentrations of antibody. The comparisons were based upon both visual assessment and photometric measurement. Coupled antisera raised by the first method gave brighter specific fluorescence than antisera raised by the second method when used in the highest concentration which did not give non-specific staining. The usefulness for detection of viral antigens of sera raised by both methods is discussed.

The smallest amount of purified antibody capable of positive weal and erythema response is of the order of 4 μg N. Antigen-antibody complexes are capable of producing specific weal and erythema reactions with antigen—antibody ratio of 1:1 or above. In these instances antibodies occupied at one or both sites are responsible for this response. Pre-formed antigen–antibody complexes are capable of mediating this response.

Antisera specific for the H antigens (flagella) of Salmonella typhi and Salmonella typhimurium have been raised in rabbits with protein extracts of acetonedried bacteria from which O antigens, inevitably present in such extracts, have been removed by precipitation with specific anti-O sera. The same procedure with Salmonella enteritidis proved unsuccessful. [ABSTRACT FROM AUTHOR]

Slowly sedimenting components with a sedimentation velocity of about 7S and reacting with specific antisera against IgM were found in six pathological human sera. Four of these came from patients with different forms of reactive macroglobulinaemia of infectious origin. Similar components were produced from purified IgM preparations by bacterial action, and to a lesser extent by spontaneous splitting at room temperature. The conditions for such a splitting were, however, absent for most of the sera investigated in this study. The possibility that slowly sedimenting IgM-like components are artefacts, is discussed. [ABSTRACT FROM AUTHOR]

Antisera prepared in rabbits against frog oviducal homogenate produce a minimum of six to nine precipitation bands in agar-gel diffusion plates. Individual bands were isolated from several plates and injected into rabbits to produce antibodies specific to the antigen represented in the precipitate. Bands formed with antigen excess or antibody excess were effective in eliciting an immune response against the antigen. Antibodies produced against the γ-globulin portion of the precipitate were detected in the serum of one-half the rabbits injected. Rats injected with the above isolated precipitates failed to produce antisera specific to individual rabbit antibodies. Agar-gel diffusion and immunoelectrophoresis showed that isolated precipitin bands may be used for the production of specific antisera against a single antigen in a complex mixture. [ABSTRACT FROM AUTHOR]

Two types of canine antisera directed against the same antigenic determinants differ in their in vitro reactions with antigen. The precipitating type of antiserum, which does not result in complete precipitation of antigen in the region of antibody excess, is composed of precipitating and non-precipitating antibodies. This type of antiserum reaches equilibrium with antigen more slowly than the customary type of rabbit precipitating antiserum. The non-precipitating antiserum is composed almost entirely of non-precipitating antibody. The addition of the canine non-precipitating antiserum to a rabbit precipitating antiserum directed against the same antigenic determinants resulted in a simulated model of the canine precipitating type. The reactivities of these heterogeneous populations of antibodies occurring in vivo or prepared in vitro are compared in relation to the time required for the establishment of an equilibrium with antigen.

Immunizing injections of rat liver and kidney mitochondrial fractions were administered to rabbits, and the effects of injecting the antisera into rats were investigated. Rats receiving antiserum to rat liver mitochondria showed impaired clearance of sulphobromphthalein, and developed hepatic fibrosis which in one animal progressed to cirrhosis. There was no increase in albuminuria and the kidneys showed no histological differences from those of control rats receiving normal rabbit serum. Renal changes were virtually confined to rats receiving antiserum to rat kidney mitochondria : these animals excreted increased amounts of protein and showed increased cellularity of the glomeruli, tubular casts and hydropic changes in the tubular epithelium. The significance of these findings is discussed. [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]

1. A technique for the measurement of poliomyelitis virus D antigens by means of gel diffusion tests against antisera obtained from hyperimmunized calves is described in detail. 2. The precision of the method was estimated by performing several replicate tests with three or four dilutions of concentrated antigen of each of the three types. The D antigen content, if measured by means of twelve precipitation hexagons in agar can be calculated with a precision of about 10 per cent (95 per cent confidence limits). 3. The calf sera gave no precipitation with C antigens in the concentrations suitable for measuring D antigens. 4. The local reference antigens, the D antigen concentrations of which were adjusted to equal that of the reference antigens used in the Glaxo Laboratories, contained C as well as D antigens. This was observed in gel diffusion tests with specific anti-C sera from guinea-pigs. 5. The guinea-pig anti-C sera showed a precipitation with the immune calf sera as well as with normal calf serum. This indicated the presence of anti-calf antibodies in the guinea-pig sera, presumably elicited by traces of calf proteins in the vaccines. These antibodies might give rise to non-specific precipitation if antisera used in the test for D antigen were obtained from animal species other than those used to provide components in the tissue culture medium from which the antigens were derived. [ABSTRACT FROM AUTHOR]

1. Using chromatography on DEAE-cellulose, a neutral, low ionic strength extract of human grey matter has been separated into fractions of proteins with basic and progressively acidic groups. 2. The reactions of each group with rabbit antiserum against the original extract, in double-diffusion tests, suggest the presence of a minimum total of thirteen distinct antigens between them. These results are supported by immunoelectrophoretic findings, which indicate the basic group to contain five, and the progressively acidic group seven to eight distinct antigens. These antigens do not appear to be human serum proteins. 3. Antigens belonging to the BE class (resistant to boiling and relatively soluble in ethanol) are present among the progressively acidic proteins, and possibly among the basic proteins also.

Attempts were made to produce antisera to solubilized tissue fractions rich in basement membranes and reticulin. Murine tissue fractions solubilized with sodium hydroxide elicited precipitating antibodies upon injection into rabbits. Although no nephrotoxic effect was observed upon injecting the rabbit antisera into mice, the antisera were fixed to the glomerular basement membrane, and not elsewhere, within 5 minutes of injection and remained fixed for at least 3 weeks. Specificity studies suggested that in addition to unique antigens, reticulin and epithelial basement membranes share a common antigen which is responsible for the similar in vitro immunofluorescence produced by antisera to tissue fractions rich in one or the other of these components. [ABSTRACT FROM AUTHOR]

The immunological characteristics of antigens extracted from X-irradiated rabbit testes were studied. Antigen from tissue obtained 10 or 42 days after 2000 r was immunologically less active than antigen extracted from 2-day post-2000 r or from non-irradiated testes. Irradiated testis antigen was not of greater sensitivity in detecting natural antibodies of whole body or testis-irradiated animals than antigen obtained from non-irradiated tissue. Thus, no evidence was obtained to indicate that radiation-altered tissue acquired new antigenic determinants which were immunologically foreign to its host. In addition, relative immunological unresponsiveness of the rabbit to its own tissue did not seem to be broken by irradiation. Therefore, irradiation did not induce autoantibody formation either by altering tissue antigens or by breaking down the unresponsiveness of the antibody-forming system. [ABSTRACT FROM AUTHOR]

Two types of antibodies directed against biochemically different antigens have been found in antisera produced by hyperimmunizing rabbits with whole homogenates of chicken or quail brain. DEAE-cellulose column chromatography was used to separate the two antibodies which, based on differences in sedimentation rate and susceptibility to inactivation by 2-mercaptoethanol, were identified as belonging to the 7S and 19S classes respectively. Although both antibodies fix complement in the presence of whole brain homogenates, the 19S fraction reacts only with the chloroform—methanol extractable portion while the 7S fraction reacts only with the residue remaining after such extraction. It is concluded that the 19S and 7S antibodies are directed against lipid or lipoprotein and protein antigens respectively. [ABSTRACT FROM AUTHOR]

A precise method has been used to investigate the kinetics and inhibition of isoimmune cytolysis of mouse lymphocytes. Target cells were labelled with 51Cr and lysis was estimated by measurement of the radioisotope released in a non-sedimentable form. Kinetic studies under conditions of limited complement or limited antiserum reveal a close similarity with rabbit anti-sheep haemolysis, and suggest that at least some of the molecular events causing lysis are the same in both cases. With the antisera studied, C′ probably requires a bimolecular antibody complex in order to bind to a cell, but the overall order of reaction, with respect to antiserum, may not be two. The observed variations in lytic response are believed to be due to the effects of antigen density and the presence in antisera of antibodies made of different kinds of immunoglobulins having the same or different specificities. Inhibition of lysis by syngeneic unlabelled lymphocytes is proposed as a standard in a quantitative assay for histocompatibility antigens. [ABSTRACT FROM AUTHOR]

Mice paralysed by bovine serum albumin gradually recover immunological reactivity. Recovery has been mapped at various ages by challenge with antigen in Freund's adjuvant, and comparing the resulting antigen binding capacity of the serum with that of normal controls. Recovery proceeds more rapidly in young than in old animals, irrespective of the duration of previous exposure to antigen. This is considered to confirm the importance of cell turnover in recovery. In harmony with this hypothesis, irradiation proved capable of deleting an existing state of paralysis without appreciably enhancing recovery. A cell transfer test has been used to detect antigen in paralysed animals, supplemented by measurement of radioactivity from [131] labelled antigen. Antigen can be detected throughout the lag period preceding the start of recovery in quantities probably sufficient to account for the lag. [ABSTRACT FROM AUTHOR]

Immune complexes composed of two insoluble and two soluble antigens and their corresponding antibodies of rabbit origin were separated by electrophoresis in agar gel performed at 56°. Antibodies could then be demonstrated in the cathodal part of the electrophoretic field; they formed precipitation lines with goat antiserum to rabbit serum. The separation of soluble antigens could be demonstrated by the precipitation reaction with their corresponding antisera. [ABSTRACT FROM AUTHOR]