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The cross-reactivity of heterologous erythrocytes was measured at the level of the B and the T cell. B cells produced antibody with restricted cross-reactivity as compared with the cross-reactivity of T cells, assayed by their ability to help B cells in a primary anti-sheep erythrocyte immune response. Helper function was maximally elicited with low antigen doses. The data suggest that the T-cell antigen receptors for erythrocyte antigens differ in specificity from those of B cells and that the sensitivity of T cells to antigen may be very great. [ABSTRACT FROM AUTHOR]

The results suggest that the anaemia in mice associated with the injection of C. parvum is mediated by an immune mechanism. Survival studies of 51Cr-labelled red blood cells (RBC) in splenectomized and unsplenectomized mice show that mice immunized with C. parvum three times, clear RBC faster than unimmunized mice. However, hyperimmunized mice stabilized their haemoglobin concentrations above their minimum level and did not clear 51Cr-labelled RBC faster than unimmunized mice. All mice produced antibody against C. parvum but remained direct Coombs'-negative. Serum from immunized animals did not contain opsonins for -51Cr-labelled RBC. The reticulocytosis, normal osmotic fragility tests and normal survival times of 51Cr-labelled RBC from C. parvum-immunized mice all suggest that the main direct influence of the micro-organism is extrinsic of the RBC. Antigens from the micro-organism were adsorbed by erythrocytes in vitro. It is hypothesized that the anaemia is caused by destruction of RBC brought about by antibody reacting with bacteria that have been adsorbed by RBC. [ABSTRACT FROM AUTHOR]

The effect of procarbazine and cyclophosphamide on the formation of antibodies to sheep red blood cells in mice is investigated. Cyclophosphamide was found to have the best immunosuppressive effect when given after antigen; recovery from drug treatment was relatively fast; under experimental conditions that favour cyclophosphamide action (antigen prior to drug) IgM formation was found to be depressed and prolonged; IgG formation was markedly depressed or altogether missing. Procarbazine was found to act best when given prior to antigen; recovery was slow; under experimental conditions that favour procarbazine action (drug prior to antigen) the induction period was prolonged. IgM titres were slightly and IgG titres markedly depressed. Under all conditions tested procarbazine had little effect on the onset of IgG production. [ABSTRACT FROM AUTHOR]

The reactions of two monoclonal IgM lambda cold agglutinins, Sch and Sher, have been studied in detail with human and animal erythrocyte antigens. Although they were unusual in having lambda and not kappa polypeptide chains, they could be assigned to the anti I and anti-Pr1 groups of cold agglutinins. The findings with serum Sher indicated that the Pr1 antigen may be more complex than previously thought. The occurrence of unexpectedly large numbers of specificities among monoclonal anti-I, anti-i and anti-Pr antibodies is discussed and it is suggested that each monoclonal antibody may recognize only a limited portion of a complex red cell antigen. [ABSTRACT FROM AUTHOR]

Guinea-pig erythrocytes are cleared very rapidly from the circulating blood of 10-day-old rabbits; in contrast, sheep and mouse erythrocytes are cleared twelve to thirteen times more slowly, while rat erythrocytes are cleared six times more slowly, The differential rates of blood clearance of these xenogeneic erythrocytes largely substantiate differences between low titres of haemagglutinins or haemolysins, either naturally acquired or of maternal origin. Interference by such antibody in induction of the plaque-forming cell response and the minimum time antigen must persist in the circulation for successful induction of a haemolytic antibody-forming cell response are discussed. [ABSTRACT FROM AUTHOR]

The ability of rabbits to respond to relatively high doses of sheep erythrocytes appears within 5 days after birth Young rabbits exhibited an apparent sequential maturation of responsiveness to sheep, mouse, rat and guinea-pig erythrocyte antigens. The delay in response to these antigens can be correlated with different levels of specific haemagglutinin, presumably of maternal origin, in the circulation of young rabbits. [ABSTRACT FROM AUTHOR]

Surface immunoglobulin can be demonstrated on almost all sheep erythrocyte rosette-forming cells in CBA and AKR mice, either by inhibition of antigen binding by anti-immunoglobulin or by immunofluorescence. When surface immunoglobulin on a rosette-forming cell is `capped' by anti-immunoglobulin, the bound red cells migrate with the immunoglobulin, demonstrating that the receptor-combining site and immunoglobulin antigenic determinants share the same mobile unit in the membrane. These observations strongly support the concept that the antigen receptor is surface immunoglobulin (at least for B cells), and illustrate the usefulness of the `capping' technique for studying the associations between membrane elements. [ABSTRACT FROM AUTHOR]

The progeny of mothers subjected to marginal lipotrope or moderately low protein diets exhibit deficient humoral immune responses. Serum antibody titres and numbers of antibody-forming cells to sheep erythrocytes (SRBC) are lower in the offspring of mothers whose diets were limited in either percentage protein or percentage lipotropes than in offspring whose mothers were fed a complete diet. Immune lymphocyte stimulation by SRBC antigens was marginal and not measurably different between the progeny of malnourished mothers and controls. The in vitro response of' spleen cells from offspring of nutritionally deprived mothers to the T-cell mitogen phytohaemagglutinin (PHA) is considerably less than that of controls. Thymus cells from marginal lipotrope offspring respond well to PHA, suggesting that PHA-reactive cell migration may be inhibited in these animals. These results, obtained using a T-dependent antigen (SRBC) and a T-dependent mitogen (PHA), support the conclusion that maternal dietary deficiency can adversely affect T-cell function in rat offspring. [ABSTRACT FROM AUTHOR]

The regulation of the immune responses by antibody administered passively on peritoneal exudate cells (PEG) was studied in guinea-pigs. The immunogenicity of sheep red blood cells (SRBC) associated with PEC for the induction of delayed hypersensitivity (DH) to soluble erythrocyte antigen was enhanced when PEG were incubated with anti-SRBG antibody. In contrast, the antibody response to SRBC was depressed. This phenomenon was only observed with specific antibody and was partially blocked when PEG covered with anti- SRBC antibody were incubated with rabbit anti-guinea-pig globulin serum. Comparable amounts of anti-SRBC antibody injected separately had no enhancing effect. Anti-SRBC sera from which the cytophilic antibody activity had been removed were inactive. PEG-associated SRBC were usually more immunogenic than the same amount of `free' SRBC for the induction of delayed hypersensitivity and always more immunogenic for antibody production. These observations suggested that macrophage cytophilic antibody might be involved in the regulation of the immune response and play a role in the preferential induction of delayed hypersensitivity in the studied system. [ABSTRACT FROM AUTHOR]

Antibody reacting against syngeneic erythrocytes (RBC) was elicited in several strains of mice by immunizing the mice with rat RBC. In nearly all mice sustained production of antibody reacting against syngeneic RBC required that the mice be continually stimulated with rat RBC. We suggest that this is further evidence that naturally-occurring immunological tolerance can be terminated temporarily by immunization with antigens cross-reacting with self. The results support the concept that clones of antibody-producing cells potentially reactive against self, exist in the normal mouse. [ABSTRACT FROM AUTHOR]

In contrast to MN receptors, Pr1 and Pr2 antigens of isolated glycoprotein of erythrocytes are not inactivated by acylation of free amino groups. By introduction of easily leaving groups, e.g. trifluoroacetic residues, inactivation of the MN activity can be made reversible. No differences between Pr1 and Pr2 antigens could be found with the reactions described in this paper. Acylation m aqueous phase (buffer/anhydrous acetic acid) and m organic phase (pyridine/ anhydrous acetic acid) gave identical results qualitatively and quantitatively. It was not possible to split the N-acetic binding neither under mild alkaline conditions nor with aminoacylase. 4-5 amino groups out of 11 could be acylated, whereas succinylation in aquous phase (buffer/ anhydrous succinic acid) resulted in modification of only one free amino group, the calculations being based on a molecular weight of 30 000. Following succinylation MN activity was also lost, whereas the antigenic activity of Pr1 and Pr2 remained unchanged. The appearance of a negative charge instead of the positive charge of the free amino group on the glycoprotein did not affect Pr1 or Pr2 activity m any way. If glycoprotein was esterified under conditions which cause esterification of free sialic acid to a wide extent neither MN activity nor Pr1/Pr2 activity was altered. [ABSTRACT FROM AUTHOR]

Naturally occurring titres of heterophil haemagglutinins against rat erythrocytes are related to ABO blood groups, being higher in people of blood groups O and A and lower in people of blood groups B and AB. Comparable titres of haemagglutinin activity can be demonstrated against human AB erythrocytes as well as rat erythrocytes. Activity against rat erythrocytes can be absorbed with human AB erythrocytes and vice versa. Furthermore, after absorption of heterophil antibody with human A or B erythrocytes, heat elution allows partial recovery of the antibody which reacts with rat erythrocytes. A rise in titre of heterophil antibody comparable to that occurring after renal transplantation was demonstrated after surgical operations but only where blood transfusions were given. It is postulated that these rises in antibody titre result from exposure to non-HL-A isoantigens which crossreact with human A and B substance and rat erythrocyte antigens. These antibodies can be distinguished from Forsmann and infectious mononucleosis antibodies, are absent in placental cord blood, occur naturally as IgM and respond to a secondary challenge as IgG, except in immunosuppressed renal transplant patients, where both IgG and IgM responses occur. [ABSTRACT FROM AUTHOR]

A significant increase in titre of human heterophil antibodies against rat erythrocytes was demonstrated in eighteen of twenty-eight patients after renal transplantation. Although this rise in titre preceded an acute rejection episode in the renal graft in some instances, it is considered that this relationship was coincidental. There is no relationship between the numbers of such rejection episodes or the degree of incompatibility for HL-A antigens between donor and recipient and the titres of heterophil antibodies after transplantation. [ABSTRACT FROM AUTHOR]

Homocytotropic antibody induced with ragweed and timothy pollen extract has been produced in non-atopic dogs. Characterization of the antibody was carried out by direct skin tests, passive transfer, tanned red cell haemagglutination, Sephadex filtration and immunoelectrophoresis. Two types of antibody were induced. A 7S antibody which had skin sensitizing characteristics and a 19S antibody which was the haemagglutinating antibody and 2ME sensitive. The findings are similar to that seen in spontaneous canine atopy except that the dogs did not respond with respiratory or systemic symptoms after antigenic challenge. [ABSTRACT FROM AUTHOR]

The onset of ability to produce haemolytic antibody to sheep erythrocytes does not appear in hamsters until 11 days after birth. However, when mouse erythrocytes were used as antigen an earlier onset of immunocompetence at 6 days after birth was detected. The hamster can make haemolytic antibody only 9.5 days after the appearance of lymphocytes in the thymus—this is sooner than any other species. This capacity is compared with other species on the basis of physiological development by age-equivalence. [ABSTRACT FROM AUTHOR]

A graft-versus-host reaction was induced in F1 hybrid mice by the inoculation of spleen cells from one of the parental strains. One week later cultures for primary immunization in vitro against sheep red cells were initiated with the spleen cells of the recipients. It was found that the antibody response against sheep red cells was considerably inhibited in spleen cells from mice subjected to a graft- versus-host reaction as compared to the response of spleen cells from untreated mice. Furthermore, when spleen cells from animals subjected to a graft-versus-host reaction were cultured together with spleen cells from normal animals or animals preimmunized to sheep red cells, the antibody responses of the latter two cell populations were inhibited. The relation between these findings and the antigenic competition phenomenon is discussed and it is suggested that antigenic competition is caused by an non- specific inhibition of the antibody response, caused by secretion of locally acting factors or by inhibition of normal cells by direct contact with ‘active’ cells, stimulated by antigen. [ABSTRACT FROM AUTHOR]

Sephadex will adsorb protein non-covalently and this can be detected by mixed agglutination reactions with antibody and antigen coated red cells. Rheumatoid factor will adsorb to Sephadex and can be demonstrated by immunoglobulin coated red cells. BioGel particles show considerably less nonspecific adsorption of protein and this makes them more suitable for the demonstration of antibody on antigen coated particles by means of antiglobulin reagents. Using a two step antiglobulin reaction it was possible to detect IgG, IgM and IgD anti-insulin antibody. [ABSTRACT FROM AUTHOR]

The method of preparation of partially purified [125I]IgM anti-D and of subunits of 180,000, 90,000 and 40,000 molecular weight with anti-D activity is described. It was found that approximately 11,500 whole IgM anti-D molecules would bind to each red cell of phenotype R1R2, On the other hand, approximately 31,000 molecules of both whole IgG anti-D and an IgM anti-D subunit of molecular weight 40,000 would bind to the same cell. The following possibilities are proposed to explain these findings: (1) IgM anti-D molecules bound to the red cell sterically hinder binding of other IgM antibody molecules to closely adjacent D-antigen sites and (2) some D-antigen sites are too far below the surface of the red cell to permit binding by the large IgM molecule. The average values of the equilibrium constants (K0) of two different examples of IgM anti-D were 1.7 × 109 and 2.5 × 109 litres/mole, and of the IgG antibodies obtained from the same plasmas were 9- and 32-fold lower respectively. The K0 of the IgM subunits was on average 23-fold lower than that of the whole IgM and it is concluded that IgM anti-D binds bivalently to each red cell but that the binding energy of one of the sites is relatively small. [ABSTRACT FROM AUTHOR]

The concept that antigen has a continuous role in the recruitment and differentiation of immune progenitor cells was tested with optimum and suboptimum doses of heterologous erythrocytes in mice. These studies further evaluated an immune cell maturation scheme in which continuous antigenic stimulation is required for both the recruitment of ‘antigen-sensitive units’ and the expansive proliferation of a distinct sensitized cell compartment, which undergoes irreversible differentiation to functional antibody-forming cells. Haemolytic plaque-forming cell capacity during both the primary and secondary immune reactions were studied, both in the intact animal and with the spleen cell transfer technique. This in vivo culture technique was used to measure the sensitized cell compartment in the absence of existing antibody regulatory mechanisms. The results clearly demonstrate a higher detectable secondary immune capacity in the suboptimum antigen dose group than in the optimum antigen dose group. This was demonstrated for both the 19S and 7S cellular responses, as well as with humoral antibody levels measured in the spleen cell recipient mice. It can be concluded that in the presence of a suboptimum dose of antigen, which rapidly diminishes during the early intervals of the primary response, there is adequate recruitment with subsequent preservation or rescue from antigen-mediated depletion of the sensitized cell compartment, at the expense of the detectable primary response. [ABSTRACT FROM AUTHOR]

The effect of a single subcutaneous dose of hydrocortisone (730 mg/kg ∼ 21-day LD50) on the haemolysin response of mice to sheep erythrocyte antigen was examined. Hydrocortisone was administered once at times varying from 5 days before immunization with sheep erythrocytes to 9 days after antigen. Total suppression of the 7S haemolysin titre was brought about by treatment with single dose of 750 mg/kg in the period 5 days prior to antigen until 2 days after antigen; at the same time, the titre of 19S haemolysin exceeded the control 19S titre. Microplaque assay on the 5th day failed to confirm total suppression of 7S antibody synthesis, as 4 per cent of the splenic plaque-forming cells produced 7S. The same assay failed to verify the augmentation of 19S production on a cellular level, as the number of 19S plaque-forming was significantly decreased. Hydrocortisone could be shown to influence the _morphology of the 19S antibody producing cells by increasing the percentage of mature cell types. A selective depressing activity by hydrocortisone on 7S memory was found. The theoretical implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Lepisosteus osseus possesses high titres of natural agglutinins to human group A (HuA) erythrocytes and to Salmonella typhosa 'O' (STO) antigens. Moderate natural haemagglutinin titres to sheep red blood cells (SRBC) were noted. Cold agglutinin (4°) titres exceeded agglutinin titres recorded at 25°. Immunization with STO antigen resulted in a marked increase in corresponding anti-STO agglutinins, as well as increases in anti-HuA and anti-SRBC agglutinins. Animals immunized with SRBC antigen produced elevated anti-SRBC agglutinin titres, while immunization with HuA erythrocytes produced no changes in agglutinin levels. Immunization with STO following previous injections with HuA resulted in increases of corresponding anti-STO agglutinins, as well as marked increases in anti-HuA agglutinins. Absorption studies indicated that anti-STO agglutinins were not cross reactive against HuA antigens. Natural and immune antibodies were all of the same macromolecular class. Rises in antibody titres were reflected in both cold and warm agglutinins. Animals maintained for long periods at low temperatures exhibited preferential ability for increased synthesis of cold agglutinins to HuA. Natural haemolytic titres, as measured by an SRBC indicator system, were also elevated as compared to mammalian systems. These titres increased with stimulation with SRBC. Results suggest that the high levels of natural haemolytic and agglutinin activity in this species are of immunogenic origin. [ABSTRACT FROM AUTHOR]

Initiation of an immune response to sheep erythrocytes by the transplantation of spleen cells into irradiated recipients is thought to involve an interaction between two functionally different lymphoid cells. If such populations actually exist, it should be possible to separate them from each other and to study the properties of each type of cell. To this end, mouse spleen cells have been fractionated using equilibrium centrifugation in density gradients of Ficoll. Two functionally different populations of spleen cells were obtained. Neither population was active in initiating an immune response by itself on transplantation into irradiated mice, but mixtures of the two populations were. One population of spleen cells was also active when transplanted together with normal mouse bone marrow and the other population was active when mixed with thymus cells from normal mice. These results indicate that two different spleen cells must interact to initiate an immune response in vivo. The specificity of the cell that synergizes with thymus ceils was investigated further and shown to have antigen-specific receptors on its surface. These cells can be recognized by their ability to make rosettes with erythrocyte antigens. Thus, the ‘background’ rosette-forming cells found in unimmunized mice are actually progenitors of antibody-producing cells. [ABSTRACT FROM AUTHOR]

Normal serum samples from the Australian freshwater catfish, Tandanus tandanus (Mitchell), were shown to have haemolytic and haemagglutinating activity for a variety of animal erythrocytes. Specific agglutinins were present for the erythrocytes of most animals tested but some cross-reactivity of agglutinins was demonstrated. Evidence was obtained that the greater part of the human erythrocyte agglutinating activity was directed toward antigens of the ABH system. Whereas some sera clearly showed anti-H or anti-A and B agglutination patterns, others agglutinated equally erythrocytes of different groups. ABO erythrocytes were agglutinated to higher titres than were erythrocytes of the 'Bombay' phenotype, salivas from secretors inhibited the agglutination of erythrocytes carrying appropriate ABH antigens, whereas salivas from non-secretors did not and absorptions of sera often revealed ABH specific agglutinins. While some sera agglutinated equally ABO erythrocytes because they contained a mixture of agglutinins with different ABH specificities, other serum samples containing one predominating species of agglutinin caused equal agglutination of O, A, AB and B erythrocytes. The specificity of this latter type of agglutinin is discussed. [ABSTRACT FROM AUTHOR]

A soluble antigen has been obtained from sheep erythrocytes by sonication. This material is not sedimented by centrifugation by 3.5 × 106 g minutes. but is excluded from Sephadex G-200 gel. The soluble preparation elicits an antibody response in vivo and reacts with antibody prepared against intact sheep erythrocytes. The soluble antigen is as effective as erythrocytes in inducing delayed-type hypersensitivity and in eliciting the delayed-type reaction. [ABSTRACT FROM AUTHOR]

Lectins from the seeds of Euonymus europeaus and Dolichos biflorus as well as lipopolysaccharides from enterobacteria, have been used for linking proteins to red cells as a means of detecting antibody. Bovine serum albumin, human serum albumin, rabbit IgA, Salmonella adelaide, flagellin, and tetanus and diphtheria toxoids were covalently coupled to these linking agents by means of toluene-di-isocyanate, to give stable non-agglutinating reagents capable of coating red cells with antigen. Lipopolysaccharide from an Rd rough strain of Shigella flexneri was a particularly useful linking agent as human serum containing antibody to the smooth form of this organism did not react with the Rd lipopolysaccharide. The test is capable of detecting about 100 ng antibody/ml. [ABSTRACT FROM AUTHOR]

The mixed antiglobulin reaction has been used to demonstrate the Gross (G) antigen on a tissue culture cell monolayer, and with greater sensitivity than could be achieved by indirect immunofluorescence. Erythrocytes from NZB/Bl mice, showing positive direct antiglobulin tests, when treated with an anti-mouse immunoglobulin serum, provided a convenient source of indicator cells for the reaction. [ABSTRACT FROM AUTHOR]

Following oral infection of a 12-week-old chicken with an overnight broth culture of Salm. gallinarum, serum was removed on the 9th day and tested for the presence of haemagglutinating antibody using erythrocytes modified with a Westphal lipopolysaccharide extract of Salm. gallinarum. The serum was found to have a high titre and on ’Sephadex’ fractionation and ultracentrifugal preparation showed a marked IgM response. Spectrophotometric measurements were made of interactions between the O-antigen extract and the whole serum or its component IgM and IgG fractions. Hapten-antibody complexes were measurable with the whole serum and the IgM fraction, but no complexes were detected with the IgG fraction under the conditions of the experiment.

The concurrent action of complement and antiserum globulin (anti-SG) antibodies on red cells coated with an antigen and sensitized with the specific antibodies was studied. The occurrence of haemolysis in such systems is dependent on the haemolytic properties of the anti-SG antibodies used in the test. The haemolytic properties of the immunoglobulins that sensitize the coated cells have no apparent role in the development of the lysis. The method of titration of antibodies by passive haemolysis can be rendered more sensitive and can be applied to detect antibodies that do not fix complement, if a suitable anti-SG serum is permitted to react with the immunoglobulins that have combined with the antigen coating the erythrocytes. Under standard conditions reproducible titrations can be performed to detect 0·009 μg N Ab/ml with an error equal to 10 per cent.

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]

Mice thymectomized within 24 hours of birth were immunized by intraperitoneal injection of sheep erythrocytes at 6 weeks of age and again 60 days later. Serum haemolysin activity was measured at various times following antigen injection. The serum haemolysin was fractionated by sucrose gradient centrifugation into two classes. A time-course of the antibody response was constructed for total, 19S and 7S haemolysins. Both reduction in circulating antibody activity and delay in reaching maximal levels were observed.

Studies with rabbit antisera have revealed the presence of three distinct agglutinogens on the surface of mouse ascites tumour cells and ‘L’ cells. The T-antigen is the dominant agglutinogen on the mouse nucleated cells and is absent from mouse and sheep erythrocytes; the F-antigen is common to the mouse nucleated cells and sheep erythrocytes but absent from mouse erythrocytes; the C-antigen is common to the surface of mouse nucleated cells and erythrocytes but absent from sheep erythrocytes. The C-antigen is not the major agglutinogen of the mouse erythrocyte. Methods are described for the independent assay of the T, C and F antigens and their occurrence on a number of mouse tumour cells described. [ABSTRACT FROM AUTHOR]

Spleen cells from non-immunized mice were mixed with sheep red blood cells and incubated in diffusion chambers implanted in the mouse peritoneal cavity. At 6 and 7 days an increase was noted in the proportion of plaque-forming cells in the chambers and this was accompanied by the appearance of haemagglutinin and haemolysin in the chamber fluid. Excess antigen was not observed to depress this proportion of plaque-forming cells. [ABSTRACT FROM AUTHOR]

Purified antibodies to tetanus toxoid have been separated from serum γG-globulins and from low molecular weight fragments of urinary γ-globulins (Fu fragments). Isolated Fu antibodies have been shown to be of low molecular weight and appear to be bivalent; they agglutinate antigen-coated red cells, fix complement and contain an average of five disulphide bonds, one of which is more labile than the remaining four. The antibody activity of the molecule is destroyed by reduction of this labile disulphide bond. These results are discussed in the light of current concepts of antibody structure. [ABSTRACT FROM AUTHOR]

Several parameters such as dosage and schedule of antigen injection were examined. The effect of a single intraperitoneal or intravenous injection of various amounts of sheep red cells (SRBC) was studied over a dose range of more than 105. Approximately 5 × 107 SRBC was the optimal amount of antigen beyond which no further increase in titre occurred. Multiple injections and overloading the organism with large amounts of SRBC did not result in a depression of the immune response. Immunization with SRBC incorporated into incomplete Freund's adjuvant was slightly inferior to immunization with SRBC suspended in saline. The response depended on the antigen concentration in the adjuvant mixture rather than on the absolute amount of antigen injected. Early in immunization antibodies against SRBC belong to the 19S class and change gradually into 7S antibodies. 19S and 7S haemolytic activity was mercaptoethanol-sensitive. From absorption experiments it was concluded that the mouse antibody against SRBC has specificities other than anti-Forssman, anti-mononucleosis and anti-serum sickness and is directed against an isophilic heat-labile antigen(s). [ABSTRACT FROM AUTHOR]

A/Jax and C57BL mice were immunized with either human red blood cells or with both human cells and (A/Jax × C57BL)F1 hybrid spleen cells, and sensitized spleen cells were transferred to isogeneic or hybrid hosts. Antihuman haemagglutinin titres were lower in hosts receiving C57BL rather than A/Jax cells, were lower in hybrid than isogeneic hosts, and were lower in animals receiving doubly- than in those receiving singly-sensitized cells. The results can be best interpreted on the basis of competition of antigens, rather than by postulating allergic death of sensitized lymphoid cells exposed to an overwhelming dose of antigen. Competition of antigen was found m occur in Ajax and C57BL mice actively immunized with both human red cells and hybrid spleen cells, when antibody titres were compared with those of singly-immunized controls. Anti-human haemagglutinin titres in actively immunized hybrid mice were intermediate between those of the parental strains, suggesting that differences in titres between the parental strains were due to physiological differences in the antibody-forming system, rather than to the more poorly reacting strain sharing antigenic determinants with and therefore being partially tolerant to human red cells. [ABSTRACT FROM AUTHOR]

Mixed haemadsorption should be regarded as an application of the mixed antiglobulin reaction to situations where the antigen is sessile on a glass surface. Antibody attached to the antigen when exposing the latter to an antiserum is traced by red cells carrying an antiglobulin layer which makes them adsorb to the antibody. The indicator cells are prepared by coating them first with a layer of γ-globulin from the animal species, the antibody globulin of which they are intended to trace, and then with a layer of the corresponding antigtobulin. The most effective indicator cells were obtained by attaching antibody to natural receptors on the red cells to achieve their first coating of γ-globulin. The preparation of indicator cells for tracing antibodies from a number of species, including human, is described. The mixed haemadsorption technique is highly specific and has a sensitivity which is comparable to that of the most sensitive serological techniques. Test procedures adapted for different purposes are outlined and a number of applications to experimental and clinical problems are reviewed. [ABSTRACT FROM AUTHOR]

Differences in reactions in the mixed agglutination test with anti-A sera from different individuals were found to be unrelated to the titre against human or pig A red cells or simply to the presence or absence of either 7S or 19S antibody. It was found that 7S anti-A was capable of producing mixed agglutination, but that 19S fractions more frequently produced mixed agglutination than the 7S fraction from the same serum. Treatment of sera with 2-mercapto-ethanol abolished the capacity to produce mixed agglutination from some sera, whilst other sera were unaffected. Some anti-A sera gave the same titre of mixed agglutination with both secretor and non-secretor buccal cells, but other sera reacted more strongly with secretor than with non-secretor cells. Production of mixed agglutination by 7S antibodies was also demonstrated with fractions of rabbit antisera against Forssman antigen and against bovine red cells. [ABSTRACT FROM AUTHOR]

A study was made of the antigenicity of soluble and cell-attached common antigen obtained from enteric bacteria. This antigen becomes readily attached to erythrocytes, which become agglutinable in the presence of the corresponding antibody. Although the common antigen is extractable from numerous species and serogroups of Enterobacteriaceae, in only a few, notably Escherichia coli O14, does it engender antibodies in the rabbit upon intravenous injection. It is shown that antibodies against the common antigen are produced after intravenous injection of cell-attached antigen from E. coli O111, Salmonella spp. and Shigella spp. but not, or only slightly, after administration of soluble antigen. Antibodies are also formed after injection of antigen attached to C3H mouse fibroblast (L) cells. Intravenous injection of equivalent amounts of common antigen from E. coli O14, E. coli O111, S. typhi-murium and S. flexneri results in antibody formation against the common antigen only with that from E. coli O14. [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]

A new red cell sensitizing antigen (called antigen '44') has been found in streptococci of Lancefield groups, D, F, H, K, M and Q and in many viridans streptococci, but only in strains producing the Hickey (polyglycerophosphate) antigen. It possibly occurs as a determinant of a complex of which the Hickey antigen is also a part. Both antigens are released in large amounts into the culture medium but are present also in the cell wall, in which they may constitute a 'filler' material. [ABSTRACT FROM AUTHOR]

The uptake of haemolytic fowl C' by fowl anti-BSA and BSA has been detected and measured with sheep red cells sensitized with fowl haemolysin. It was, however, very small compared with the amount of guinea-pig C' taken up by similar amounts of rabbit anti-BSA, and not directly related to the amount of precipitating antibody in any particular antiserum; nor did it occur when preformed resuspended specific precipitates were added to the C' solution. Fixation of guinea-pig C' by fowl anti-BSA could not be demonstrated, either with fresh antiserum, or with heated antiserum in the presence of normal fresh fowl serum. Uptake of haemolytic fowl C' was shown to coincide with a great increase in specific precipitation when this was done in 0.9 per cent NaCl. This increase was produced partly by the addition of a non-specific serum factor, and partly by more complete precipitation of the antibody. Specific precipitates formed in 0.9 per cent NaCl in the presence of fresh normal fowl serum (NFS) contained much larger amounts of a macroglobin component than precipitates formed in the absence of NFS or in 8 per cent NaCl. [ABSTRACT FROM AUTHOR]

131I-labelled anti-Rho (D) antibody was made to react with suspensions of spermatozoa and red blood cells from Rho (D) positive and negative donors at 37° for 1 hour. Spermatozoa from Rho (D) positive donors did not take up more 131I anti-Rho (D) than was bound by spermatozoa from Rho (D) negative donors. The mean value for Rho (D) positive donors was 0.0142 ± 0.0061 X 10-2 μg. nitrogen per 106 spermatozoa, and the comparable value for Rho (D) negative donors was 0.0189 ± 0.0130 X 10-2 μg. nitrogen per 10-6 spermatozoa. Red blood cells from Rho (D) positive donors took up thirty to forty times more 131I anti- Rho (D) antibody than was taken up by spermatozoa from Rho (D) positive donors, whereas red blood cells from Rho (D) negative donors took up only 0.5 to 1.7 times more 131I anti-donors. Pre-treatment of spermatozoa with trypsin increased by two to three times the quantity of 131I anti- Rho (D) antibody bound to spermatozoa from both Rho (D) positive and negative donors. The quantitative increase was similar for spermatozoa from Rho (D) positive and negative donors. No evidence for the presence of Rho (D) antigen on human spermatozoa was obtained using 131I anti- Rho (D). [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]

The value of sera containing immuno-conglutinin or conglutinin in demonstrating complement-binding by human blood group iso-antibodies was investigated. Provided that a suitable complement was used, positive results were obtained with all human sera which had been shown by other methods to contain complement-binding antibodies. When human red cells were sensitized with human complement-binding blood group antibody and then treated with horse serum as a source of complement, the strongest reactions were obtained with rabbit immuno-conglutinin; the reactions with bovine serum (containing conglutinin) were distinctly weaker, and the reactions with human sera containing immuno-conglutinin were weaker still. When rabbit complement was used instead of horse complement, stronger reactions were obtained, but it was difficult to avoid ‘false positive’ reactions. When human complement was used, good reactions were obtained with rabbit immuno-conglutinin, but the reactions with bovine conglutinin and human immuno-conglutinin were completely negative. [ABSTRACT FROM AUTHOR]

Treponemes are shown to be about twenty times as sensitive to immobilizing antibody at 35° as at 25° C., so it was conjectured that our former technique of preliminary incubation of treponema suspension and test serum merely served to accustom the treponemes to the 35° temperature in the environment. Experiment confirmed that a preliminary incubation of the suspension alone, in bulk, was an adequate substitute; thus, the technique has been simplified. Different strongly reactive sera or pools appear to act similarly, as they should if they were merely different dilutions of the same active agent. A different value from 2 may sometimes be more suitable for the slope coefficient ϒ of log t in regressions versus log D, where D is the 50 per cent immobilizing dilution; but 2 may be used as approximation for ϒ in correction factors applied to observations made at only moderately different times. Three World Health Organization TPI controlserum preparations were tested simultaneously with five of our own strongly reactive rabbit-serum pools in a series of experiments, yielding fairly reliable estimates of their relative potencies. This permitted making a blend as a replica (ALB1) for replacement of our nearly exhausted supply of the second international TPI control serum (WHO2W) that we had obtained in whole-serum form. The third international control serum (WHO3) was shown to have about 73 per cent of the potency of the WHO2W control serum, whose potency we had taken as standard, p°= 100. It was shown that WHO3, which is composed of human serum, is similar in action to rabbit serum pools; hence the latter appear adequate for use as standards of reference in testing human sera. [ABSTRACT FROM AUTHOR]

Whilst the ability of isoantibodies to agglutinate mouse red cells in saline medium does depend in part upon the properties of the antibody molecules, the concentration of antigen upon the red cell and certain genetically determined properties of the cell surface are of even greater importance. The red cells of mice of any strain will give positive results in the human serum:dextran system whilst reactions in a saline medium are unusual. In this paper the term ‘incomplete antibody’ is used to denote antibodies that can only be detected in conjunction with other antibodies in the human serum:dextran system. The time of appearance of antibodies to homografts of an ascites sarcoma, an ascites leukosis and a solid mammary carcinoma has been studied. Incomplete antibody as defined above has been detected by two methods. In the blocking test incomplete antibody is allowed to react with red cells before contact with complete antibody, a positive result being observed as a fall in titre when the treated red cells are subsequently exposed to complete antibody. In the second type of test, the synergic test, incomplete antibody is mixed with suitably diluted complete antibody and fitrated against suitable red cells, a positive result being a significant rise in titre as compared with complete antibody mixed with normal serum. Antibodies produced by the antigen donors did not give significant blocking or synergic effects. Incomplete antibodies are sometimes detectible on the third day and with regularity on the fourth day. Antibodies ‘complete’ for A strain red cells may appear at any time from the fifth day onwards. There is sometimes a drop in fitre of varying duration on the sixth day. This corresponds with the commencement of marked inflammatory changes on the graft bed. The maximum titre is attained at about two weeks. Antibodies may disappear in under three months or persist as long as a year. The function of antibodies in homograft reactions varies with the target tissue. However, they appear before the onset of anatomical signs of homograff response regardless of the type of target cell. [ABSTRACT FROM AUTHOR]