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A pentraxin was isolated from acute-phase guinea-pig serum by calcium-dependent affinity chromatography on agarose. It was immunochemically identical to guinea-pig amyloid P component and therefore has been called guinea-pig serum amyloid P component (SAP). Guinea-pig SAP has an apparent MW of between 265,000 and 300,000 by different techniques, and is composed of 10 non-covalently associated subunits arranged in two pentameric annular discs interacting face-to-face. It is apparently composed of two types of subunit, which run as a closely spaced doublet on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least one type of subunit is glycosylated. The serum concentration was 16 ± 4 mg/l in outbred animals, rising to 25±4 mg/l in an acute-phase response. Binding to agarose correlated with the agarose pyruvate content and was completely abolished by diazomethane treatment of the agarose, which methylates the pyruvate caboxylic moiety. Binding was also inhibited in the presence of free methyl 4,6-o-(carboxyethylidine)-beta-D-galactopyranoside. No protein resembling C-reactive protein (CRP) was obtained by calcium-dependent affinity chromatography of acute-phase guinea-pig serum on phosphorylcholine (PC)-Sepharose, and it not clear whether a counterpart of CRP exists in this species. [ABSTRACT FROM AUTHOR]

Primary antibody response in vitro by human peripheral blood mononuclear cells (PBMC) against sheep red blood cells (SRBC) was investigated by the method of haemolytic colonies in soft agar. The response was modulated by two antagonistic populations of surface-adherent cells (AC) that could be enriched by physical means using differential adhesion to the plastic. Firmly adherent cells (AC/FA), which remained surface stuck after 18 hr, cocultured with AC-depleted PBMC, significantly inhibited the production of haemolytic colonies; AC/NR cells, which were released after this interval, strongly increased the response. The inhibition by AC/FA cells was mainly due to production of peroxides and the stimulation by AC/NR to the synthesis of arachidonic acid derivatives. When the two subpopulations were present contemporarily at the concentration shown in PBMC suspensions, the suppressive action of AC/FA overwhelmed the enhancing activity of AC/ NR cells. Functional, histochemical and immunochemical data suggest that the AC/FA fraction is mainly composed of typical phagocytosing cells, whereas the AC/NR fraction contains cells of uncertain classification that do not exhibit active phagocytic activity. [ABSTRACT FROM AUTHOR]

A protein exhibiting immunochemical cross-reactivity with the murine α1-pregnancy-associated protein (α1-PAP) has been detected in the sera of female rats. The protein has an α1-electrophoretic mobility, an estimated molecular weight of 150,000 and is readily detectable in the sera of non-pregnant female rats of each strain examined. During pregnancy, the serum concentration increases up to five-fold, reaching maximal levels at the same gestational stage as α1-PAP in the mouse. Immunohistochemical studies revealed a staining pattern for the rat protein similar to that seen for α1-PAP in the mouse, with positive cells being observed in lumbar lymph nodes, the lamina propria of gut mucosa, Peyer's patches, intracellularly in some hepatocytes and, during pregnancy, also in the placenta and metrial gland. On the basis of these immunochemical, physicochemical and immunohistochemical findings, it is proposed that the protein detected in rat sera represents the analogue of the murine α1-PAP, and that the rat strains examined more closely resemble high endogenous α1-PAP-producer mouse strains. It is proposed that α1-PAP, and a number of other rodent pregnancy proteins recently described, may be used in functional studies of human α2-PAG. [ABSTRACT FROM AUTHOR]

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (γ,κ) tumour cells with spleen cells from (BALB/c x C57Bl/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-ε-3.57, NP-ε-15,88, NP-ε-91,58 and NP-ε-95,31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) indicated that NP-ε-15,88, NP-ε-91,58 and NP-ε-95.31 all possessed λ1 (or possibly λ3) light chains; and that NP-ε-3,57 possessed λ2 light chains; NP-&elipson;-95,31 also expressed the P3-X20 derived, MOPC-21 κ light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-ε-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-ε- aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells. [ABSTRACT FROM AUTHOR]

An antigenic heterogeneity among IgD myeloma proteins was tested by immunoelectrophoresis and double immunodiffusion in agar with four kinds of anti-delta(Fc) antisera produced by immunization with isolated Fc fragments of IgD myeloma proteins. According to antigenicity of the Fc fragment, lgD myeloma proteins were divided into two different groups. Anti-delta(Fc)-Tl (S.T.) antiserum, absorbed with the Y.S. myeloma protein or serum, either gave a faint precipitin line or failed to react against the isolated IgD myeloma proteins or sera. With the papain-digested lgD mycloma proteins and sera, anti-del-ta(Fc)-T1 antiserum either gave heavy precipitin lines or failed to react. Of twelve papain-digested sera containing lgD myeloma proteins tested, nine (75%) showed positive precipitin lines using anti-delta(Fc)-T1 antiserum. No relationship was found between the two groups of myeloma proteins with respect to IgD levels. SDS polyacrylamide gel electrophoresis of the IgD myeloma proteins (S.T. and Y.S.) showed no difference in the molecular weights of the whole myeloma proteins, and their light and heavy chains. Polyacrylamide gel electrophoresis of the lgD myeloma proteins (ST. and Y.SJ, after treatment with papain, revealed almost the same patterns. [ABSTRACT FROM AUTHOR]

Immunochemical studies were made on the antibodies induced in rabbits against poly(ADPribose) and naturally-occurring antibodies in patients with systemic lupus erythematosus. Antibodies against poly(ADP-ribose) could also be induced in rabbits by oligo(ADP-ribose) associated with rat liver histones and by a complex of poly(ADP-ribose) with methylated bovine serum albumin (MBSA). The two types of antibody were inhibited to the same extent by poly(ADP-ribose). However, the antibody induced by oligo(ADP-ribose) associated with histones was inhibited by oligo(ADP-ribose) with an average chain length of 4 ADP-ribosyl units and by phosphoribosyl adenosine monophosphate (PR-AMP) but not by mono ADP-ribose, whereas that induced by poly(ADP-ribose) was practically not inhibited by these related compounds even in excess amounts. The sera of ten cases of systemic lupus erythematosus showing high antibody activity against poly(ADP-ribose) were also examined immunochemically. It was found that the antibodies of three patients showed a similar inhibitory pattern to that of antibody induced in rabbits by oligo(ADP-ribose) associated with histones, those of three patients showed a similar pattern to that of antibody produced in rabbits by poly(ADP-ribose), and the remainder did not show either pattern. These findings suggest that oligo(ADPribose) associated with histories may serve as antigen to elicit naturally-occurring antibodies to poly(ADPribose) in patients with systemic lupus erythematosus. [ABSTRACT FROM AUTHOR]

Antisera were produced which recognized specifically native type 1 and type II collagens and proteoglycan. These were used in immunofluorescence studies to investigate the distribution of collagens and proteoglycan in intervertebral discs from adult and newborn pigs. Cervical, thoracic and him- bar discs gave similar staining patterns. In the adult, the outer 1 mm of the annulus fibrosus resembled a perichondrium and was negative for type II collagen. The inner regions of the annulus contained proteoglycan and both types of collagen, but these molecules appeared to have separate distributions. The nucleus showed no staining for type I collagen. Newborn pig discs differed from those of the adult in that type II collagen was restricted to the central notochord and to a narrow zone surrounding it. The newborn annulus was negative for type Ii collagen but reacted strongly with antibodies to both type I collagen and proteoglycan. It is suggested that during development of the pig annulus fibrosus, cells producing type II collagen may migrate into this area from the central regions. [ABSTRACT FROM AUTHOR]

Basement membrane (type IV) collagens were extracted from a mouse tumour with acetic acid and from human placenta after limited enzymatic digestion. Antisera were produced against both collagens in rabbits and guinea-pigs and examined by various assays. These antisera were found to be specific for basement membrane collagen and showed little or no cross-reactions with the interstitial collagens, types I, II and III or with human placenta collagen consisting of αA and αB chains. Varying degrees of cross-reaction were observed between antisera to human and mouse type IV collagen. Immunochemical analyses demonstrated the presence of three distinct determinants in the tumour type IV collagen. Rabbit antisera against this antigen reacted with either collagenase-resistant segments or with a collagenous, disulphide-bonded segment (P3). Guinea-pig antisera recognized primarily antigenic determinants in the P3 segment. Antisera to placenta type IV collagen reacted with another collagenous, pepsin fragment (P1) which lacks disulphide bonds. These antisera showed complete cross-reaction with collagenous αI(IV) chains prepared from pepsin-digests of human placenta and bovine lens capsule. [ABSTRACT FROM AUTHOR]

The initiating complement component (C1q) in the classical pathway of 730 subjects and the first essential component (C3) in the alternative pathway of 461 subjects in Japan were examined. The study population consisted of normal healthy newborns, infants, children, adults and the old (from birth up to 75 years of age). In cord sera, both C1q and C3 were about 60 % to the total mean level. At 3 days of age, C1q markedly increased to the mean level which remained relatively invariable up to 40 years of age. And above 40 years, the C1q level increased steadily with age up to about 75 years of age. C3 reached the mean level at about I month of age, and was highest during infancy. This level declined at about puberty and then continued to increase steadily with age up to around 55 years. In normal healthy subjects, a moderate positive rank correlation was found between C1q and C3 amounts. No significant differences of C1q and C3 levels were evident between male and female. No C1q was demonstrable in the colostrum from which lipids were previously removed, but after concentration by precipitation in a chelating agent with a low ionic strength, C1q, measured immuno-chemically, was detectable at concentrations of 300 ng/ml of colostrum. C3 was also detected at concentrations of about 200 μg/ml of colostrum. [ABSTRACT FROM AUTHOR]

Microscopic, histochemical and ultrastructural techniques were used to define characteristics of high endothelial venules (HEV) in rat lymphatic tissues. This endothelium contained acetyl esterase and acid hydrolase activities which were not altered by lymphocyte depletion. No immunoglobulins were detected on luminal surfaces of HEV by fluorescent antibody staining. Only minor structural differences were seen between HEV within lymph nodes and Peyer's patches. At both sites, high endothelial cells were linked together by macular junctional complexes and interlocking basal foot processes. Endothelial cell cytoplasm moulded about surfaces of lymphocytes migrating through the venular wall, and flocculant deposits of basement membrane formed over lymphocytes penetrating the basal lamina. The endothelium was ensheathed by three to five layers of overlapping reticular cell plates and connective tissue. Each plate was linked to the reticular meshwork of the node by collagen bundles and anchoring filaments which inserted into the plate's external limiting membrane. This permitted individual plates to separate or approximate each other as tissue and intravascular pressure varied, and lymphocytes moved across the sheath by insinuating themselves into gaps between overlapping plates. This composite structure of the HEV wall appeared to facilitate lymphocyte entry into the node and minimized vascular leakage. [ABSTRACT FROM AUTHOR]

Immunochemical studies of the intestinal secretory immune system of the chicken have led to further characterization of lgA in bile, intestinal contents and serum. A component was detected in late Sephadex G-200 fractions of caecal and intestinal contents which showed partial identity with bile, intestinal and a high molecular weight fraction of serum IgA. This component showed similar sedimentation characteristics to bovine serum albumin in sucrose density gradients, a fast electrophoretic mobility on polyacrylamide gel and is a possible analogue of mammalian secretory component (SC). Fractionation of serum from birds affected with infectious synovitis revealed two molecular classes of IgA. Comparative double diffusion studies produced a reaction of complete identity between bile IgA and high molecular weight serum IgA (15S) and partial identity with low molecular weight serum IgA (7S), suggesting a lack of an SC determinant on the latter. A spur of partial identity between 15S and 7S serum IgA was also observed. Although no direct structural homology with mammalian or human IgA could be demonstrated by immunological cross-reactivity, the similarities of molecular characteristics, particularly emphasized by the presence of a secretory component, favour a functional analogy between the secretory immune system of the fowl and mammalian species. [ABSTRACT FROM AUTHOR]

In order to characterize the precise structure Within human and rabbit IgG molecules against Which ‘general’ rheumatoid factors are directed, an immunochemical comparison has been made of the effects of the selective substitution of specific amino acid side-chains on various types of antigenicity exhibited by human and rabbit IgG. The ε-amino groups of lysine residues have been substituted by citraconylation and carbamylation; whilst tyrosine residues have been substituted by nitration with tetranitromethane. In this manner, evidence has been obtained which indicates that the autoantigenic determinants of human IgG are structurally distinct from species-specific ones and from certain Fc-located allotypic markers (Gm(a) and Gm(x)). It is also concluded that lysine residues are probably not in the site of IgG reactivity with ‘general’ rheumatoid, in contrast to tyrosine residues which appear to be implicated in the activity of human but not rabbit IgG. [ABSTRACT FROM AUTHOR]

A computer model for simulation of the interactions between a macromolecular antigen and its corresponding IgG has been developed. Thc model takes all possible immune complexes into account, and it calculates the most probable immune complex distribution patterns on the basis of basic thermodynamic principles from the valences and initial concentrations of antigen and antibody, respectively, together with an association constant assumed to be common to all mutual interactions. In antigen excess small antigen-rich complexes are predicted. At or near equivalence a rich variety of relatively small complexes is predicted, while in antibody excess complexes of the type AgAbn are found to be the most probable. By further assuming that the precipitate consists of antibody excess complexes, a precipitin curve can be calculated. The agreement between calculated results and experimentally obtained data is found to be good. It is of special interest that this theory implies that the outcome of immunochemical interactions depend equally well on the concentrations of antigen and of antibody. [ABSTRACT FROM AUTHOR]

Electrophoretic analysis of the serum from a patient Im suffering from a multiple myeloma revealed the existence of two abnormal bands. Purification of these two fractions on a DEAE-cellulose column showed that both fractions had the same sedimentation coefficient (7S), belonged to the same class (IgG), and subclass (γ1) and bore the same allotype (Gm1). However, the heavy, (H) chains of one immunoglobulin were associated with type κ light (L) chains whereas those of the other were associated with type λ L chains. Three individual antigenic determinants were found in these two proteins. One was common to the H chains of both proteins and the other two were each specific to one of the L chains. These results suggest that the myeloma cells of patient (Im) were able to produce at least three polypeptide chains, one H chain and two different types of L chains. [ABSTRACT FROM AUTHOR]

In earlier experiments we showed that lightly hapten-coupled bovine serum albumin (e.g. DNP5-BSA) elicited mainly IgG anti-DNP antibodies and concomitant immunological memory in mice, whereas DNP50-BSA induced a primary IgM response, little IgG antibody and poor memory. Although the latter are characteristic of T cell-independent antibody responses, antibody formation to DNP50-BSA was found to be highly thymus-dependent. In the present study the metabolism of DNP5-BSA and DNP50-BSA was investigated. In vivo DNP50-BSA was rapidly cleared from the bloodstream, but persisted at much higher levels in the liver and spleen than DNP5-BSA. DNP50BSA was taken up more efficiently by macrophage cultures than DNP5-BSA, but released comparatively slowly. Analysis of culture supernatants showed that macrophages degrade both antigens to the same degree. We conclude that heavy dinitrophenylation decreases the susceptibility of DNP-BSA to degradation by lysosomal proteases, but the relationship between the metabolic behaviour of various DNP-BSA conjugates and their immunogenic properties is at present unclear. [ABSTRACT FROM AUTHOR]

Oxazolone-specific cytophilic antibodies in the sera of non-immune CBA mice and mice contact-sensitized with oxazolone, were studied with a rosette test employing peritoneal exudate macrophages and oxazolone-coupled sheep erythrocytes. Macrophage rosettes, produced by direct or indirect cytophilic antibodies, were found to depend on optimally hapten-coupled erythrocytes. Sera obtained 1 week after contact sensitization with oxazolone contained principally 7S IgG2a cytophilic antibodies. Monomeric 7S IgM antibodies cytophilic for macrophages may have been present as well. Primary contact sensitization and boosting was found uniquely to lead to high titres of hyperimmune oxazolone cytophilic antibodies predominantly binding to trypsin-sensitive macrophage receptors. It has been previously shown that specifically sensitized macrophages mediate a component of delayed skin reactions in mice contact sensitized with oxazolone. Since these reactions can be transferred by immune serum or by normal macrophages coated with immune serum, and since acquisition of this passive sensitization can be abolished by prior trypsinization of these cells, it is suggested that 7S IgG2a and/or 7S IgM cytophilic oxazolone antibodies attaching to trypsin-sensitive macrophage receptors, mediate the specific macrophage component of contact sensitivity in mice. [ABSTRACT FROM AUTHOR]

Partial paralysis of the antibody response in guinea-pigs produced by administration of an excessive dose of ovalbumin was accompanied by the formation of non-precipitating IgGI and IgG2 antibodies. Immunochemical properties of these non-precipitating antibodies were compared with those of the precipitating antibodies obtained from animals given a normal dose of ovalbumin. Non-precipitating IgG2 antibodies did not fix complement and non-precipitating IgGI antibodies elicited PCA but with less efficiency than precipitating EgGI antibodies. On the other hand, the ammonium sulphate globulin precipitation technique for the determination of antibody affinity revealed that precipitating and non-precipitating antibodies were similar with regard to valency and average affinity for the antigen. These results are compatible with the hypothesis that non-precipitating antibodies result from a limited recognition of multiple antigenic determinants on ovalbumin molecules. [ABSTRACT FROM AUTHOR]

We have recently described a cytoplasmic form of CD32 (FcγRII) within the vast majority of normal human peripheral blood lymphocytes (PBL) including T cells. The function of cytoplasmic CD32 is not known. These flow cytometric studies were conducted using single cell suspensions of PBL that had been pre-fixed and permeabilized using methanol/triton-X-100. In this study we have attempted to visualize cytoplasmic CD32 by immunocytochemistry using normal PBL processed in various ways and have also looked for CD32 within tissue lymphocytes. Weak cytoplasmic CD32 staining was observed in paraffin sections of normal lymphocytes but only when sections were microwave treated. The intensity of staining for CD32 did however, appear to be much stronger within infiltrating lymphocytes found in autoimmune diseases or in rejecting allografts: an observation that suggests that up-regulation of cytoplasmic CD32 may occur when T cells become activated in vivo. Microwave treatment of PBL suspensions was shown to disrupt the outer cell membrane, thus effectively permeabilizing the cell and allowing for the detection of cytoplasmic components, like CD32, by flow cytometry. Microwave treatment may, therefore, afford an alternative method for cell permeabilization and may prove to be a useful method for the study of cytoplasmic molecules in cell suspensions and in paraffin-embedded tissues. [ABSTRACT FROM AUTHOR]

Three monoclonal antibodies which recognize rabbit leucocytes have been characterized by immunofluorescence staining of a variety of cell populations and also by immunochemical techniques. The evidence obtained suggests that these antibodies recognize the rabbit equivalents of the CD58/LFA-3 (VC21), CD43/leukosialin (L11/135) and CD9 (MM2/57) antigens. A fourth antibody, RPN3/57, recognizes an antigen expressed strongly on T cells, thymocytes and neutrophils and at lower levels on platelets. It has not, however been possible to characterize the antigen recognized by RPN3/57 in molecular terms. Both L11/135 and RPN3/57 are useful reagents for the detection of T cells both by flow cytometry and by immunohistochemistry. [ABSTRACT FROM AUTHOR]

The Leu-19 (CD56) antigen, which is recognized by anti-Leu-19 and NKH-1 monoclonal antibody, is a 200,000-220,000 molecular weight (MW) glycoprotein that is expressed predominantly on human natural killer (NK) cells that mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity. However, cross-reactivity of this antibody has been observed in lung cancer, and in muscle and neural tissues. In the present study, we used the immunoperoxidase technique to examine the expression of Leu-19 antigen in human thyroid epithelial cells. In normal thyroid tissues (n=4), thyroid tissues from Graves' patients (n = 7) and benign thyroid tumours (n = 7), thyroid epithelial cells expressed Leu-19 antigen in all cases. In thyroid papillary carcinoma (n=6) there was no expression in four cases. This staining pattern of anti-Leu-19 antibody is similar to that of antithyroid peroxidase antibody. These findings implicate that the expression of Leu-19 antigen is closely related to the differentiation of thyroid epithelial cells. [ABSTRACT FROM AUTHOR]

A new bovine B-cell differentiation antigen is described that is detected by three monoclonal antibodies (mAb). The antigen is not an immunoglobulin and is precipitated from peripheral B cells as a molecule with an approximate molecular weight (MW) of 120,000 or 145,000 before and after reduction, respectively. Data obtained from two-colour cytofluorimetry and immunohistochemistry confirmed that the antigen was found only on mature B cells and on cells with dendritic morphology in the follicles of the organized lymphoid tissues. Its level of expression is directly correlated with that of IgM on peripheral blood B cells and Theileria parva-transformed B cells. The marker was also expressed on the peripheral cells which expressed surface IgG. Based on the antigen's cellular distribution, biochemistry and histochemistry, it is considered to be analogous to the human CD21 antigen. [ABSTRACT FROM AUTHOR]

The molecular nature and cellular localization of Thy-1 antigen in human renal tissue were studied. Strong immunohistochemical staining was observed in frozen sections of human kidney using monoclonal anti-human Thy-1 antibody; this reaction was almost completely abolished by pretreating the kidney section with phosphatidyl inositol (PI)-specific phospholipase C (PI-PLC). Immunohistochemical analysis revealed that the Thy-1 antigen is localized on the proximal tubular epithelial cells and the Bowman's capsule of the glomerulus. Northern blot analysis of renal mRNA using a cloned human Thy-1 gene revealed the presence of human Thy-1 mRNA of a similar size to the one in human brain. When a human kidney cDNA library was greened with the same probe, a cDNA of human Thy-1 was isolated. Moreover, human Thy-1 protein with a molecular weight (MW) of 21,000 was detected in renal tissue by gel electrophoresis and Western blot analysis using monoclonal anti-human Thy-1 antibody. These data demonstrate for the first time the production of human Thy-1 as a PI-anchored protein with a unique cellular location in human renal tissue. [ABSTRACT FROM AUTHOR]

The immunochemistry of antibody binding to solid-phase immobilized antigen is reviewed. Experimental data are compared with different theoretical models of reaction mechanisms at solid-liquid interfaces. It was found that reactions at the solid-liquid interface can become limited by the diffusion rate due to depletion of reactants close to the surface, even though the intrinsic bimolecular reaction at the surface is reaction-rate limited. The forward reaction-rate constant decreases with increasing concentration of bound antibodies at the surface, and when not limited by diffusion the forward reaction rate can be more than 1000-fold slower than the corresponding reaction in a liquid solution. Some evidence is presented which indicates that the stability of these complexes can be due to attractive lateral interactions between bound antibodies.

The physicochemical features, biosynthesis and glycosylation of sheep class II molecules were investigated using a panel of seven monoclonal antibodies. The class II molecules recognized by different monoclonal antibodies could be differentiated using SDS-PAGE. Two monoclonal antibodies, SBU.H 42-20 and 49-1, reacted with dissociated sheep class II molecules and recognized epitopes on class II alpha and beta polypeptides, respectively. The structure of the sheep class II heterodimer differed from that of mouse and man in that it was unstable in the presence of 1% SDS at 20° and, following reduction, sheep beta polypeptides displayed a marked increase in MW, resulting in the apparent co-migration of reduced alpha and beta polypeptides on SDS-PAGE. This phenomenon was not seen using sheep class II molecules synthesized in the presence of tunicamycin. Pulse-chase analyses of biosynthetically labelled sheep class II molecules suggested the rapid association and glycosylation of sheep class II alpha and beta polypeptides during synthesis. Both alpha and beta polypeptides of sheep class II molecules carried N-linked oligosaccharides of MW 6000 and 3,000, respectively. However, unlike human class II oligosaccharides, these were exclusively of the complex or sialylated type. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to compare the monoclonal antibody (Mab) binding patterns of various tissue macrophages with each other and with blood monocytes. To allow recovery from the effects of the isolation procedure, or to obtain purified populations, macrophages were cultured for 24 hr and 48 hr. For comparison, blood monocytes were also cultured for 24 hr and 48 hr. Mab binding to individual cells, detected by the biotin avidin immunoperoxidase method, was quantified cytophotometrically and the results expressed as the median of the specific mean absorbance per 0.25 µm² cell surface area or as specific integrated absorbance per cell. Analysis of the quantitative data in relation to the results of subjective evaluation of the peroxidase reaction product, demonstrating Mab binding to cells, yielded three classes for description of the intensity of antigen expression by cells: weak (specific mean absorbance per unit cell surface less than 007), moderate (values between 007 and 014), and intense (values more than 014). No matter how the results were expressed, comparison of the Mab binding patterns of macrophages with those of blood monocytes showed that spleen macrophages bound significantly less F4/80 and more M5/l 14 (Ia antigen). Kupffer cells and skin macrophages bound either approximately the same amount or considerably less of the various Mabs than monocytes did. Pulmonary tissue and alveolar macrophages bound significantly more 30.G.12 (leucocyte antigen), M3/38 (Mac-2 antigen), and M3/84 (Mac-3 antigen) and comparable amounts or considerably less of the other Mabs than the monocytes did. Peritoneal macrophages bound significantly more F4/80, M1/70 (complement receptor III), and 2.4.0.2. (Fe receptor II) and comparable amounts or considerably less of the other Mabs than monocytes did. It is concluded that macrophages from different organs and different anatomical sites within one organ differ from one another, for example, peritoneal macrophages do not resemble any other population of macrophages and alveolar macrophages do not resemble pulmonary tissue macrophages, and differentiation of blood monocytes into tissue macrophages does not show a distinct pattern. [ABSTRACT FROM AUTHOR]

Our recent identification of chondroitin sulphate E-containing mast calls (E-MC) in the human colonic mucosa is extended here to the human gastric mucosa by using a combination of both biochemical and immunochemical approaches. Most of the mast cells in human gastric biopsies, which were located mainly around small blood vessels in the submucosa, showed various degrees of degranulation and were granular when stained by monoclonal antibody against chondroitin sulphate proteoglycan. The human gastric mucosa biopsies incorporated (35S)-sulphate into proteoglycans. Cells in the tissues which were histamine-positive also incorporated (35S). The 35S proteoglycans, which were either left associated with the tissue or released into the medium, were found not to be heparin but chondroitin sulphate E. Incubation of the human gastric mucosa biopsies in the presence of anti-human IgE revealed significant enhancement in the release of both (35S)-chondroitin sulphate E proteoglycan and histamine. [ABSTRACT FROM AUTHOR]

The studies reported here demonstrated that adult chickens, when immunized with bovine serum albumin, respond with the production of serum antibodies which will mediate passive cutaneous anaphylactic (PCA) reactions in 10-day-old chicks but not in adults. This difference is at least in part due to differences in histamine sensitivity between chicks and adults. The molecule responsible for this PCA activity was indistinguishable from the predominant 7S chicken immunoglobulin antigenically and by a combination of gel filtration on Sephadex G-200 and ion exchange chromatography on DEAE-cellulose. However, the PCA activity is apparently biologically separable from portions of the 7S serum immunoglobulin population since this activity has never been elicited by immunoglobulins derived from yolk. On the basis of stability to denaturing treatments and short tissue fixation time periods, the chicken PCA-mediating antibodies appear to resemble the γG1 homocytotrophic antibodies of mice and guinea-pigs instead of the IgE-type reagins found in some mammals. Therefore, PCA-mediating anti-bodies appear to be a subpopulation of the chicken 7S immunoglobulins, distinguishable by its inability to pass into the egg yolk but indistinguishable by standard chemical and immunochemical criteria. [ABSTRACT FROM AUTHOR]

Antibody production in the African clawed toad (Xenopus laevis) against Salmonella flagella and the immunochemical properties of the antibodies were studied. The titre of the serum antibodies increased slowly and reached a maximum at 3 weeks after immunization and they were detectable even 20 weeks after antigenic stimulation. No protein components were electrophoretically demonstrated at the region corresponding to the IgG of mammalian antibodies. Two types of antibodies were found; the one produced early after immunization was heat- labile and the other produced later was heat-stable. The former was of low molecular weight (7S component) and the latter was macromolecular (19S component). Both antibodies moved electrophoretically to the cathode and were sensitive to 2-mercaptoethanol (2-ME) treatment, and their antigenicities were not identical. The low molecular weight antibody did not fix complement in the presence of antigen, in contrast to the macromolecular antibody. The low molecular weight antibody did not correspond to the IgG type of mammalian antibodies in the following properties; 2-ME sensitivity, heat-sensitivity, electrophoretic mobility and absence of complement-fixing ability. [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]

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]

A highly reproducible method for obtaining a relatively homogeneous preparation of keyhole limpet haemocyanin (KLH) is described. This method employs ion-exchange chromatography on DEAE-cellulose followed by gel filtration on agarose beads. Analysis by immunoelectrophoresis in agar, electrophoresis in polyacrylamide gels, analytical ultracentrifugation and double diffusion in agar indicated that the purified preparation contained one major antigenic component, whereas the crude material contained multiple antigenic components. The crude KLH was found to be a more potent immunogen than the purified preparation upon primary stimulation of rabbits. [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]

Rat brain myelin purified by ultracentrifiagation in a sucrose gradient was solubilized with lysolecithin. The water soluble protein-lipid complexes obtained were antigenic and encephalitogenic and in a complement fixation system reacted more efficiently with anti-myelin sera than intact myelin itself. Increasing the lysolecithin/myelin ratio above the minimum required to render myelin completely soluble reduced the amount of complement fixed. This suggests that solubilization increases the number of accessible antigenic sites, while further addition of lysolecithin to the molecular surface interferes with its antibody combining capacity. Following isopycnic centrifugation the maximum complement-fixing activity was present in a fraction having a density of 1.04 at 10°, corresponding to one of three peaks of galactocerebroside concentration. Antibody to lysolecithinized myelin reacts with the myelin moiety of the complex and not with lysolecithin, since it can be removed by absorption with particulate myelin and it will not react with lysolecithinized red cell ghosts. Anti-rat myelin antiserum cross-reacts with lysolecithinized myelin from other mammalian species; to a lesser degree with frog, and only minimally with dogfish myelin. It is suggested that solubilization by lysolecithin may prove a convenient starting point for immunochemical studies on myelin by providing lipid-protein complexes which reflect the structure of the native material. [ABSTRACT FROM AUTHOR]

(1) Immunological reactivity to sheep red blood cells was tested in three congenic resistant strains of mice and was found to vary according to the H-2 locus and its alleles. The reactivity of the strains in decreasing order is: A. CA, A/Sn, A. BY. (2) Non-specific immunostimulation with Brucella increases the immune response to sheep red blood cells in the A. CA and A/Sn strains but not in strain A. BY. This immunostimulation with Brucella is H-2 dependent. (3) Methylcholanthrene has an immunodepressive effect on the immunological reactivity to sheep red blood cell antigens in the three strains of mice, each strain being equally affected. [ABSTRACT FROM AUTHOR]

A single antigen-antibody complex of Toxoplasma gondii was isolated using irnmunochemical techniques. Subsequent to isolation, a monospecific antibody was produced against the antigen component in this complex. [ABSTRACT FROM AUTHOR]

Rabbit thyroglobulin was enzymatically digested with trypsin, and fragments were isolated through gel filtration. A 10-hour enzyme digest was studied in detail. Upon examination in a synthetic-boundary cell in the ultracentrifuge it revealed a predominantly slow sedimenting component with a 1 S value. The various fragments of this digest were tested against three different antisera, i.e. heteroantisera to whole thyroglobulin, and m fragments of thyroglobulin, and an isoantiserum, containing autoantibodies, to whole thyroglobulin. The thyroglobulin fragments reacted strongly with the heteroantisera, especially with the antiserum to thyroglobulin fragments, but the same fragments reacted very poorly with the autoantibodies. The former antiserum revealed six lines of precipitation whereas the latter antiserurn showed only two lines. These observations supported the hypothesis that a lesser number of antigenic determinants on the thyroglobulin molecule were active against autoantibodies as compared to the heteroantibodies. [ABSTRACT FROM AUTHOR]

Measurements of the nine components of complement in the serums of 16 members of a kindred have established the diagnosis of hereditary deficiency of the second component of complement (C2). The autosomal recessive mode of inheritance resembles that of previously described families with C2 deficiency. Both C2 activity determinations with a stoichiometric haemolytic assay and C2 protein measurements with electroimmunodiffusion against antibody monospecific for C2 detect the heterozygous deficient state. Antigenic analysis, in vitro reconstitution experiments, and the constant ratio of C2 function to C2 protein indicate that the C2 synthesized by heterozygotes is indistinguishable from normal human C2. Studies of neonatal homozygous deficient serum and maternal heterozygous deficient serum show that transplacental passage of C2 does not occur. C2 deficiency in this family is not associated with clinical defects in host resistance. [ABSTRACT FROM AUTHOR]

The present study of human colostrum and human milk showed that normal milk contains a considerable amount of free secretory piece in addition to IgA-bound secretory piece. This free S-piece does not show complete, but only partial identity with bound S-piece. An antiserum to the specific determinants of this free S-piece could be prepared, which antiserum will no longer react with bound S-piece. It is argued that the molecular weight of free S-piece occurring in human milk is approximately 90,000, and it seems likely that free S-piece is present in a tetrameric form. The antigenic differences between free and bound S-piece are probably due to differences in quaternary structure between the tetrameric free form and the IgA-bound form. [ABSTRACT FROM AUTHOR]

The physical and chemical nature of the bovine brain BE antigen was studied, along with antigenic studies to verify the organ-specificity characteristics. These preparations were found to be quite heterogeneous physico-chemically, containing nucleic acids, carbohydrates, protein and possible lipid constituents. The extinction coefficients were found to be 12·;0 (at 280 my) and 2·;2 (at 540 mμ, biuret). Ultracentrifugal analysis revealed three major components, with extra- polated sedimentation coefficients of 8·60S, 2·20S and 1·80S. Electrophoretic analysis showed six (or more) components. Because of the heterogeneity chemical classification of the actual brain-specific antigen was determined by degradative studies, using chymotrypsin and periodate. Based on thermostabiity, a carbohydrate content of 4·6 percent, and destruction by dilute chymotrypsin, the brain-specific antigenic component has been tentatively classified as a mucoprotein.

The immunization of hybrid rabbits under subtropical climatic conditions, confirmed previous findings indicating the existence of common 'O' protein determinants of Salmonella typhi, Salmonella enteritidis and Salmonella typhimurium. The antisera from these rabbits however, cross-reacted differently from those previously, under European climatic conditions, with the same Salmonella species. The previously found pattern of stronger precipitation with heterologous than with homologous antigens of the sera to S. enteritidis was not obtained in the present experiments, and the sera to S. typhi—in contrast to the previous results—reacted in agar gel with the antigens of S. enteritidis and Salmonella dublin. [ABSTRACT FROM AUTHOR]

Shigella flexneri rough mutants were divided into rough serotypes 1, 2, 3 and 4 by studying the homologous and heterologous reactions of their lipopolysaccharide components in a complement fixation test. The four serotypes corresponded with the classification of these lipopolysaccharides into four chemotypes on the basis of their qualitative sugar composition. As the molecular structures of the four chemotypes have been elucidated, the proposed classification of Sh. flexneri R-mutants was related to their chemical structure as well as their serological behaviour. [ABSTRACT FROM AUTHOR]

The synthetic polypeptide-antibody precipitin curves indicate the presence of multiple antigenic systems in the polypeptides: a typical precipitin curve can be analyzed into a minimum of four discrete antigen-antibody systems. Prolonging the course of immunization brings out the weaker antigenic systems and accounts for the observed broadening of the precipitin curve. Longer periods of incubation of the antigen-antibody reaction (20 days) also enhance the precipitation of the weaker antigenic systems. Dilution of the antibody results in a non-linear change in precipitable antibody concentration. Increase in the viscosity of the reaction medium with sucrose up to approximately 5 cp at 4° has little effect on the precipitin reaction. [ABSTRACT FROM AUTHOR]

water soluble extract of rat bone marrow was compared with similar extracts of several other organs of the reticuloendothelial system by simple electrophoresis in agar gel and by immunoelectrophoretic analysis followed by selective staining of chemical and enzymatically active constituents. Antiserum prepared against the soluble bone marrow extract showed extensive cross-reactions with extracts of the other organs used in the study; the extracts possessing the most antigenic constituents in common with marrow were those of spleen and peritoneal exudate cells. Immunoelectrophoretic analysis of the marrow extract, combined with absorption of the anti-marrow serum with extracts of the other organs, permitted the identification of the antigens common to marrow, spleen, peritoneal exudate cells, lymph node, thymus, liver and heart. Every antigen detected in bone marrow was found in at least one other organ extract and several of these antigens were found to possess enzymatic activities. Transferrin was the only serum protein identifiable in large amount in extracts of well washed bone marrow cells. [ABSTRACT FROM AUTHOR]

Antiserum prepared by immunization of rabbits with chymotrypsinogen A reacted in equal titre with the proenzyme and alpha, beta, delta and gamma chymotrypsin in serological tests. Inactivation of the four enzymes by diisopropylfluorophosphate did not diminish their precipitability. Immunodiffusion tests in gel plates with the proenzyme produced a single precipitin band which merged without spur formation with the bands produced by each of the native and inactivated enzymes. The antiserum partially inhibited activation of the zymogen by trypsin. The proteolytic activity of each of the four chymotrypsins was also inhibited by the antiserum. [ABSTRACT FROM AUTHOR]

Human γ globulins are shown by means of special antisera and papain digestion to consist of three to five different species which share only some of their determinants. A minimum total number of eight determinants is suggested. Only four of these determinants are possessed by the bulk of the serum globulins and one of these four determinants is shown to be shared with the cryoglobulin of a Waldenstrom serum. This cryoglobulin is shown to have a further minimum of two determinants shared with one of the subsidiary γ globulins occurring in normal human sera, as well as at least one specific determinant not found in normal sera. Other macroglobulinaemic sera are shown to have variable amounts of globulins antigenically related to this cryoglobulin and even normal sera appear to have traces of such macroglobulins. All γ globulins appear to be split more readily than other serum proteins, but the Waldenström macroglobulin more readily still. It is suggested that the slight forking of the γ-globulin line often seen in immunoelectrophoretic patterns near the cathode side may be due to the existence of antigenically related, but not identical, γ globulins whose range of electrophoretic mobilities does not quite overlap there and may vary in different subjects. The previously suggested spontaneous or enzymatic cleavage remains another possible explanation. [ABSTRACT FROM AUTHOR]

Fowl antibodies to rabbit γ globulin, bovine serum albumin and to ε toxin were studied by means of their reactions with rabbit antisera to fowl globulin. Specific precipitates redissolved either in antigen excess or in solutions of lower salt concentration than those in which they had been formed were used to measure the sedimentation and diffusion coefficients and the electrophoretic mobilities of the soluble complexes of fowl antibody with antigen. Fowl antiserum to any one antigen was found to contain two types of homologous antibodies, derived from different constituents of serum, having the same electrophoretic mobility but giving two distinct bands of precipitation with rabbit antifowl-globulin serum. The values obtained for the molecular weights of soluble complexes indicate that one type of antibody has a molecular weight of about 600,000 and the other 180,000, and also suggest that the latter type combined with only one molecule of antigen in antigen excess. Both types of antibody were precipitated by antigen in 0.9 and 9 per cent NaCl; the larger precipitates formed at the higher salt concentration contained more of the low molecular weight antibody, together with another component that was neither antigen nor antibody. [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]

Monoclonal antibodies have been produced that bind to four different antigens expressed on the surface of ovine lymphocytes as well as to a variety of other ovine cell types. These antigens have been characterized with respect to their tissue distribution and immunochemistry. The timing of appearance of these antigens within the ovine embryo is reported. [ABSTRACT FROM AUTHOR]

Vaccination is considered the most effective medical intervention. In the last century it eliminated or eradicated most of the infectious diseases responsible for much of human morbidity and mortality during recorded history. Most of the vaccines have been developed following the principles set by Pasteur to isolate, inactivate and inject the microorganism causing disease. While these technologies have been very successful in the past and will continue to dominate the short term future of vaccination, the progress in the understanding of the immune system and the availability of the genetic code of microorganisms provide new opportunities allowing to develop vaccines against diseases that have been difficult to tackle following Pasteur's principles. Today novel vaccines can be designed from the genome of microorganisms (reverse vaccinology) and can be made more potent by using novel adjuvants and immunostimulatory molecules targeting the innate immune system. In general, novel vaccines also have an excellent safety profile. The possibility to manipulate the immune system by powerful immunostimulatory molecules also opens the way to tackle by therapeutic vaccination chronic infectious diseases like HCV, HIV and cancer. [ABSTRACT FROM AUTHOR]

Editorial. Comments on the application of immunological techniques to problems of clinical diagnosis. Need of a ready supply of appropriate serological reagents; Complexity of immunochemical reagents; Recommendations for specifications for anti-immunoglobulin antisera for use in gel diffusion analysis and in immunofluorescence.