Hemagglutination Inhibition Experiments Research Articles

IMMUNOCHEMICAL OBSERVATIONS ON THE HUMAN BLOOD GROUP P SYSTEM

Haemagglutination inhibition experiments were carried out with anti-P1, anti-Pk and anti-P sera in an attempt to increase understanding of the chemical, genetical and serological relationships within the P system. The test-substances comprised a glycoprotein with human blood group P1 and Pk activity isolated from sheep hydatid cyst fluid, fragments isolated from the partial acid hydrolysis products of the P1Pk active glycoprotein, glycolipids, monosaccharides and di- and oligo-saccharides of known structure. The trisaccharide alphaGal(1 leads to 4)betaGal(1 leads to 4)GlcNAc isolated from the glycoprotein hydrolysis products, and earlier established as the P1 determinant (Cory et al., 1974), was the only low molecular weight compound that gave strong inhibition with human, rabbit and goat anti-P1 sera. A disaccharide alphaGal(1 leads to 4)Gal, also isolated from the glycoprotein hydrolysis products, failed to react with anti-P1 reagents but inhibited human anti-Pk sera as strongly as the trisaccharide. The glycolipid alphaGal(1 leads to 4)betaGal(1 leads to 4)Glc-Cer (Ceramide trihexoside) and other oligosaccharides containing alphaGal(1 leads to 4)Gal at the non-reducing terminal were also strong inhibitors of anti-Pk sera. Oligosaccharides with terminal alpha-galactosyl residues joined in other positional linkages gave definite, although less strong, inhibition. The inhibition results suggest a close structural relationship between the P1 and Pk determinants and indicate that the specificity of anti-Pk sera is less closely delineated than that of anti-P1. Human anti-P sera differed markedly from anti-P1 and anti-Pk and were not inhibited by any of the compounds containing alpha-galactosyl residues. The glycolipid betaGalNAc(1 leads to 3)alphaGal(1 leads to 4)betaGal(1 leads to 4)Glc-Cer (globoside) strongly inhibited the anti-P sera. The inhibition of anti-Pk and anti-P sera by ceramide trihexoside and globoside, respectively, confirms the observations of Naiki & Marcus (1974) and supports their conclusions that Pk is the precursor of P. The genetic relationship of the P1 antigen to P and Pk is not clear but biosynthetic pathways are discussed that might explain the absence of P1, Pk and P antigens in individuals of the p phenotype.

Common enterobacterial antigen in Yersinia enterocolitica.

Production of the common enterobacterial antigen (CA) by strains of Yersinia enterocolitica (Y.e.) serotypes 3 and 9 (Winblad), by strains of 5 different Y.e. serogroups (Wauters) and various other bacteria was examined. Antibody against CA was raised by immunization of rabbits with E. coli O 14. Extract prepared from S. typhimurium was used for the sensitization of sheep erythrocytes with CA. Absorption and haemagglutination inhibition experiments showed that CA could be detected in heat extracts from all Y.e. strains examined, and in that from Yersinia pseudotuberculosis. CA was not detected in extracts from Pasteurella multocida, Francisella tularensis, Brucella abortus, Acinetobacter calcoaceticus or Pseudomonas aeruginosa. Anti-CA antibodies could not be demonstrated in serum from rabbits immunized with Y.e. bacteria, but were demonstrated in serum from rabbits immunized with a CA-containing fraction of the Y.e. extract. The possibility of participation of anti-CA antibody in indirect haemagglutination tests for detection of antibody to Y.e. O antigens is emphasized.

Catfish antibodies to blood group substances. II. Cross-reacting anti-A and B antibodies.

Abstract. A sample of normal serum and some anti‐B secretor saliva antiserum, both obtained from the Australian freshwater catfish Tandanus tandanus Mitchell, were used in haemagglutination inhibition experiments with human secretor and non‐secretor fluids, blood group substances and some simple sugars. These experiments, together with immunodiffusion studies, clearly showed that the sera examined contained antibodies reacting with both the A and B, but not with the H, blood group antigens.The results obtained are discussed with particular reference to the findings and speculations of others who have studied such cross‐reacting antisera.

Catfish Antibodies to Blood Group Substances

A sample of normal serum and some anti-B secretor saliva antiserum, both obtained from the Australian freshwater catfish Tandanus tandanus Mitchell, were used in haemagglutination inhibition experiments with human secretor and non-secretor fluids, blood group substances and some simple sugars. These experiments, together with immunodiffusion studies, clearly showed that the sera examined contained antibodies reacting with both the A and B, but not with the H, blood group antigens. The results obtained are discussed with particular reference to the findings and speculations of others who have studied such cross-reacting antisera.

Bacteriophage attachment sites, serological specificity, and chemical composition of the lipopolysaccharides of semirough and rough mutants of Salmonella typhimurium.

Extracted lipopolysaccharides (LPS) from one smooth, one semirough, and five rough mutants of Salmonella typhimurium LT2 or LT7, for which the chemical structure of the polysaccharide chain had been elucidated by using methylation analysis, were characterized with passive hemagglutination inhibition and phage inactivation experiments. Each addition of a sugar residue to a LPS from chemotype Rc was reflected in changed serological reactivity and phage-inhibiting activity of a collection of bacteriophages of the isolated LPS. Thus, certain criteria can be established for a classification of rough mutants of S. typhimurium. The observation that the serological RII specificity corresponds to a completed common core polysaccharide was verified. The serological RI specificity was found in LPS with terminal d-galactose I residues. One of the mutants, SL733, yielded a LPS which cross-reacted with anti-O5 factor serum although the polysaccharide was virtually free from contaminating O-specific material. The O5 reactivity was destroyed by alkaline treatment of SL733 LPS. The smooth- and rough-specific Felix O-l (FO) and the rough-specific 6SR and Br2 phages were shown to have their receptors in the LPS. There was a good correlation between the adsorption rate constant to whole cells and the phage inhibiting activity of isolated LPS suggesting that the LPS exert the major influence on the attachment of these phages to the bacteria. The polysaccharide structures in the LPS necessary for attachment of the 6SR and Br2 phages were defined. It was found that measuring the phage-inhibiting properties of isolated LPS as PhI(50) (LPS concentration required to inactivate 50% of the phages under defined conditions) was a more sensitive method for a characterization of the LPS than the serological and chemical assays used.

Toxic Constituents of Neisseria gonorrhoeae

IN 1959, Tauber and Garson reported the extraction and isolation of a lipopolysaccharide endotoxin from whole cells of Neisseria gonorrhoeae1. Maeland has reported the extraction of endotoxin of N. gonorrhoeae by several methods and has shown that the endotoxin contains at least two antigenic determinants as revealed by haemagglutination inhibition and absorption experiments. One determinant is a polysaccharide and the other a protein2.

Immunological studies in ulcerative colitis. IV. Origin of autoantibodies.

The incidence and height of antibody titers to colon, assayed by indirect hemagglutination with a heat stable colon extract from germ free rats, is significantly higher in sera from patients with ulcerative colitis than in those from healthy controls or from patients with amebic liver abscess or dysentery. While sera from ulcerative colitis patients and controls are indistinguishable in regard to incidence and height of antibody titers to Forsman antigen, Staphylococcus aureus S 209, Clostridium difficile, and several common strains of E. coli, they have elevated titers and increased incidence of antibodies to a heat stable antigen of E. coli O14. Patients with amebic dysentery have normal titers of such antibodies. Absorption of patients' sera with E. coli O14 antigen inhibits the colon directed hemagglutination reaction in approximately 30% of the cases tested. Likewise, the anti-E. coli O14 reaction can sometimes be inhibited with the colon extract. Other E. coli strains and other bacteria are inactive or have only weak inhibitory activity. Hemagglutination inhibition experiments show that germ free rat colon and E. coli O14 contain common structures, depicted by antibodies in the patients' sera. This pattern of reactivity closely resembles that seen in rats made autoimmune to colon by injection of newborn rabbit colon. E. coli O14 is known to carry a heterogenetic antigen present in lower concentration (or activity) in most Enterobacteriaceae. Hemagglutination inhibition experiments with rabbit antisera to E. coli O14 suggest that the antigen common for E. coli O14 and colon is related to this heterogenetic antigen. The findings imply that this antigen, which is constantly present in low concentrations in the human colon, may give rise to anticolon antibody formation in ulcerative colitis through breakage of tolerance. Since this antigen is present in healthy individuals as well, additional factors are required to explain the induction of anti-colon autoimmunity in ulcerative colitis.

Disaggregated Human Erythrocyte Membranes and Blood Group Activity

Summary Twice lyophilized human erythrocyte membranes were solubilized or disaggregated with an anionic detergent, sodium duodecyl sulfate, and the latter completely removed by anionic exchange chromatography with Dowex 2. Gel filtration of the solubilized material in detergent showed multiple peaks indicating a diversity of molecular size. Gel filtration of the solubilized material after detergent removal showed a single sharp peak coming off with the void volume (molecular weight equal or more than 300,000). After detergent removal, blood group A, B and O (H) activities were shown to be present by hemagglutination inhibition experiments. M and N activities were also seen, but not Rh antigen activity.

Autoantibodies to Colon in Rats and Human Ulcerative Colitis: Cross Reactivity with Escherichia coli O:4 Antigen

SummaryAutoantibodies to germ free rat colon were produced in rats by repeated intraperitoneal injections with newborn rabbit colon in Freund's complete adjuvant. Antibodies were assayed by means of indirect hemagglutination of sheep erythrocytes sensitized with a heat stable phenol-water extract of germ free rat colon or by fluorescent antibody staining of rat colon sections. The response of rat colon injected rats was less intense. A few of the rats injected with adjuvant alone also developed an anti-colon titer. Hemagglutination inhibition experiments demonstrated that the antibodies were specific for gastrointestinal antigen and represented autoantibodies in a true sense. They were found to be specific for at least three different colon determinants: one unique for germ free rat colon, one common for rat and rabbit colon, and one common for both colons and a polysaccharide from Eschericria coli O:14, grown on synthetic medium. No or only very weak cross reactions appeared with similar antigens extract...

Studies of urinary tract infections in infancy and childhood: VII. The relation of E. coli antibodies in pyelonephritis as measured by homologous and common (Kunin) antigens

Summary By means of a hemagglutination test theresponse to an antigen (CA), common to most E. coli O types and to several other Enterobacteriaceae , has been followed in 22 patients with pyelonephritis. The results have been compared to simultaneous determinations of the highly type-specific homologous coli antibody reaction. A titer to CA exceeding 1:16—the highest titer found in 36 uninfected controls—was observed in only seven patients. Two patients showed an elevation in the CA-antibody titer at the same time as the maximum rise in the homologous antibody titer, though at a much lower and less significant level. Relatively high titers of CA-antibodies were found in two patients with frequently relapsing pyelonephritis. In hemagglutination inhibition experiments,employing CA as inhibitor, slight inhibition was observed in 16 of the 28 serum samples tested, while strong inhibition was found in all when the reaction was inhibited with the homologous antigen. It is concluded that homologous antigencan not be substituted by CA in the study of the serologic reaction of patients with parenchymal E. coli urinary tract infections. Further, it does not seem necessary in clinical studies to take special precautions to neutralize antibodies to CA before estimations of the type-specific antibodies are performed.

Immunological studies in ulcerative colitis. II. "Colon" antigen and human blood group A- and H-like antigens in germfree rats.

Sera from patients with ulcerative colitis contain antibodies which hemagglutinate sheep red cells, sensitized with phenol-water extracts from. colon, cecum, or feces of germfree rats. Minor concentrations of such antibodies are also present in a certain fraction of normal human sera. Hemagglutination and hemagglutination inhibition experiments with human erythrocytes and with the rat extracts showed that the latter contained an antigen similar to human blood group A antigen. In contrast, a blood group B-like antigen could not be detected in these extracts. However, experiments with eel serum indicated that these extracts also contained an antigen similar to the H antigen of the human ABO system. Absorption of ulcerative colitis sera with human A(1) erythrocytes but not that with B or O erythrocytes gave, in a few cases, a slight reduction of the hemagglutinating titers against rat cecum-sensitized sheep erythrocytes. In contrast, this treatment considerably reduced such titers when found in sera from healthy persons or from patients with unrelated diseases. It could be concluded that the rat extracts also contained a "colon" antigen, detected with antibodies, present at elevated titers, in the sera of ulcerative colitis patients, but not in those of the controls. This colon antigen is immunologically distinct from the blood group antigens studied. Hemagglutination inhibition experiments indicated that A, H and colon antigen were widely distributed throughout the gastrointestinal tract of the germfree rats. The colon antigen was found to be enriched in the extracts from colon, cecum, and feces. Fluorescent antibody staining provided evidence that both the colon antigen and the A antigen were present in similar sites of the colon and cecum mucosa, particularly in goblet cells of the crypts, and in the mucus.

A study of the immunological determination of pituitary gonadotrophin in urine.

ABSTRACT An antiserum to chorionic gonadotrophin (HCG) has been studied in agar diffusion, haemagglutination and haemagglutination inhibition experiments. The anti-HCG serum contained antibodies to inert urinary proteins and serum proteins as well as a »hormone-specific« antibody. Absorption of the anti-HCG serum with small amounts of inert urinary protein removed precipitins to non-hormonal protein, without altering the haemagglutinating titre of the antiserum. There was no difference between absorbed and unabsorbed anti-HCG serum in haemagglutination inhibition reactions with inert and gonadotrophic urinary extracts. When larger amounts of inert protein were used for absorption there was a fall in the haemagglutinating titre of the anti-serum but it was shown that this was due to removal of »hormone specific« antibodies. Urinary extracts were obtained from patients with various endocrine disorders, from patients with normal pituitary and gonadal function and from menopausal subjects. The results of bioassay and immunoassay of these extracts were compared. It was found that many of the extracts, although potent in the immunoassay, were biologically inactive. It is concluded that a specific or non-specific reaction can occur between nonhormonal urinary proteins and the antihormone. The anti-HCG serum used in the present investigation, although successfully used for pregnancy diagnosis and HCG assay, was unsuitable for urinary gonadotrophin estimation in non-pregnant individuals.

SELECTIVE DESTRUCTION BY PSEUDOMONAS AERUGINOSA OF COMMON ANTIGEN OF ENTEROBACTERIACEAE.

Whang, H. Y. (Children's Hospital, Buffalo, N.Y.), and E. Neter. Selective destruction by Pseudomonas aeruginosa of common antigen of Enterobacteriaceae. J. Bacteriol. 88:1244-1248. 1964.-Supernatant fluids and filtrates from several strains of Pseudomonas aeruginosa, when incubated with common antigen from various enteric bacteria, will produce selective destruction of this antigen, leaving intact the simultaneously present O antigen. This effect is evidenced from hemagglutination, hemolysis, antibody absorption, hemagglutination inhibition, and immunization experiments. The Pseudomonas factor is heat-labile, being destroyed by heating at 100 C for 10 min. The action of this factor on the antigen is time-dependent and temperature-dependent, requiring incubation for 18 to 72 hr and progressing more rapidly at 50 or 56 C than at 37 C. The specificity of the reaction is indicated by the fact that supernatant fluids from cultures of Staphylococcus aureus and Bacillus subtilis, as well as several enzymes, such as pancreatic protease, trypsin, and lipase, have no effect on the common antigen. Utilization of the Pseudomonas factor, conceivably an enzyme, may aid in the eventual elucidation of the chemical nature of the common antigen of enteric bacteria.

Immunogenicity and Specificity of Collagen

Summary Acid-soluble collagen (procollagen), collagen treated with hydroxylamine (“hydroxam-collagen”) and parent gelatine of calf origin, both from skin and joint capsule-periarticular connective tissue, were used to immunize rabbits. These preparations and similar ones prepared from rabbits were used to test the resultant antisera in passive hemagglutination, along with ovalbumin and bovine γ-globulin as specificity controls. The various preparations were examined for their content of hydroxyproline, tyrosine, hexoses, uronic acid and nitrogen. Procollagen induced mainly the formation of species-specific collagen antibodies, reacting also with hydroxam-collagen and parent gelatine of the same species. Hydroxam-collagen induced the formation of antibodies with a new specificity, that against a collagen preparation after hydroxylamine treatment. Hyperimmune sera reacted only with “hydroxam”-collagens. Early immune sera, however, showed cross-reactions with parent gelatine. Parent gelatine induced formation of antibodies against collagen preparations and their derivatives, having no species specificity and showing a broad spectrum of reactivity with gelatine and collagens. Used in serologic testing, however, parent gelatine also displayed properties of species specificity. No differences in specificity were found between preparations made from skin and from joint capsule-periarticular connective tissue. Hemagglutination-inhibition experiments were performed in addition. The results confirmed the observations made in direct hemagglutination: Hemagglutinating systems of anti-procollagen sera and calf-collagen preparations were inhibited only by species-specific (calf) parent gelatine and hydroxam-collagen. Hemagglutinating systems of early anti-hydroxam-collagen sera and hydroxamcollagen were inhibited significantly by hydroxam-collagen, less distinctly by parent gelatine. Identical systems prepared with hyperimmune sera, however, were inhibited only by hydroxam-collagen. Hemagglutinating systems of anti-parent gelatine sera and parent gelatine or hydroxam collagen of different origin were inhibited by all collagen preparations or collagen derivatives independently of their species or tissue origin. The results obtained are discussed in view of our present knowledge about the structure of collagen or its derivatives.

Demonstration of an epithelial antigen in colon by means of fluorescent antibodies from children with ulcerative colitis.

Thirteen sera from children with ulcerative colitis were examined for antibodies reacting with constituents of human colonic tissue by means of immunofluorescent methods. 3 out of 10 sera reacted positively when tested by the direct staining method while 6 out of 13 reacted positively when tested by the indirect method with conjugates of rabbit anti-human gamma globulin. The specificity of the reactions could be confirmed by inhibition tests. 16 sera from healthy children and adults yielded completely negative results. The staining capacity of various sera was correlated to their hemagglutinating titer when they were tested with sheep erythrocytes, coated with phenol-water extract of human colon. Absorption experiments indicated that the stainable antigen was also present in the extracts used for the hemagglutination experiments. In unfixed tissue sections, fluorescent antibodies were adsorbed onto the epithelial cells of the mucosa. Adsorption on epithelial basement membranes could not be demonstrated. Fluorescent H agglutinins, isolated from eel serum, were adsorbed onto the same mucosal structures of human colon (blood group O) as the antibodies in the sera of patients with ulcerative colitis. However, any immunological relationship between H substance and the colonic antigen of ulcerative colitis could be ruled out by cross-inhibition and hemagglutination inhibition experiments. Fluorescent serum from patients with rheumatoid arthritis also stained sections of human colon but the localization of the stainable antigens was different from that visualized with the ulcerative colitis sera. Inhibition experiments indicated that the rheumatoid arthritis serum contained antibodies staining colon antigens different from those reacting with antibodies in the ulcerative colitis sera. Sera from patients with systemic lupus erythematosus or with the nephrotic syndrome, which all hemagglutinated erythrocytes coated with colon extract, did not stain the sections of the colon tissue either because of suboptimal antibody concentration or because of a difference in type or localization of the antigen.