Titration of Cholera Antitoxin in Human Sera by Microhemagglutination with Formalinized Erythrocytes

Microtiter hemagglutination tests employing formalinized sheep erythrocytes sensitized with either crude or purified cholera toxin were used to assay the cholera antitoxin content of human sera. Comparable results were obtained with either crude or purified toxin-sensitized cells with the exception of two sera that gave unusually high hemagglutination titers with the crude toxin. Sera from 13 convalescent cholera patients showed a high degree of correlation between antitoxin levels as determined in vitro by the hemagglutination test and in vivo by the skin permeability factor neutralization test. Fourfold or greater rises in antitoxin levels between acute and convalescent sera were detected in 9 of 15 patients with bacteriologically proven cholera. No significant increases in titer were observed in 14 cases of noncholera diarrhea. Cholera antitoxin was detected by hemagglutination in only 1 of 33 sera, obtained from eight countries, containing vibriocidal antibodies. Formalinized sheep erythrocytes sensitized with toxin and stored at 4 C in the presence of 1:10,000 thimerosal were stable and sensitive for at least 6 months (the longest time tested).     

Echinococcus granulosus: host lymphocyte transformation by parasite antigens.

Lymphocyte transformation, measured by in vitro tritiated thymidine incorporation, and indirect hemagglutination tests were carried out on hydatid patients and normal individuals using sheep and human hydatid fluid or scolex antigens. The hydatid patients showed statistically significant lymphocyte transformation with human and sheep hydatid fluid or scolex antigens when compared to normal individuals. The indirect hemagglutination tests resulted in high titers of antibody with sheep or human hydatid fluid antigens, while very low titers were obtained with scolex antigens. Unlike in the indirect hemagglutination test, the source of the antigen, scolex or fluid, was not of consequence in the lymphocyte transformation test. Furthermore, there was no correlation between the results of the serologic and lymphocyte transformation tests, since some patients with very high lymphocyte stimulation indices produced low indirect hemagglutination titers and vice versa. Similar results were obtained from rabbits which were immunized with sheep hydatid fluid or scolex extracts. The skin tests were of the immediate type of hypersensitivity reactions. Delayed skin reactions did not occur in spite of the presence of sensitized lymphocytes in the blood of the immunized rabbits.     

Titration of tetanus antitoxin by passive hemagglutination. I. Titration of guinea-pig antitoxins at various periods of immunization.

An improved technique for passive hemagglutination (HA) for titration of tetanus antitoxin was described. The use of highly purified tetanus toxoid and of improved diluent increased the specificity and reproducibility of the test. Several hundreds of specimens of guinea-pig serum taken at various stages of immunization were titrated by HA and toxin neutralization (NT) in mice. The ratio of HA to NT titers varied significantly depending on the immunization stage; higher at early stages and lower at later stages. The high HA/NT ratio was not due to the IgM antitoxin, which is very rare in guinea pigs. The variation in discrepancy between HA and NT titers decreased considerably by grouping the serum specimens with respect to the stage of immunization. Thus, it is possible to predict the in vivo titer of a tetanus antitoxin accurately enough for clinical study. The HA test may be useful as an alternative method for titrating tetanus antitoxin in the field trials. Moreover, it can be used for the study of characteristics of antitoxins.     

Evaluation of the anti-rubella immunity levels on a lot of 5,000 sera from women at procreative age, tested by HAI, in Romania

From the study performed on a lot of 5030 sera, prelevated from women between 15-40 years old, tested by hemagglutination inhibition reaction (HAI), divided in 5 age groups of 5 years each, resulted that the percentage of women that can be considered immunologically protected against rubella (with HAI antibody titers greater than or equal to 1/20) varied between 79.5% (15-20 years of age group), and 75.3% (31-35 years of age group). The average percentage for the total lot was 76.7. The geometrical means (MGx/divided by SG) of the individual HAI titers ranged between extreme values of 54.2 (x/divided by 4.1) for the Group I, and 40.7 (x/divided by 3.8) for the Group IV, presenting the value of 44.4 (x/divided by 3.9) for the total lot. The covering coefficients of the protection limit, estimated at the level of geometrical means of antibody titers, ranged between 2.7 and 2.0, according to the age group, while the estimations made at the levels of the limits of the statistical range of one geometric standard deviation (MG x/divided by SG), presented values comprised between 11-8 and 0.7-0.5, respectively. On the basis of some theoretical mathematical models proposed in the literature (and using data resulted from the study performed), an attempt was made to approximate the rate of fetal exposure as well as the presumptive risk of congenital rubella syndrome (to 0/0000 live-born).     

Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide

Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.     

In vitro binding ofBifidobacterium bifidum DSM 20082 to mucosal glycoproteins and hemagglutinating activity

Adhesive properties ofBifidobacterium bifidum strain DSM 20082 were studied by the hemagglutination test and an enzyme-linked immunosorbent assay (ELISA).B. bifidum caused agglutination of human A, B, and O erythrocytes and rabbit erythrocytes, but the interactions were not specific of blood group antigens. The hemagglutination was inhibited by porcine gastric mucin and rat intestinal and colonic mucin.B. bidifum was shown to adhere to different immobilized mucosal glycoproteins and to glycophorin A, a specific erythrocyte membrane glycoprotein. The data obtained with many glycosylated components indicated thatB. bifidum receptors involved in the hemagglutination test were not the same as those that adhere to mucus glycoproteins. The results suggest that the mucosal preparations contain receptors for specific bacterial adhesins, but their structures remain to be determined.     

Titration of tetanus antitoxin by passive hemagglutination. II. Serological characteristics of antitoxin production in rabbits and monkeys.

1) Production of tetanus antitoxin in rabbits and monkeys was followed by passive hemagglutination (HA) and toxin-neutralization (TN) tests. The HA activity was observed in both IgM and IgG in both animal species. 2) In rabbits, IgM antitoxin was detected as early as in 7 days, reached the maximum titer in 10--14 days, and disappeared in 3 weeks after the primary immunization. Antitoxin of IgG class was detected in 10 days, and increased gradually. The ratio of HA/TN titers ("serum ratio") was high at an early stage of primary immunization and approached the unity in 3--4 weeks. Unlike the case of guinea pigs, IgM was found to contribute greatly to this high level of ratio. Besides, most rabbits produced IgG antitoxin of high ratios at early stages of immunization. 3) The immune response of monkeys showed a pattern very similar to that of rabbits except a few days' delay in the time course of antitoxin titers. No IgG antitoxin with a high serum ratio was demonstrated. Therefore, the high serum ratio of early sera could be accounted for mainly by IgM. 4) In response to the secondary immunization, no IgM antitoxin was detected in either animal species. 5) No definite correlation between serum ratio and avidity in terms of "dilution ratio" was demonstrated. However, both the dilution ratio and serum ratio were high at an early stage of immunization and gradually decreased, though the magnitudes of the ratios were variable depending on individual animals.     

Problems posed by the preparation of specific anti-cytomegalovirus immunoglobulins

The authors describe the preparation of a first batch of intravenous cytomegalovirus (CMV) immune globulin at the Nancy Regional blood transfusion centre. Immune plasmas were selected from 3 640 healthy volunteer blood donors on the basis of CF antibody titers to CMV (Kolmer's method modified) of, at least, 1:8; plasmas from approximately 10% of the donors were therefore selected. The 68 liters of pooled immune plasma had à CF antibody titer of 1:16 (CMV antibody titers of 1: 10 000 and 1: 640 when tested in the ELISA assay and passive hemagglutination assay respectively). Intravenous immune globulin was produced from pooled plasma by Cohn fractionation and treatment with pepsin at pH 4; 4.8 liters of immune globulin were prepared and divided in 96 doses of 50 ml each. The final product was found to have a CMV antibody titer of 1: 32 (CF) 1: 50 000 (ELISA) or 1: 2 560 (passive hemagglutination). Recent reports on the preparation of CMV immune globulin are briefly reviewed.     

Expression of virulence-related properties by, and intestinal adhesiveness of, Vibrio mimicus strains isolated from aquatic environments.

A study of the major pathogenic characteristics of Vibrio mimicus was carried out with 77 strains isolated from aquatic environments in Okayama, Japan. Of the strains tested, 96% demonstrated in vitro adherence to the rabbit intestinal mucosa, of which 36, 20, and 43% belonged to the strongly, moderately, and weakly adhesive groups, respectively. Of the 27 strains which appeared to be enterotoxigenic in the experiments using rabbit ileal loops, 74% belonged to the strongly adhesive group. All strains of V. mimicus at early log phase showed cell-mediated hemagglutination, and 70% of strongly hemagglutinative strains belonged to the strongly adhesive group, implying a possible correlation between cell-mediated hemagglutination and bacterial adherence. However, no significant correlation could be detected in the production of putative exocellular pathogenic factors and bacterial adherence or enterotoxigenicity.     

Cimulative hemagglutination by Mycoplasma pneumoniae and other agglutinins

John, T. Jacob (University of Colorado School of Medicine, Denver), Marlene Stahl, and Vincent A. Fulginiti. Cumulative hemagglutination by Mycoplasma pneumoniae and other agglutinins. J. Bacteriol. 92:1002-1004. 1966.-The phenomenon of cumulative hemagglutination, or hemagglutination by two agglutinins, each in subagglutinating concentration, was demonstrated by use of four different systems, namely, horse serum and Mycoplasma pneumoniae, horse serum and measles antigen, M. pneumoniae and measles antigen, and parainfluenza 2 virus and M. pneumoniae. Cumulative hemagglutination appears to be the mechanism by which a horse-serum diluent causes high hemagglutination titers of M. pneumoniae, since both contain hemagglutinins against vervet erythrocytes. It was also shown that antibodies against either one of the two antigens may cause inhibition of such hemagglutination.     

Alteration in immune complex glomerulonephritis by arachidonic acid

We employed a model of immune complex glomerulonephritis produced in mice by the daily injection of apoferritin to study the effect of treatment with arachidonic acid (AA). Apoferritin injections produced demonstrable glomerular damage by light microscopy associated with deposition of immunoglobulin along peripheral capillary loops. Treatment with AA 100 micrograms daily resulted in significantly less glomerular damage and a shift inthe location of immune complex deposition to the mesangium. The amount of anti-apoferritin antibody was determined by hemagglutination and found to be significantly decreased in mice treated with AA. Separate studies employing this dose of AA revealed that the number of IgM antibody producing cells to SRBC was not altered by AA.     

Titration of Bactericidal Activity against Smooth and Rough Strains of Salmonella which Are Resistant in Isotonic Medium

Bactericidal factors in antisera against various chemotype strains of Salmonella were assayed by means of the spot method. Certain smooth and rough strains were resistant to the killing effect of immune mouse sera when the activity was assayed in an isotonic salt medium and guinea pig complement was used. However, the sensitivity was found to be increased in various degrees by assaying it in a medium containing hypotonic concentrations of NaCl or tris(hydroxymethyl)-aminomethane-HCl (Tris-HCl). Keeping the resistant bacteria in hypotonic solutions before or after treatment with complement increased the titer of antiserum, indicating that the hypotonic solution increases the sensitivity of bacterial cells to the antiserum and/or complement. The optimal salt concentration for the assay of serum-sensitive and -resistant smooth strains was 0.02 M NaCl. With Ra through Rd chemotype strains, 0.1 m NaCl before complement treatment and 0.05 m NaCl after the treatment was the best. Isotonic medium was necessary for the titration of the Re chemotype strain. Specificity of the killing effect which was assayed by the hypotonic spot method was shown by adsorption studies on the immune sera. Use of C4-deficient guinea pig complement resulted in low titers against certain strains of bacteria and high titers were restored by the addition of the C4 component of complement. These results indicate that serologically specific bactericidal factors including antibody can be assayed by the spot method using hypotonic NaCl or Tris-HCl.     

Use of Stable, Sensitized Cells in Indirect Micro Hemagglutination Test for Amebiasis

Human "O" cells were fixed with pyruvic aldehyde, treated with tannic acid, and fixed with glutaraldehyde. The cells were sensitized with amoeba antigen and stored in a refrigerator. The sensitized cells were used periodically for the indirect hemagglutination test with a battery of sera from patients with intestinal amebiasis and confirmed and unconfirmed amebic liver abscess, and also from negative controls. The same battery was tested with cells sensitized with different batches of antigen and also with fresh sheep cells. None of the cells showed any reaction with negative control sera. The fixed cells remained sensitive and stable throughout the study. Reproducibility of the titers with the fixed cells within each day and from day to day was satisfactory. The titers with fixed human "O" cells were slightly lower than were the titers with fresh sheep cells. The advantages of using stable, sensitized cells are pointed out.     

Epidemiology of toxoplasmosis in Chile. VI. Prevalence of human infection, investigated by means of an indirect hemagglutination test, in regions VII, VIII and IX

In presecuting the investigations on the epidemiology of toxoplasmosis in Chile, a new series of serological surveys has been performed during 1982-1989 in 10 urban and 25 periurban-rural localities from the regions VII, VIII and IX of the country (34 degrees 41'-39 degrees 38' South lat.). In 9,758, randomly selected apparently healthy persons, and indirect hemagglutination test (IHAT) for toxoplasmosis was carried out. The age of these individuals (4,203 males and 5,555 females) varied between 4 and 84 years. The examined persons represent a 0.33% of the total population of the three studied regions. IHAT titers of 1: > or = 16 were regarded as positive. The global prevalence for positive IHAT was 45.5% (50.5% in men and 41.7% in women). A higher proportion of positive tests was observed in urban areas (47.0%) than in periurban-rural sections (33.3%). An increasing prevalence with age was also observed. Only 5 (0.05%) persons had IHAT titers higher than 1:1000.     

Norovirus P particle, a novel platform for vaccine development and antibody production

The norovirus P particle is an octahedral nanoparticle formed by 24 copies of the protrusion (P) domain of the norovirus capsid protein. This P particle is easily produced in Escherichia coli, extremely stable, and highly immunogenic. There are three surface loops per P domain, making a total of 72 loops per particle, and these are potential sites for foreign antigen presentation for immune enhancement. To prove this concept, a small peptide (His tag, 7 amino acids [aa]) and a large antigen (rotavirus VP8, 159 aa) were inserted into one of the loops. Neither insertion affects P particle formation, while both antigens were presented well on the P particle surface. The immune-enhancement effect of the P particle was demonstrated by significantly increased antibody titers induced by the P particle-presented antigens compared to the titers induced by free antigens. In addition, the measured neutralization antibody titers and levels of protection against rotavirus shedding in mice immunized with the VP8 chimeric P particles were significantly higher than those of mice immunized with the free VP8 antigen. Sera from P particle-VP8 chimera-vaccinated animals also blocked norovirus virus-like particle (VLP) binding to the histo-blood group antigen (HBGA) receptors. From these data, the P particle appears to be an excellent vaccine platform for antigen presentation. The readily available three surface loops and the great capacity for foreign antigen insertion make this platform attractive for wide application in vaccine development and antibody production. The P particle-VP8 chimeras may serve as a dual vaccine against both rotavirus and norovirus.     

In vitro adherence of bovine erythrocytes infected with Babesia bovis to thrombospondin and laminin

This study was undertaken to examine how eight putative adhesive agents bound to plastic surfaces affected the capacity of bovine erythrocytes, infected with either virulent or avirulent strains of Babesia bovis, to adhere in vitro. Thrombospondin (TSP) induced B. bovis-infected bovine erythrocytes to adhere and adherence was augmented when the infected blood was cultured for 24 h before the assay. Moreover, TSP also caused erythrocytes infected with avirulent strains of B. bovis to adhere to plastic in vitro. Laminin promoted the adherence of infected, and to a lesser extent, of uninfected erythrocytes.     

Role of C-terminal region of HA-33 component of botulinum toxin in hemagglutination.

Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not. Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains. The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes. These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin. Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain.     

Alteration in immune complex glomerulonephritis by arachidonic acid

We employed a model of immune complex glomerulonephritis produced in mice by the daily injection of apoferritin to study the effect of treatment with arachidonic acid (AA). Apoferritin injections produced demonstrable glomerular damage by light microscopy associated with deposition of immunoglobulin along peripheral capillary loops. Treatment with AA 100μg daily resulted in significantly less glomerular damage and a shift in the location of immune complex deposition to the mesanguim. The amount of anti-apoferritin antibody was determined by hemagglutination and found to be significantly decreased in mice treated with AA. Separate studies employing this dose of AA revealed that the number of IgM antibody producing cells to SRBC was not altered by AA.     

Hemagglutination properties of Enterococcus

In total, 86 enterococcal strains including representatives of most of the described species were tested for the ability to agglutinate human, sheep, and rabbit erythrocytes. Five strains did not react with any of the erythrocytes tested, and 81 (94.2%) strains agglutinated only rabbit erythrocytes. The hemagglutination titers ranged from 2 to 64. Loss of the hemagglutination activity was observed when rabbit erythrocytes were treated with trypsin or neuraminidase. Trypsin treatment of the bacterial suspensions also caused loss of the agglutination ability. On the other hand, heat treatment of bacterial suspensions increased the efficiency of the interactions, and higher titers were obtained. Assays for inhibition of hemagglutination were performed with -d-fucose, -d-galactose, -d-galactose, d-glucose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetylneuraminic acid, N-acetylneuraminic acid-lactose, and fetuin. Only fetuin was able to inhibit the hemagglutination reactions. The results showed that hemagglutination properties are common to the different enterococcal species tested. They also suggest that enterococci possess hemagglutinins of proteic and non-proteic nature that are involved in the attachment to sialic acid-containing receptors on the surface of rabbit erythrocytes.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.