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).     

Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enteroxigenic Escherichia coli

The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated. The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1. A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors. However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1. Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone. These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination.     

Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin.

The purified progenitor toxin of Clostridium botulinum type C strain 6814 (C-6814) forms a large complex composed of 150-kDa neurotoxin (NT), 130-kDa nontoxic-nonhemagglutinin (NTNHA), and hemagglutinin (HA) components. The HA component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26-21 and 17 kDa. We isolated the HA subcomponents from the progenitor toxin by chromatography in the presence of denaturants. The isolated HA subcomponents, designated as i-HA-33, i-HA-55, i-HA-70 and i-HA-33/17, were nearly homogeneous on SDS/PAGE, but the HA-17 and HA-26-21 components were not purified. Some HA subcomponents, designated as f-HA-33 and f-HA-33/17 complex, existed free of the progenitor toxin in the culture medium and they were separately purified. Every HA subcomponent so far isolated shows binding activity to erythrocytes. The hemagglutination activities of each HA subcomponent had a titer of 25 for the f-HA-33/17 complex, and below 23 for the other f- and i-HA subcomponents, while the parent progenitor L toxin was 28. The reconstitution of various combinations of f- and i-HA subcomponents was attempted via mixing and tested for hemagglutination activity. When the i-HA-33/17 complex and i-HA-55 were mixed, the hemagglutination activity was recovered to a titer of 29, which was slightly higher than that of the parent toxin. These data imply that a combination of at least HA-33, -17 and -55 subcomponents is required for full hemagglutination activity of the botulinum progenitor toxin, but each single HA subcomponent shows weak or no aggregation of erythrocytes.     

Molecular characterization of the type-specific gamma-determinant located on the adenovirus fiber.

The fiber knob carries the type-specific gamma-antigen which can be demonstrated in hemagglutination inhibition tests. In order to characterize the gamma-determinant we selected subgenus DI adenovirus serotypes 9 and 19 (Ad9 and Ad19) which exhibited 29 amino acid exchanges in the knob domain. Like all subgenus DI adenoviruses they showed a complete hemagglutination pattern with rat and human erythrocytes. We constructed a total of 14 chimeric Ad9/Ad19 and Ad19/Ad9 fiber proteins, which possessed fiber knobs with progressively exchanged Ad9 and Ad19 amino acids. Furthermore, we created 39 fiber proteins with distinct amino acid exchanges in the knob regions by primer-directed mutagenesis. The proteins were expressed in Escherichia coli and tested in hemagglutination and hemagglutination inhibition tests. From our results we can conclude that the type-specific gamma-determinant is not restricted to a distinct region on the adenovirus fiber knob but is composed of at least 17 amino acids. Most of the amino acids contributing to the Ad9 and Ad19 gamma-determinants are located on the fiber knob loops.     

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.     

Mode of Action of an Inhibitor from Agar on Growth and Hemagglutination of Group A Arboviruses.

Colón, Julio I. (Fort Detrick, Frederick, Md.), Jane B. Idoine, Orville M. Brand, and Richard D. Costlow. Mode of action of an inhibitor from agar on growth and hemagglutination of group A arboviruses. J. Bacteriol. 90:172-179. 1965.-A polysaccharide obtained from agar, and having properties similar to a previously described sulfated polysaccharide, was observed to inhibit growth and hemagglutination of some group A arboviruses. The evidence presented confirms that the inhibitory activity, in part, is the result of direct interaction between the agar polysaccharide (AP) and free virus particles. Additional evidence indicates that inhibition of viral growth also occurs as the result of interaction between AP and the chick-fibroblast cells used for propagation of the virus. The possibility was considered, therefore, that at least two different inhibitors could be present in AP-one that reacts directly with the virus particle and another that reacts with host cells. AP does not induce the production of interferon in the test system used.     

MEASUREMENT OF INFLUENZA VIRUS-ANTIBODY REACTION BY QUANTITATIVE ELECTRON MICROSCOPY

Measurement of the weight of individual virus particles from untreated and antibody-treated populations was made by quantitative electron microscopy. The weight of antibody bound depended on the concentration of antibody in solution. One population of viruses exposed to an antibody concentration which resulted in 95% inhibition of hemagglutination showed a mass increase of 55%, corresponding to an absolute increase of 9.0 x 10^-17 g in the median value. Another population, whose hemagglutination inhibition assay was 64%, showed a 39% increase in mass corresponding to an absolute median increase of 7.3 x 10^-17 g. The larger viruses in each population bound a greater absolute amount of antibody than did the smaller ones, but the latter bound relatively more antibody in proportion to their mass. No cross-reactivity was found between the antibody to influenza A/PR8 and the influenza strain B/LEE. Influenza A/PR8 controls exposed to nonspecific gamma-globulin displayed a significant weight loss, at least in part owing to loss from the core, as judged from the electron micrographs.     

Serological studies with the ficin-antificin system.

Rabbit antisera prepared against ficin reacted with it in a series of serologic tests. Upon immunodiffusion analysis, ficin was found to consist of at least nine antigenic components. Ficin will adsorb spontaneously to erythrocytes which can be used in passive hemagglutination tests. The enzymatic activity of ficin was not abolished by antificin sera.     

Inactivation of Encephalomyocarditis Virus in Aerosols: Fate of Virus Protein and Ribonucleic Acid

After aerosolization at relative humidities of 50% or lower, encephalomyocarditis virus is rapidly inactivated. In this process the protein coat of the virion is damaged. This appears as a loss of hemagglutination activity and loss of affinity for hemagglutination inhibiting antibodies. The ribonucleic acid of the virus retains its infectivity but it becomes susceptible to ribonuclease. It sediments in sucrose gradients when centrifuged at high speed with the same velocity as free infectious ribonucleic acid extracted with phenol from intact encephalomyocarditis virus.     

Heterogeneity of antibody response to Salmonella lipopolysaccharide measured by passive hemagglutination and hemolysis in mice

The complement-requiring passive hemolysis test with Salmonella typhimurium lipopolysaccharide-coated sheep erythrocytes is more sensitive for antibodies directed against the lipopolysaccharide than is the passive hemagglutination test. The hemagglutinating and hemolyzing antibodies produced in Swiss mice by hyperimmunization, either with or without Freund's adjuvant, were distributed in both the light and heavy fractions isolated by sucrose density gradient fractionation and gel filtration. IgM fractions, whether tested by hemagglutination or hemolysis, were sensitive to 2-mercaptoethanol (0.15 m). On the other hand, IgG hemolytic antibodies were more sensitive to 2-mercaptoethanol than were IgG hemagglutinating antibodies. The resistance of IgG hemagglutinating activity amounted to about 72 to 95% of the total IgG recovered, whereas the resistant portion of the IgG hemolytic activity was approximately 40 to 53%. It is suggested that, although mercaptoethanol sensitivity is not a definitive test for IgM antibody, its use in connection with the hemagglutination test gives at least an approximation of the IgG antibody, whereas the hemolysis test gives a better approximation of maximal measurable antibody against Salmonella lipopolysaccharides.     

A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.     

Hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

The hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin (LTc-B) produced by chicken enterotoxigenic Escherichia coli was studied by hemagglutination and hemagglutination inhibition. No or weak hemagglutination of intact human erythrocytes was found by the LTc-B at the highest concentration used, whereas strong hemagglutination of both neuraminidase- and pronase-treated human erythrocytes was found. Enhancement in hemagglutination of treated human erythrocytes induced by the LTc-B was over 2 to 120-fold for type A and B erythrocytes and over 8-fold for type O erythrocytes, respectively. With intact and treated sheep erythrocytes, on the other hand, no hemagglutination was found by the LTc-B at the highest concentration used. Hemagglutination of pronase-treated human type B erythrocytes by the LTc-B was inhibited by methyl-alpha-D-galactopyranoside, galactose, melibiose, hog A + H, asialo-bovine salivary mucin and asialo-thyroglobulin among mono-, di- and polysaccharides and glycoproteins used as inhibitors. These results suggest that the LTc-B is a galactose-specific bacterial lectin.     

Evaluation of a Hemagglutination Test for Human Leptospirosis

An indirect hemagglutination test for the diagnosis of leptospirosis is described; the test uses a soluble antigen from serotype patoc to sensitize sheep erythrocytes which are then fixed with glutaraldehyde. Evaluation of this procedure indicates that it is more reliable than the conventional macroscopic agglutination test and, in contrast with both microscopic and macroscopic agglutination tests, is positive only with sera from persons with current leptospiral illness. The test is simple and convenient and sensitized fixed cells may be stored for at least a year. In comparison with the macroscopic and microscopic tests, only a single antigen is required.     

A novel erythrocyte-based immunoassay for simultaneous detection of both antimycobacterial antibody response and mycobacterial antigen in human serum samples of pulmonary tuberculosis and a control group of patients using 'a single probe'

A modified passive hemagglutination using double aldehyde stabilized cells (tanned sheep erythrocytes treated with glutaraldehyde and pyruvic aldehyde) was evaluated for detection of both antimycobacterial antibodies and circulating mycobacterial antigens simultaneously in human serum samples from patients with pulmonary tuberculosis (n=40) and a control group (n=44). Double aldehyde stabilized cells sensitized with an optimum dose of 200 microg mL(-1) of sonicate extract of Mycobacterium tuberculosis antigens was used as single probe to detect both antibodies and antigen, respectively, by passive hemagglutination and passive hemagglutination inhibition. The sensitivity limit of passive hemagglutination inhibition was determined to be 280 ng mL(-1) using a dose-response curve. Sensitivity of passive hemagglutination and passive hemagglutination inhibition, respectively, was 90% and 52.5%, and specificity was 91% and 100%. Although passive hemagglutination and passive hemagglutination inhibition need further evaluation, these erythrocyte-based immunoassays are potentially advantageous, especially as double aldehyde stabilized sensitized cells could be used as a single probe for detection of both antibodies and antigen. In addition, erythrocyte-based immunoassays are rapid, simple and cost-effective with a high degree of sensitivity.     

Release of bound immunoglobulin from SSPE brain by acid elution.

SSPE brain homogenate extracted at pH 7.4 yields immunoglobulin with a 4- to 5-fold greater hemagglutination inhibition activity per microgram of IgG than serum from the same patient. Serial washing of the homogenate results in a low level steady-state release of IgG. Elution of the washed sediment with pH 2.5, 0.1 M glycine buffer results in a 2- to 3-fold increase in recovery of hemagglutination inhibition activity with a greater hemagglutination inhibition activity per milligram of IgG than the IgG recovered by phosphate-saline extraction at pH 7.4.     

Detection of Foot-and-Mouth Disease Virus Antibodies: I. “Passive” Hemagglutination Test

A passive hemagglutination test has been developed to detect and measure foot-and-mouth disease virus (FMDV) antibody by using glutaraldehyde as a coupling reagent. An optimal concentration of 10 to 40 mug of virus per ml with 0.25% glutaraldehyde at 25 C for 1 hr was established for the sensitization of sheep erythrocytes. A reaction time of 18 hr at 4 C or 2 hr at 37 C induced good agglutination in the presence of specific antibody. Sensitization was carried out in phosphate buffer, whereas agglutination and preadsorption of nonspecific agglutinins from sera were performed in gelatin (0.1%, w/v)-stabilized, phosphate-buffered saline. An optimal pH of 7.2 was also established for all reactions. Antibodies derived from guinea pigs hyperimmunized by infecting with FMDV, types A, O, and C were both virus-and type-specific. Preliminary experiments showed that strain A-119 and strain A-24 Cruzeiro could also be distinguished by hemagglutination. Parallel hemagglutination and complement-fixation tests showed the former to be two to four times more sensitive than the latter.     

Immunodiagnostic tests for hydatidosis in sheep: an evaluation of double diffusion, immunoelectrophoresis, indirect hemagglutination, and intradermal tests

Immunoelectrophoresis (IEP), double diffusion (DD5), indirect hemagglutination (IHA), and intradermal (ID) tests were evaluated to determine their ability to detect echinococcosis in sheep. Four sheep were infected per os with approximately 4,00, 1-wk-old eggs of Echinococcus gradulosus; four more sheep were similarly infected with approximately 3,000 1-wk-old eggs of Taenia hydatigena, and two additional sheep were used as uninfected controls. Blood samples were collected from each sheep prior to infection, at 2 and 4 wk postinoculation, and monthly thereafter for 1 yr. Serum from each blood sample was tested by IEP and DD5 for antiantigen "5" activity and by IHA for Echinococcus-specific hemagglutination activity. Following the last blood collection, an ID test for echinococcosis was performed on each sheep, after which all sheep were necropsied, and the type, location, and size of all larval tapeworms recorded. The DD5 test was found to be more sensitive and at least as specific as IEP in detecting echinococcosis in sheep. The IHA test approached the specificity and sensitivity pattern of DD5 and IEP if a titer of greater than or equal to 1:1,024 was considered positive. The ID test supported DD5 and IEP results but demonstrated a lack of specifiity. Necropsy data verified that all sheep were infected according to the experimental design. We conclude that DD5 reliably detects echinococcosis in experimentally infected sheep, and that further research is warranted to evaluate this test for detecting echinococcosis in naturally infected sheep.     

Purification, characterization, and cDNA cloning of alpha-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera

We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS-PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4-20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal alpha-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl(2). cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca(2+) but no hemagglutination.     

几种水稻植株中血凝活性物质的分布与变化及其稻胚凝集素的比较

开花前的水稻旗叶提取液中不含有血凝活力,但开花后旗叶提取液中能测得血凝活力,并随着开花后天数增加而逐渐升高。雄蕊和成熟花药中无血凝活力,开花前和开花早期的子房提取液中有血凝活性,开花5d后子房或胚乳中测不到血凝活性,但胚中血凝活性随种子发育而明显增加。分别从“寒丰”和“双丰一号”水稻成熟胚中分离得到凝集素,用电泳、免疫学方法和血凝试验表明两者的分子特性和血凝性质完全相同。     

Rapid, Sensitive Assay for Staphylococcal Enterotoxin and a Comparison of Serological Methods

Reversed passive hemagglutination was used to assay enterotoxin in culture filtrates and in food samples. With cells tanned and then sensitized with antitoxin globulin and preserved with either formaldehyde or pyruvic aldehyde, as little as 0.0007 mug of enterotoxin was detectable. The results of hemagglutination tests compared well with those obtained by quantitative precipitin tests or by immunodiffusion, but hemagglutination was 50 to 100 times more sensitive than the immunodiffusion technique. In addition, results of the hemagglutination test were available within a few hours, and neither elimination of interfering proteins from food extracts nor concentration of the sample, both of which are necessary for immunodiffusion, was required for this procedure.