Direct Hemagglutination Research Articles

Precipitating and Agglutinating Activity in Duck Anti-Soluble Protein Immune Sera

Ducks were inoculated parenterally, up to 4 times, with bovine serum albumin (BSA), B gamma globulin (BGG), or horse gamma globulin (HGG). Their sera were tested in immunodiffusion tests for precipitins. Only BSA and BGG induced precipitins. Only 7 of 443 sera tested, obtained from 3 of 62 inoculated ducks, developed a precipitin line with homologous antigen. All 7 sera were obtained from the earliest bleeding (7 days) after inoculation. Sera were also tested for agglutinins in direct passive hemagglutination (DPHA) and direct red-cell-linked antigen tests (DRCLAT) and for nonagglutinating antibodies in indirect red-cell-linked antigen tests (IRCLAT). No duck had a passive-hemagglutination-demonstrable primary immune response. Demonstrable DPHA titers after subsequent inoculations were very low compared with responses to these antigens of other species noted by other workers. Duck immune response was greatly diversified: all inoculation regimens that induced agglutinins in some ducks left others completely unstimulated. Precipitins and agglutinins of the same ducks correlated well: those 7 sera that had precipitins also had the highest DPHA titers for the 3 donor ducks. However, hemagglutinating titer and presence of precipitins in sera of different ducks correlated poorly. Increasing the age of the ducks at the first inoculation from 6 to 10 weeks increased the number of responders and DPHA titers of their sera. Non-agglutinating antibodies were demonstrated in anti-BSA sera: in IRCLAT, the sera had titers 2-to-256-fold higher than the sera in DPHA or DRCLAT had. Duck immunoglobulins were deficient in those immunological reactions (precipitation and agglutination) that require functional bivalency.

Detection of ABO group-specific substances on the red cells of the cynomolgus monkey.

Several attempts were made to demonstrate the presence of ABO blood group substances on the red blood cells (RBC) of the cynomolgus monkey. Neither the direct hemagglutination test nor the antibody absorption test was successful in detecting these substances from whole RBC, the stromata or the butanol extracts of RBC. By the heat elution of antibodies, however, the presence of ABH substances was demonstrated on RBC of every phenotype without fail. In all cases, there was a reciprocal relationship between the ABH substances of RBC and of saliva and the anti-A and anti-B antibodies in serum, being in good agreement with Landsteiner's rule. Accordingly, it can be concluded that a small amount of ABH substances must be present on RBC of the cynomolgus monkey.

Escherichia coli O151:K−:H10 („KRIM”) als erreger von darminfektionen

The first time infections by Escherichia coli O151:K-:H10 were reported from Japan and the Soviet Union. 1972 it was included into the international antigenic scheme. Since 1976 these germs were occasionally demonstrated in sporadically occured acute enteritis in the G.D.R. In autumn 1979 60 E. coli 0151 strains were isolated from accumulating cases of gastroenteritis in the Rostock district. These infections mainly concerned young children, but also adults. Microbiological properties of 87 isolates of this serogroup are described. Biological examination of these strains did not result in symptoms for invasive activity or enterotoxin production. An indirect reference to the existence of fimbrial antigens could be obtained by the Evans' method of direct hemagglutination. A corresponding hemagglutination pattern was found out in 60 E. coli O151 strains tested: mannoseresistent hemagglutination of bovine and chicken erythrocytes. Hemagglutination pattern, association of these strains with enteritis and antigenic relations to Salmonellae motivated the recommendation to integrate these germs into the group of facultatively enteropathogenic Escherichiae coli described by Czirók and Evans. E. coli O151 strains show biochemical particularities which can be useful for identification of these strains. E. coli O151:K−:H10 wurde 1968 in Japan sowie in der Sowjetunion zum ersten Male beschrieben. 1972 erfolgte die Aufnahme in das internationale Antigenschema. In der DDR wurden diese Keime seit 1976 bei sporadischen Fällen akuter Darminfektionen gelegentlich nachgewiesen. Im Herbst 1979 wurden bei gehäuft auftretenden Gastroenteritiden im Bezirk Rostock 60 E. coli 0151-Stämme isoliert. Diese Infektionen betrafen überwiegend Kleinkinder, z.T. jedoch auch Erwachsene. Für 87 Isolate dieser Serogruppe werden die mikrobiologischen Eigenschaften beschrieben. Bei der biologischen Prüfung dieser Stämme konnte kein Anhalt für Invasivität oder Enterotoxinbildung erhalten werden. Mit Hilfe der direkten Hämagglutination nach Evans konnte ein indirekter Hinweis auf das Vorliegen von Fimbrien-Antigenen erhalten werden. Bei 60 geprüften E. coli 0151-Stämmen wurde ein übereinstimmendes Hämagglutinationsmuster festgestellt: eine mannoseresistente Hämagglutination von Rinder- und Hühner-Erythrozyten. Dieses Hämagglutinationsmuster, die Assoziation dieser Stämme mit Darmerkrankungen und die Antigenbeziehungen zu Salmonellen führen zu dem Vorschlag, diese Erreger in die von Czirók u. Evans beschriebene Gruppe der fakultativ enteropathogenen E. coli einzuordnen. E. coli 0151-Stämme besitzen biochemische Besonderheiten, die für eine Identifizierung der Stämme nützlich sein können.

Antibodies to Toxoplasma gondii in equids in north India.

The prevalence of antibodies to Toxoplasma gondii was investigated among equids in 3 localities of north India, using the direct haemagglutination test. Of the 603 animals sampled, titres ranging from 1:8 to 1:512 were found in 71 (11.8 per cent). Specific titres of 1:64 or more were found in 34 (5.6 per cent) sera. The number of positive titres at Babugarh (Uttar Pradesh) was considerably higher than at 2 other localities. Although the likelihood of positive sera appeared to increased with age, the animal's sex appeared to have little influence. Subjects with reproductive disorders or eye ailments did not have a high prevalence of positive titres compared with normal subjects.

Increased sensitivity of rosetting assay for Fc receptors obtained by using non-hemagglutinating monoclonal antibodies

The sensitivity of the conventional EA (Fc) rosetting assay for detecting Fc receptors could be significantly increased by coating sheep erythrocytes (SRBC) with high concentrations of a preparation of homogeneous monoclonal IgG2b anti-SRBC antibodies. This was made possible by virtue of the fact that the monoclonal antibody preparation used was completely incapable of directly hemagglutinating SRBC, although it possessed a high titer as assessed by direct hemolysis or direct hemagglutination. The monoclonal IgG2b-coated SRBC indicator cells were compared to SRBC coated with subhemagglutinating amounts of the IgG fraction of a hemagglutinating rabbit anti-SRBC antiserum: at high coupling concentrations, the monoclonal antibody-coupled SRBC gave consistently higher percentages of Fc rosettes, a large proportion of which were large and multi-layered, when various lymphoid cell populations and a variety of Fc receptor-positive cultured tumor cell line populations were tested. The increased sensitivity of the Fc rosette attained by this method may depend on the nature of the monoclonal anti-SRBC reagent used. Thus, indicator cells prepared by incubation with high concentrations of IgG1 subclass-specific non-hemagglutinating monoclonal anti-SRBC antibodies always gave significantly lower percentages of Fc rosettes than conventionally prepared indicator cells.

The epidemiological type identification of Serratia marcescens from outbreaks of infection in hospitals.

A study of serological, bacteriocine and phage typing of Serratia marcescens was made. Specific O-antisera of adequate titre were relatively simple to prepare but H-antisera exhibited many heterologous agglutination reactions amongst the type strains. Most of these cross-reactions were not reproduced when immobilization tests with H-sera were performed. Direct haemagglutination tests were used to establish the presence of fimbriae amongst the H-type strains and the results of agglutination tests with non-fimbriate variants of strains indicated that fimbrial antibody in high titre was present in some sera. Replicate typing of 100 pairs of cultures by the phage-typing method indicated that small variations in pattern were common and that larger variations occurred occasionally. Therefore differences in pattern of less than two strong reactions should not be taken as evidence that strains can be distinguished. Cultures of S. marcescens, 273 in total, from six outbreaks of infection in British and European hospitals were typed by O-serology, H agglutination and immobilization tests, phage typing and bacteriocine susceptibility by a cross-streaking method. The typability of strains by each method was high but the results suggested that no single method was sufficiently discriminating to be used alone for typing. Comparison of the H-type and typing patterns of members of the same O serogroup from incidents of infection showed that reliable results were obtained by H-typing or by phage and bacteriocine typing after the application of the appropriate 'difference' rule. The greatest discrimination between strains of the same O-group was obtained by the use of H-typing or phage typing.

Passiver Haemagglutinationstest für Semliki-Forest-Virus

Several methods of coupling immunoglobulins onto sheep erythrocytes have been examined with a view to demonstrate Semliki-Forest virus by use of a passive hemagglutination test. With the exception of the carbodiimid coupling reaction, most methods could be used to couple goat anti SFV IgG to erythrocytes. Best results were obtained when anti SFV goat IgG was coupled to sheep red blood cells by using 0.25% glutaraldehyde. Sheep red blood cells treated after that method could be used to identify as well as titrate SFV in any given solution. The actual mode of agglutination was made visible by electron microscopy in so far as SFV was found to form dimers of adjacent erythrocytes via a bridge formation. The passive hemagglutination offers considerable advantages compared to other methods such as the direct hemagglutination with goose erythrocytes, as formalin inactivated virus can be detected thereby. The described test therefore offers an additional approach to check the potency of inactivated togavirus vaccines. Zusammenfassung Verschiedene Methoden zur Kopplung von Anti-Semliki-Forest-Virus (SFV) Immunoglobulin an Hammelerythrozyten wurden im passiven Haemagglutinationstest geprüft. In der Mehrzahl der geprüften Systeme war eine wirkungsvolle Bindung an Hammelerythrozyten erreichbar. Beste Ergebnisse wurden jedoch erreicht, wenn Immunoglobuline mittels Glutaraldehyd (0,25%) an Erythrozyten gekoppelt wurden. Derart beladene Erythrozyten konnten sowohl zur Identifizierung als auch zur Titration von Semliki-Forest-Virus verwendet werden. Mit Hilfe elektronenoptischer Untersuchungen wurde die spezifische Bindung von SFV an Erythrozyten mit der Bildung von Brücken zwischen Erythrozyten sichtbar gemacht. Der passive HA-Test kann zur Erfassung von SFV aus Virussuspensionen ohne Vorbehandlung derselben Verwendung finden. Außerdem kann Formalin-inaktiviertes Virus quantitativ erfaßt werden, welches im aktiven HA-Test nicht nachweisbar ist. Dadurch eröffnet dieser Test möglicherweise einen neuen Weg zur Wirksamkeitsprüfung von Togavirusvakzinen aus inaktivierten Viren.

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O + erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.

In vitro direct haemagglutination by Corynebacterium ovis exotoxin

Haemagglutinating activity of toxigenic Corynebacterium ovis culture supernatants was estimated by titration in phosphate buffered saline diluent against sheep erythrocytes. The haemagglutination titre was closely related to the haemolysis and rabbit dermonecrosis titres of culture supernatants and to the toxigenicity of different C ovis strains. The substance responsible for haemagglutination was diffusible and its concentration was unrelated to cell yield in cultures. Corresponding inactivation of all three activities by antitoxin, toxoid formation with formalin, heat and Seitz filtration suggested that haemagglutination is another activity of C ovis exotoxin.

Murine antigen H-2.7: Its genetics, tissue expression, and strain distribution

Reinvestigation of alloantisera containing antibodies to murine antigen H-2.7 revealed that the crucial recombinant, A.TFR1 (H-2an1), which was reported to separate theH-2G locus from theSs-Slp loci, has ak-like instead off-like H-2.7 antigen. Therefore, the crossover position inH-2an1 and the position of the G locus in theH-2 map are now uncertain. By using the hemagglutination-serum inhibition test, anti-H-2.7 reactive substance was found to be present in normal mouse serum in a strain-specific manner. Tissue distribution study by absorption analysis indicated that H-2.7 antigen is present, in addition to RBCs, on spleen and lymph node cells, but is absent on thymus cells. Thirty B10.W congenic lines were analysed for the presence of the H-2.7 antigen. Two lines (B 10.CHA2 and B 10.KPA44) were found to be H-2.7 positive by both direct hemagglutination and absorption tests.

Direct hemagglutination technique for differentiating Bacteroides asaccharolyticus oral strains from nonoral strains

A simple and economical method for differentiating Bacteroides asaccharolyticus of oral sources from nonoral sources is described. The present data indicate that oral strains of B. asaccharolyticus strongly agglutinate sheep erythrocytes, whereas isolates from various nonoral sites typically are devoid of hemagglutination activity. The direct hemagglutination test may aid in determining the source of B. asaccharolyticus present in an infection, and thus the procedure has potential value as a means of biotyping.

Evaluation of Passive Hemagglutination Techniques in Detection of Australia Antigen (HBsAg)

Some techniques for HBsAg detection in the serum were evaluated. The direct hemagglutination test with turkey erythrocytes appears to be a suitable test for speed, simplicity, and cost.

Effect of Infectious Bursal Disease Virus on the Immunological Responsiveness of the Chicken

The immunological response of chickens infected at 28 days of age with infectious bursal disease virus (IBDV) was examined. Cellular immunity was studied by the graft-versus-host reaction and delayed type hypersensitivity skin reactions to tuberculin. Antibody responses to sheep red blood cells (S-RBC’s) were tested by a direct hemagglutination test. Circulating levels of immunoglobulins (Ig’s) IgM, IgG and IgA were monitored by the radial immunodiffusion test. The results of the graft-versus-host reaction and delayed type hypersensitivity reactions were similar for both the infected and control chicks. IBDV significantly reduced the primary antibody response to S-RBC’s, but had no marked effect on the secondary antibody response. Chicks infected with IBDV had normal levels of serum IgM and IgG, but significantly lower levels of IgA when compared to uninfected control birds.

Hemagglutination inhibition studies for the evaluation of blood group antigens in ethanol soluble substances (ESS) obtained from human, baboon and vervet monkey red blood cells.

Soluble blood group substances, isolated from the red blood cells of humans, baboons, and vervet monkeys by ethanol extraction, possessed serologically active specificities for the following antigens: A, B, H, Lea, LebL, P, P19 Pk and I. Human red blood cells lacking any of these specificities by the direct hemagglutination test also lacked the related antigens in their soluble extract. The only exception was in "Bombay" Oh cells, from which soluble H substance could be readily isolated. Soluble substances obtained from baboon and vervet monkey red blood cells, which lack the human variety of A, B, and H antigens on their red blood cells, inhibited both human and lectin anti-H reagents. The detection of "hidden" H activity in Oh cells will pose some important questions regarding membrane characteristics and the role of immune surveilance.

Hemagglutinins in the mucus of pacific hagfish, Eptatretus stoutii

1. 1. A disc electrophoretic and immunologic comparison was made of Pacific hagfish ( Eptatretus stoutii) serum and a concentrated, water-soluble fraction of mucus. 2. 2. Usually 12–15 protein components were obtained by disc electrophoresis of mucus and the profile was similar in many regions to that of serum. 3. 3. Usually at least two and sometimes four proteins of mucus could be shown to be immunologically related by double diffusion. 4. 4. Natural hemagglutinins which were found in mucus by direct hemagglutination were also shown to have heat-sensitivity similar to hemagglutinins in serum.

A Tanned Cell Haemagglutination Test for the Detection of Hepatitis‐Associated‐Antigen (Au‐Ag) and Antibody (Anti‐Au)

Summary. A tanned cell haemagglutination technique using inactivated Au‐Ag to sensitize human ‘O’ Rh negative crythrocytes is described. The method may be used to detect Au‐Ag by haemagglutination inhibition (HI) and anti‐Au by direct haemagglutination (HA). Sensitivity and specificity arc demonstrated and its application to mass donor screening is considered.

Replication of avian infectious bronchitis virus in African green monkey kidney cell line VERO.

Avian infectious bronchitis virus (IBV), a coronavirus, requires initial isolation in, and adaptation to, chicken embryos (CE) before transfer to primary avian cell and chicken tracheal organ cultures. These are the only presently known cell cultures in which IBV replicates and produces cytopathic effects (c.p.e.) in serial passage (Estola, 1966; Cunningham, 1970). Monkey kidney cells have been reported (Fahey & Crawley, 1956; Steele & Luginbuhl, 1964) to support relication of IBV without c.p.e. when first inoculated with virus propagated in CE. Attempts apparently were not made to passage the virus in these cells. Direct haemagglutination (HA) is not a normal property of IBV (Biswal, Nazerian & Cunningham, 1966) or of the human coronaviruses (Kapikian, 1969). However, human coronaviruses OC 38 and OC 43 adapted to suckling mouse brain (McIntosh et al. 1969) cause direct HA (Kaye & Dowdle, 1969) and produce syncytia and plaques in African green monkey kidney and BSC-1 cells (Bruckova, cited by Bradburne & Tyrrell, 1971).

The immunosuppressive capacity of alloantisera in mice, including sera directed primarily against thymic antigens

Alloantisera prepared in inbred mice and directed against H-2 histocompatibility differences or against the thymic antigens θC3H and TL (thymus leukemia) were assayed for their immunosuppressive action when injected in mice against whom the alloantisera were directed. There was a significant depression of the reactions to bovine serum albumin (BSA) and sheep erythrocytes (SRBC) as determined for BSA by passive hemagglutination and by direct hemagglutination of SRBC. Plaque production (measured by the Jerne plaque assay) against SRBC was also inhibited. The mice exhibited leukopenia, and in one experiment there was a significant increase in the number of takes of syngeneic tumor transplants in antiserum-treated mice given a subthreshold inoculum of tumor cells.

Hemagglutination and cytotoxic studies of H-2. I. H-2.1 and related specificities in the EK crossover regions.

H-2 Specificities 23, 25, 11, 1, and 5, when arranged in this order, form an inclusion or subtype system, with 23 showing the narrowest distribution. Three of these specificities, 23, 25, and 11, are assigned to the K crossover region; the other two usually are assigned to the E region. Because of ambiguities in regard to 1 and 5, these specificities were selected for detailed study. Our panel of strains included two, PL/J and SM/J, with new alleles designated, respectively, H-2u and H-2v. Included also was strain B10. Y, previously typed as H-2q, but which now has an allele similar to or identical with H-2p. We provisionally call it pa. The panel did not include n in terms of which H-2.1 was originally defined. However, alleles p and n are similar, and our anti-p and anti-pa sera contained antibodies with reactions closely approximating those expected of an anti-1. Recipients for the production of antisera were selected to exclude as many antibodies as possible other than 1. Antisera were analyzed by direct hemagglutination and cytotoxicity against standard panels of cells. Many were also analyzed by in vivo hemagglutination absorption, and some, by quantitative in vitro cytotoxic absorption. Antisera made against alleles p, pa, s, v, and o were 1-like, in the range of their reactions, but showed individual differences. An antiserum made against allele q also contained a 1-like antibody, although it was complicated by the presence of anti-11. Antisera made with p as one parent of the recipient and with s as the donor (and also in the reverse combination) contained 1-like antibodies. We refer to these as blocked anti-1 sera. All unblocked anti-p or anti-pa sera reacted by hemagglutination with s, and one was fully absorbed by s. The individual differences in the reaction patterns of the antisera were largely confined to the 1-positive, 23-negative alleles. We refer to these as 1-intermediate alleles. Reactions with these alleles were often weak. With the 1-positive, 23-positive alleles (designated 1-complete alleles), except for some weak reactions by the blocked sera, the reactions were consistently strong. The differences in strength of reactivity between 1-intermediate and 1-complete alleles were particularly clear in the cytotoxic absorption tests. We suggest as a possible explanation a unidirectional cross reaction between 1 and 23. Crossover allele o received 1, but not 23, from 1-complete allele k; as would be predicted, it behaved like a 1-intermediate. We suggest that the variability of the reactions of the 1-intermediate alleles reflects mutational variation in their 1 sites, sufficient similarity remaining so that cross reactions occur, however. This hypothesis also explains the formation of 1-like antibodies in blocked combinations. The 1-complete alleles, because their EK regions trace through successive

H-2 types of translocation stocks T(2;9)138Ca, T(9;13)-190Ca and an H-2 recombinant.

H-2 alleles of translocation stocks T138 and T190 were analyzed in the direct hemagglutination test and by in vivo absorption with oligospecific antisera. The alleles of both stocks were found to be indistinguishable from the H-2q allele of strain DBA/1. This conclusion was confirmed by an F1 test with skin grafts. The possible origin of the H-2q allele of the translocation stocks and its possible relationship to H-2q alleles of other strains are discussed. An H-2 recombinant which arose by crossing over between H-2a of B10.A and H-2q of T138 was also analyzed. The recombinant has a new combination of H-2 antigens which we propose to call H-2y. The H-2y allele has its D end derived from H-2a and its K end derived from H-2q. Factors controlling antigens H-2.17 and H-2.30 are placed by this recombinant in the opposite sides from Slp in the H-2 map. On the basis of these and other data, it is hypothesized that H-2m allele of strain AKR.M might have also arisen by crossing over between alleles H-2k of strain AKR. and H-2q of noninbred strain M.