Direct Hemagglutination Test Research Articles

Properties of lectin from tissue culture of Ungernia victoris and its ultrasonic extraction

The aim was to investigate biomass extracts of U. victoris strain UV-2 tissue culture for the content of lectins, to study their general characteristics and to optimize the method of ultrasonic extraction to obtain a soluble form of the lectin. Methods. Tissue culture method, direct hemagglutination test for detection of lectin activity (LA), determination of lectin carbohydrate specificity, ultrasonic extraction method. The results. Lectin activity was detected in the soluble (supernatant) and insoluble (sediment) fractions of the extract of biomass U. victoris tissue culture. In the soluble fraction LA was significantly less compared to that in the sediment, which indicated the presence of the membrane-bound form of the lectin. LA was characterized by pronounced species specificity: the reaction with mouse erythrocytes was the most intense. The study of carbohydrate specificity revealed a weak affinity for monosugars (galactose and galactosamine) and pronounced suppression of LA in the case of polysaccharides - hyaluronic acid, heparin and mucin. The possibilities of the ultrasound extraction method for separation the detected lectin from the the cell surface in the sediment fraction were investigated and the extraction procedure was optimized. The transition of LA into the soluble phase under the influence of ultrasound depended on the following parameters: the concentration and volume of the extract, as well as the time of exposure to ultrasound. It was established that the LA of the soluble phase doubled compared to the initial one with an extract concentration of 50 mg/ml and a volume of 20 ml under the action of ultrasound 40 kHz/70 W in the interval of 15-45 minutes. Longer ultrasound treatment had a negative effect on LA. Conclusions. Lectin activity of U. victoris tissue culture biomass extracts was discovered for the first time, which can become a promising source of lectin with a wide spectrum of pharmacological properties. The general characteristics of the lectin were given, and its species and carbohydrate specificity was revealed. The lectin was generally characterized and its species and carbohydrate specificity were established. Possibilities of ultrasound extraction were used and the procedure was optimized to obtain a soluble form of the lectin, necessary for its further fundamental and practical research.

An optimized microplate-based method to evaluate complement-dependent hemolysis mediated by intravenous immunoglobulins (IVIG)

Hemolytic reactions can cause serious complications after administration of Intravenous Immunoglobulin (IVIG), due to passive transfer of anti-A and anti-B IgG antibodies (isoagglutinins). A maximum allowable amount of isoagglutinins is established in the US and EU for licensed IVIG, as measured by a specified direct hemagglutination test (DHAT). Despite this limit, reports of hemolysis have increased over time, raising the question of how well the DHAT predicts clinically significant hemolysis. This study was undertaken to develop a microplate-based complement-dependent hemolysis assay (CDHA) that reproducibly measures functional hemolytic activity of IVIG, for assessment of IVIG products. An IVIG working reference reagent (NIBSC 14/160) was qualified as an assay control and for quantitation purposes. Hemolytic activities of 36 IVIG product lots encompassing seven brands and including 6 clinically hemolytic lots were measured. Hemolytic activity varied among IVIG product brands, and to a lesser extent, from lot-to-lot for individual brands. Correlation between the CDHA and DHAT was not robust which may reflect imprecision of the DHAT method or additional variables that influence complement-dependent hemolysis after opsonization. In conclusion, the CDHA provides a simple, specific, and sensitive tool for IVIG product characterization and investigation of hemolytic events by manufacturers, researchers, and regulatory authorities.

Development of a Complement-Mediated Hemolysis Assay for Immune Globulin Intravenous Products and Characterization of Lots Associated with Clinical Hemolysis

Introduction: Immune Globulin Intravenous (Human) (IGIV), as known as Intravenous Gamma-Globulin (IVIg), are therapeutic polyclonal immunoglobulin preparations manufactured from pooled human plasma. Initially licensed for replacement therapy of primary immune deficiencies, IGIV indications have expanded to include various autoimmune and inflammatory conditions. Hemolytic reactions associated with IGIV infusion were historically considered as uncommon, but seem to be more frequent over the past 5-6 years, coinciding with widespread manufacturing changes to increase IgG yield, and use of higher dosages. Anti-A and anti-B IgG isoagglutinins in the IGIV preparations are believed to mediate these events. In 2014, FDA co-organized a workshop to discuss strategies to address hemolytic complications of IGIV infusions (Scott 2015). Although US products all comply with an anti-A and anti-B specification of ≤1:64, using the European Pharmacopoeia direct hemagglutination test (DHAT) (Thorp 2010), hemolytic events have persisted. The DHAT is semi-quantitative, imprecise, and does not measure hemolytic function. However, hemolysin tests have been difficult to standardize, especially for highly concentrated IGIV products (Scott 2015).This study reports development of a complement-dependent hemolysis assay (CDHA) suitable for testing IGIV products. We also tested a positive control preparation that is available from NIBSC (code 14/160), consisting of an IGIV lot that caused clinical hemolysis. Finally, we demonstrated differences among IGIV brands with respect to hemolytic activity.Methods: To provide consistent reagents, serum from whole blood donations were frozen as a large volume source of complement and a droplet-freezing method was used to store Red Blood Cells (Schmid 2011; Figure 1). Critical assay conditions were studied, including enzymatic treatment of RBC, removal of unbound IgG, complement source and concentration, divalent cation concentrations, and pH. Hemolytic activities of IGIV products and a positive control IGIV preparation were compared to the hemagglutination results tested using DHAT method. The CDHA was also used to evaluate hemolytic activity of IgG subclasses.Results: The most critical parameters for reproducible CDHA were complement source and preparation method, and assay pH. A pH of 6.5 (0.1M Tri-BIS buffer) was the most suitable condition for all of the tested IGIV analytes. Intra-laboratory variation was minimized by use of frozen RBCs and a consistent highly active complement preparation. The DHAT and CDHA results correlated with each other for most IGIV product lots (Figure 2). Hemolytic activity in IGIV lots varied among, in some cases within, brands. Enrichment of IgG subclass fractions from NIBSC 14/160 showed the highest hemolytic activity was present in IgG2 (Figure 3). In contrast, studies of recombinant anti-A monoclonal antibodies expressing the same anti-A F(ab'), but subclass-specific Fc receptors showed greatest activity of IgG3, consistent with evidence that IgG3 is the most potent complement-activating subclass. The role of IgG2 in hemolysis is not entirely surprising, as saccharide antigens often elicit IgG2 responses, and IgG2 can mediate complement activation (Diebolder 2014). Further subclass studies are planned.Conclusion: Hemolytic activity of IGIV products mediated by anti-A isoagglutinins can be reproducibly measured in IGIV products (Figure 4). Compared to the DHAT method, CDHA provides a continuous readout, is less variable, amenable to use of a standard curve, and more precise. Historical challenges to reproducibility are addressed through biological reagent standardization and attention to critical conditions. Studies comparing the CDHA and DHAT methods across a wider range of products and hemolytic lots will discern whether the CDHA provides a better measure of product safety than the current DHAT lot release assay. The CDHA can be helpful for characterization of new manufacturing methods and products, and investigation of hemolytic events. Studies of hemolysis-implicated lots may provide a basis for improvements in product safety. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cerebral toxoplasmosis in patients with acquired immune deficiency syndrome in the neurological emergency department of a tertiary hospital

IntroductionCerebral toxoplasmosis is the most common cause of space occupying brain lesion in patients with HIV/AIDS in Brazil. In the post-HAART era, it is responsible for high rates of morbidity and mortality worldwide. Materials and methodsThis study consists of a case series of 56 patients diagnosed with cerebral toxoplasmosis whose clinical features, brain imaging and cerebrospinal fluid aspects were analyzed. ResultsCerebral toxoplasmosis led to the diagnosis of infection by the human immunodeficiency virus (HIV) in 27 (48.2%) of the patients, while 29 (51.2%) others already knew to be HIV seropositive. However, at the time of diagnosis of cerebral toxoplasmosis, only 9 (16.6%) reported being under antiretroviral therapy and 5 (8.9%) were receiving primary prophylaxis for toxoplasmosis. Headache, strength deficit and fever were the most frequent signs and symptoms throughout the study. Fifty-three patients showed changes consistent with toxoplasmosis in CT or MRI. Thirty-four (60.7%) CSF samples were positive in the indirect haemagglutination test and for the reaction of Toxoplasma gondii IgG ELISA, while 31 (55.4%) were positive in the direct haemagglutination test. Fifty (89.3%) patients underwent first-line treatment for toxoplasmosis. ConclusionCerebral toxoplasmosis is still a very relevant neurological disease in individuals with AIDS admitted to neurology emergency departments. Early diagnosis and initiation of empiric treatment and antiretroviral therapy are important for good prognosis.

The beneficial effect of dietary zinc supplementation on anaemia and immunosuppression in Trypanosoma brucei infected rats

Zinc is an essential trace element crucial for normal development and function of cells mediating nonspecific immunity and protects bio-molecules from oxidative damage. This study was designed to assess the effects of dietary zinc supplementation on anaemia and immunity of trypanosome-infected rats. Thirty rats, divided into five groups (A–E) of 6 each, were used for the study. Parameters used to assess the effect of the supplementation are antibody response to Sheep RBC using direct haemagglutination test, parasitaemia using the rapid matching method, WBC count using the improved Neubauer haemocytometer method, haemoglobin concentration using the cynomethaemoglobin technique while PCV was determined using the microhaematocrit method. The pre-infection supplementation did not prolong the pre-patent period significantly (p > 0.05). However, it significantly (p < 0.05) increased the packed cell volume (PCV), haemoglobin (Hb) concentration, leucocyte count, and antibody titre by day 7 on the supplementation (OTS). Following infection on day 7 OTS, the PCV and Hb decreased but remained significantly (p < 0.05) higher than the infected not supplemented (INS) group, while on day 14 OTS, they maintained a significantly (p < 0.05) higher antibody titre as compared to other groups. On day 21 OTS, theweight of 8 ppm and not infected not supplemented (NINS) groups was significantly (p < 0.05) higher but the relative organ weight of their liver and spleen was significantly (p < 0.05) lower than 2 ppm, 4 ppm and INS groups. On day 21 OTS, the parasitaemia levels of INS group was significantly (p < 0.05) higher than the supplemented groups. From the results, dietary zinc supplementation can be useful in the management of anaemia and immunosupression caused by trypanosomes in rats.

Anti-A and anti-B haemagglutinin levels in intravenous immunoglobulins: Are they on the rise? A comparison of four different analysis methods and six products

Recent reports of severe haemolytic reactions upon high dose treatment with new generation intravenous immunoglobulins (IVIGs) prompted us to examine the anti-A and anti-B haemagglutinin content of these therapeutics. We compared four different test methods, namely the indirect and direct haemagglutination test as described in the European Pharmacopoiea (Ph. Eur.) and two commercial gelcard systems with the aim to define the most reliable method for a large-scale comparison of different IVIG products. Absolute titres varied when the same samples were analyzed by the four methods, while the relative ranking of six different IVIG preparations representing different manufacturing classes was identical. New generation IVIGs showed 1–2 titre steps higher anti-A titres than the older products. Haemagglutinin titres of all 48 IVIG batches analyzed were within the current Ph. Eur. specification of ≤1:64 when tested by the official pharmacopoeial method. Based on efficiency, reliability and lower costs, the direct gelcard method could be a valid alternative to the official Ph. Eur. method to serve as a limit test. However, due to the highest intermediate precision, the official Ph. Eur. method seems to be most suitable to compare haemagglutinin titres of different IVIG products.

Synthesis and pharmacological evaluation of polyfunctional benzimidazole-NSAID chimeric molecules combining anti-inflammatory, immunomodulatory and antioxidant activities

Polyfunctional compounds comprise a novel class of therapeutic agents for treatment of multifactorial diseases. The present study reports a series of benzimidazole-non-steroidal anti-inflammatory drugs (NSAIDs) conjugates (1-10) as novel polyfunctional compounds synthesized in the presence of orthophosphoric acid. The compounds were evaluated for anti-inflammatory (carageenan-induced paw edema model), immunomodulatory (direct haemagglutination test and carbon clearance index models), antioxidant (in vitro and in vivo) and for ulcerogenic effects. Each of the compound has retained the anti-inflammatory activity of the corresponding parent NSAID while exhibiting significantly reduced gastric ulcers. Additionally, the compounds are found to possess potent immunostimulatory and antioxidant activities. The compound 8 was maximally potent (antibody titre value 358.4 ± 140.21, carbon clearance index 0.053 ± 0.002 and antioxidant EC50 value 0.03 ± 0.006). These compounds, exhibiting such multiple pharmacological activities, can be taken as lead for the development of potent drugs for the treatment of chronic multifactorial diseases involving inflammation, immune system modulation and oxidative stress such as cancers. The Lipinski's parameters suggested the compounds to be bear drug like properties.

Antenatal screening forToxoplasma gondiiinfection at a tertiary care hospital in Riyadh, Saudi Arabia

BACKGROUND AND OBJECTIVES:Congenital toxoplasmosis is associated with significant morbidity and mortality. This study investigates the prevalence of toxoplasmosis among pregnant women.DESIGN AND SETTING:A retrospective study at King Khalid University Hospital, Riyadh from September 2009 to August 2010.PATIENTS AND METHODS:Laboratory data of 2176 pregnant women screened for Toxoplasma gondii in the antenatal care unit were assessed during the study period. The mean (SD) age of the women and the duration of pregnancy were 25 (7.3) years and 18 (7.7) weeks, respectively. Data were extracted for the presence or absence of anti-T gondii immunoglobulin G (IgG) and IgM antibodies.RESULTS:Of 2176 sera tested, 1351 (62%) did not show any evidence of exposure to T gondii. The remaining 825 (38%) samples tested positive for anti-T gondii IgG antibodies, and none was found to have anti-T gondii IgM antibodies in the serum. These data reveal that a significantly high number of women in the antenatal care unit at King Khalid University Hospital in Riyadh had been exposed to T gondii.CONCLUSION:A high prevalence of toxoplasmosis among pregnant women warrants multicenter community-based investigations for assessment of T gondii infection and identification of risk factors for transmission of toxoplasmosis in general, and particularly during pregnancy.

Quantitation and characterization of anti-Galalpha1-3Gal antibodies in sera of 200 healthy persons.

The natural occurrence of complement-activating anti-Galalpha1-3Gal antibodies (anti-Gal) in human serum is considered as a major obstacle to xenotransplantation. In this study we determined anti-Gal in sera of 200 healthy volunteers (100 male, 100 female) of different age groups using an IgG- and IgM-isotype specific ELISA. In addition, we used a direct hemagglutination test involving rabbit erythrocytes (E(R)), which are known for abundant surface expression of the Galalpha1-3Gal antigen. The measured arbitrary ELISA units (U) for anti-Gal ranged from 5 to 204 U (77+/-47; mean +/- 1 SD) for IgM, and from 1 to 162 U(73+/-32) for IgG. Anti-E(R) hemagglutination titers were between 1: 65536 and 1: 64 (mean 1: 703), with 75% of all serum samples being in the range of 1: 8192-1: 512. Specificity of the tests was determined by immunoabsorption of anti-Gal on Sepharose-coupled synthetic Galalpha1-3Gal antigen, which reduced ELISA as well as agglutination titers by 90% or more. Overall, inter-individual differences of both anti-Gal ELISA values and E(R) agglutination titers exceeded age-, gender-, or ABO-type related changes. A tendency was found to higher anti-Gal IgM values in women than in men (P < 0.005 by Student's t-test), whereas the gender-difference for anti-Gal IgG or E(R) titers was not significant. Application of the anti-Gal ELISA as well as the E(R) agglutination assay for pre-transplant screening of potential pig xenograft recipients is discussed.

Reaction of rheumatoid factors with IgG3 monoclonal anti-Rh(D) antibodies: more frequent reactivity to a monoclonal antibody of the Gm allotype G3m(5) in rheumatoid patients negative for G3m(5).

Human monoclonal anti-Rh(D) antibodies of known IgG isotype and Gm allotype were bound to erythrocytes and then used as the target IgG antigens for rheumatoid factors (RFs) in a direct haemagglutination test. When serum samples from patients with rheumatoid arthritis (RA) were tested for RF specificity towards these IgG monoclonal anti-D antibodies the incidence and titre of reactivity towards an IgG3 monoclonal anti-D antibody was considerably greater than for a polyclonal anti-D antibody of the same Gm allotype, G3m(5). This difference was not explained by the amount of each anti-D antibody which bound to erythrocytes. Furthermore, when patients with RA were divided into groups according to their Gm phenotype, sera from a greater proportion of patients negative for the phenotype G3m(5) reacted to the G3m(5) monoclonal anti-D antibodies than sera from those patients positive for this allotype. Analysis of RF reactivities towards two IgG3 and three IgG1 monoclonal anti-D antibodies, each with different Gm allotypic epitopes, indicated, however, that individual serum samples contained RFs with a spectrum of specificities; some sera appeared to react to a single set of Gm alleles, whereas others also reacted to isotypic or iso-allotypic epitopes, or both. Our data suggest that RFs with specificity for Gm allotypes do not arise in patients who carry that particular allotype owing to tolerance induced in fetal-neonatal life. Conversely, RFs with apparent specificity for a Gm allotype formed in patients negative for that allotype may be reacting to a closely related but different epitope. Final proof requires precise specificities for each RF formed, and IgG3 monoclonal anti-D antibodies would be useful reagents for this purpose.

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.

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.

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.

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.

Histocompatibility-2 (H-2) polymorphism in wild mice.

Red blood cells of 40 wild mice captured at four different locations in the Ann Arbor area were typed for the presence of 15 different H-2 antigens by direct hemagglutination test. Fifteen different phenotypes were found and all were different from those determined by known H-2 alleles of the laboratory mice. Great similarity between phenotypes of mice from the sane location was observed. This is interpreted as further evidence for the deme structure of natural mouse population.