Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Comparison of copper-67- and iodine-125-labeled anti-CEA monoclonal antibody biodistribution in patients with colorectal tumors.

Copper-67 has comparable beta-particle emissions to that of 131I, but it displays more favorable gamma emission characteristics for application in radioimmunotherapy (RIT). This study investigates the potential of 67Cu-labeled monoclonal antibody (MAb) 35 for RIT of colorectal carcinoma. METHODS: Biokinetics of simultaneously injected 67Cu- and 125I-labeled MAb35 were studied in six patients scheduled for surgery of primary colorectal cancer. RESULTS: Whole-body clearance (T 1/2) of 67Cu, estimated from sequential anterior and posterior whole-body scans and corrected for decay of 67Cu, was 41 hr. Serum clearance of 67Cu was faster (27.41 hr) than that of 125I (38.33 hr). Mean tumor uptake of the 67Cu-labeled compound (0.0133% ID/g) exceeded that of 125I (0.0095% ID/g), and tumor-to-blood ratios were higher for 67Cu than for 125I, with averages of 6.07 and 2.41, respectively. The average 67Cu/125I ratio was 1.9 for tumor uptake, 0.7 for blood and 2.6 for tumor-to-blood ratios. Nonspecific liver uptake of 67Cu as calculated from whole-body scans was high in four patients, up to 25% of residual whole-body activity at 48 hr, but did not increase with time. We also observed some nonspecific bowel activity, as well as moderate to high uptake in benign polyps. CONCLUSION: Copper-67-labeled MAb35 is more favorable than its radioiodine-labeled counterpart for RIT of colorectal carcinoma due to higher tumor-to-blood ratios, but the problem of nonspecific liver and bowel uptake must first be overcome. The absolute accumulation of activity in tumor remains low, however, so the probability of cure with this compound alone is questionable. The use of 67Cu as one component of a multimodality adjuvant treatment seems to remain the most appropriate application for RIT.

Evaluation of tests based on the antibody response to keyhole limpet haemocyanin and soluble egg antigen to differentiate acute and chronic human schistosomiasis mansoni

Specific IgG and IgM responses to soluble egg antigen (SEA) and keyhole limpet haemocyanin (KLH) were measured by ELISA in patients with acute and chronic schistosomiasis. The tests based upon IgM and IgG antibodies responses to KLH presented the best diagnostic discrimination, and can be used in conjunction with clinical and epidemiological data to the differential diagnosis of acute schistosomiasis.

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Diphtheria-neutralizing antibody levels in healthy adults from Rio de Janeiro, Brazil

In Brazil, until 2004, the immunization policy against diphtheria involved childhood vaccination with no official routine booster dose administered after 15 years of age. This study assessed functional antibody levels against diphtheria among blood donors. A total of 140 blood samples were collected, and diphtheria antitoxin levels were evaluated by Vero cell neutralization test. The mean age of the population was 34 years old (range: 18-61 years); 37.8% females and 62.2% males. Overall, 30.7% (95%, CI: 23.4-38.7) individuals presented neutralizing antitoxin antibody titers < 0.01 IU/ml; 42.1% (95%, CI: 34.1-50.4) showed values between 0.01-0.09 IU/ml and, 27.1% (95%, CI: 20.2-34.9) had ³ 0.1 IU/ml. In the subgroup of individuals with history of diphtheria immunization during childhood (85%), a number of 28.5% showed unprotective levels of circulating neutralizing antibody (< 0.01 IU/ml). Despite the continuous progress of immunization programs directed to Brazilian population, currently healthy adults remain susceptible to diphtheria.

Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86).

Merozoite surface protein 2 allelic variation influences the specific antibody response during acute malaria in individuals from a Brazilian endemic area

The antibody response to Plasmodium falciparum parasites of naturally infected population is critical to elucidate the role of polymorphic alleles in malaria. Thus, we evaluated the impact of antigenic diversity of repetitive and family dimorphic domains of the merozoite surface protein 2 (MSP-2) on immune response of 96 individuals living in Peixoto de Azevedo (MT-Brazil), by ELISA using recombinant MSP-2 proteins. The majority of these individuals were carrying FC27-type infections. IgG antibody responses were predominantly directed to FC27 parasites and were correlated to the extension of polymorphism presented by each MSP-2 region. This finding demonstrated the impact of the genetic polymorphism on antibody response and therefore, its importance on malaria vaccine efficacy.

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.

Active antiviral T-lymphocyte response can be redirected against tumor cells by antitumor antibody x MHC/viral peptide conjugates.

PURPOSE: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. EXPERIMENTAL DESIGN: First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice. RESULTS: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). CONCLUSION: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains

In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1·8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Antibody reactivity against potato apyrase, a protein that shares epitopes with Schistosoma mansoni ATP diphosphohydrolase isoforms, in acute and chronically infected mice, after chemotherapy and reinfection

Schistosoma mansoni ATP diphosphohydrolase isoforms and potato apyrase share conserved epitopes. By enzyme-linked immunosorbent assays, elevated levels of IgM, IgG2a and IgG1 antibody reactivity against potato apyrase were observed in S. mansoni-infected BALB/c mice during the acute phase of infection, while only IgM and IgG1 antibody reactivity levels maintained elevated during the chronic phase of infection. Antibody reactivity against potato apyrase was monitored over an 11-month period in chronically-infected mice treated with oxamniquine. Eleven months later, the level of seropositive IgM decreased significantly (~30%) compared to the level found in untreated, infected mice. The level of seropositive IgG1 decreased significantly four months after treatment (MAT) (61%) and remained at this level even after 11 months. The IgG2a reactivity against potato apyrase, although unchanged during chronic phase to 11 MAT, appeared elevated again in re-infected mice suggesting a response similar to that found during the acute phase. BALB/c mouse polyclonal anti-potato apyrase IgG reacted with soluble egg antigens probably due to the recognition of parasite ATP diphosphohydrolase. This study, for the first time, showed that the IgG2a antibody from S. mansoni-infected BALB mice cross-reacts with potato apyrase and the level of IgG2a in infected mice differentiates disease phases. The results also suggest that different conserved-epitopes contribute to the immune response in schistosomiasis.