Horseradish peroxidase cross-reacts during cytochrome oxidase histochemistry

Summary Injections of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) were made into the cortex of kittens. When cytochrome oxidase histochemistry was performed on sections taken through the injection sites staining of the WGA-HRP deposits occurred, demonstrating that WGA-HRP can cross-react during processing for cytochrome oxidase.     

Specificity of tuberculin skin test improved by BCG immunization schedule change in Japan

IntroductionTuberculin skin test (TST) has been used to diagnose tuberculosis (TB) and latent tuberculosis infection (LTBI). However, in Bacillus Calmette-Guérin (BCG) vaccinated patients, TST tends to produce false-positive results. According to the previous vaccination schedule, Japanese people were mandated to receive up to three doses of BCG-vaccine. The vaccination schedule was changed in 2003 and as per the new schedule, only infants are administered a dose of BCG vaccine. Our hypothesis is that this change can lead to a reduction in the cross-reaction to TST.MethodsWe evaluated the TST results obtained from 1097 recruits from six defense camps and 667 recruits from an air base. These TST data were divided into two groups according to the date of birth: a new group and an old group according to the BCG immunization schedule. We then analyzed positive and negative reaction of TST and erythema sizes.ResultsWe confirmed that the change in BCG-vaccination schedule significantly decreased TST false-positive reaction (P_meta = 1.4 × 10⁻¹⁸; risk ratio = 0.83; 95% confidence interval: 0.80–0.87) and erythema size (P_meta = 1.1 × 10⁻⁴; mean difference = 6.6 mm; 95% confidence interval: 3.2 mm–9.9 mm).ConclusionsWe showed the reduction in BCG cross-reaction to TST, in the new BCG vaccination schedule group, compared to the old group, we also have extracted information on the improvement in the specificity of TST for LTBI and TB diagnosis, which resulted from BCG schedule change.     

A line immunoassay utilizing recombinant nucleocapsid proteins for detection of antibodies to human coronaviruses

Most coronaviruses infecting humans cause mild diseases, whereas severe acute respiratory syndrome (SARS)-associated coronavirus is an extremely dangerous pathogen. Here, we report the development of a serologic assay for detection of antibodies to human coronaviruses (HCoVs) based on recombinant nucleocapsid (N) proteins of all known pathogenic strains (229E, NL63, OC43, HKU1, SARS). The novel immunoassay is highly useful for epidemiologic surveys, where use of nucleic acid diagnostics often is limited. Purified recombinant antigens were immobilized on nitrocellulose membranes and applied in a line immunoassay, which allows rapid detection of antibodies to 5 different HCoVs in a single experiment. For assay evaluation, serum samples from persons infected with 229E or OC43 (acute/convalescent), recovered SARS patients and healthy donors were analyzed. Screening for nucleocapsid (N)-specific immunoglobulin G (IgG) in convalescent sera reached 100% sensitivity. With this new technique, we found that recently identified NL63 and HKU1 contribute significantly to the overall spectrum of coronavirus infections. Possibly, cross-reactive antibody responses were observed using 229E and OC43 serum pairs. However, the potential of this assay could clearly be demonstrated employing SARS-positive serum samples, where nonspecific binding to nucleocapsids of other HCoVs was not observed. This coronavirus strain-specific line immunoassay represents a powerful tool for serologic diagnostics.     

Cardiovascular Anomalies Induced by Anti-Tissue Sera and Immunological Cross-Reaction

Wistar rats were administered rabbit anti-tissue serum alone or in combination with sub-teratogenic doses of anti-kidney serum (AKS) on day 9 of gestation. The results showed that the incidence of cardiovascular malformations was highest (46.7%) when a small dose (1 ml/kg) of AKS given on day 9 was followed by 9 ml/kg of anti-heart serum (AHS) on day 10. The most common cardiovascular malformations were ventricular septal defects associated with aortic arch anomalies. The spectrum of malformations did not vary with the injected sera. Immunodiffusion studies indicated that the kidney, umbilical cord and yolk sac tissues have at least one common antigen. Between the AHS and the kidney antigen, a slight precipitin band was also demonstrated. These results suggest that the synergistic effects of antisera depend on the degree of immunological cross-reaction with the yolk sac, and that the mechanisms of teratogenicity by the combined administration is caused mainly by yolk sac dysfunction.     

青霉素类药物的化学结构与其交叉过敏反应

目的 探讨青霉素类药物的化学结构与其交叉过敏反应的关系。方法 采用放射免疫吸附试验(radioallergosorbent test, RAST)方法检测133例青霉素过敏患者血清中针对4种青霉素类药物的8种特异性IgE抗体,采用RAST抑制实验和单个核细胞增殖实验研究青霉素类药物的化学结构与其交叉过敏反应的关系。结果 8种特异性IgE抗体的总阳性率为59.40%(79/133),仅对一种药物抗体呈阳性的占26.36%(35/133),不同药物间血清特异性IgE抗体存在交叉反应的占33.08%(44/133)。RAST抑制实验表明,有的病例侧链的抑制率与药物相近,母核抑制率最低;有的病例母核抑制率最高,药物及其各侧链结构的抑制率相近。细胞增殖实验显示,药物、侧链、母核均诱导外周血单个核细胞增殖。其中,药物诱导细胞增殖能力较强,而侧链诱导细胞增殖能力最弱。结论     

Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease

A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) was developed for the detection of circulating cathepsin B3 protease (CatB3) in the sera from mice experimentally infected with Fasciola gigantica and cattle naturally infected with the same parasite. The MoAb 2F9 and biotinylated rabbit polyclonal anti-recombinant CatB3 antibody were selected due to their high reactivities and specificities to F. gigantica CatB3 antigen based on indirect ELISA and immunoblotting. The lower detection limit of the sandwich ELISA assay was 10, 100 and 400 pg/ml, when applied for the detection of rCatB3 antigen and CatB3 in whole body (WB) of newly excysted juveniles (NEJ) and metacercariae (Met) of F. gigantica, respectively. This sandwich ELISA assay could detect F. gigantica infection from day 1 to 35 post infection and revealed that circulating level of CatB3 peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using serum samples from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as normal mice and hamsters. In addition, sera from cattle infected with Paramphistomum cervi, Strongylid, Trichuris sp. and Strongyloides sp., as well as sera from normal cattle were also assessed. In experimental mice, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of ELISA were 95%, 100%, 100%, 97.9%, 0%, 5.3% and 98.5%, while in natural cattle they were 96.7%, 100%, 100%, 98.5%, 0%, 3.4% and 98.9%, respectively. Hence, this assay method showed high efficient and precision for early diagnosis of fasciolosis by F. gigantica.     

PAP法和双PAP法在人淋巴瘤研究中桥抗体引起的交叉染色及消除方法

本文用琼脂免疫双扩实验和省略了第一抗体的PAP法,双PAP法研究了7份来源不同的羊抗兔IgG抗体(桥抗体)与人免疫球蛋白的交叉反应及消除方法。4份高效价的桥抗体在琼脂免疫双扩实验中与人Υ球蛋白有交叉,在双PAP法染色中与人淋巴瘤组织内免疫球蛋白有严重的交叉而致假阳性结果。其中3份抗体高浓度时在PAP法染色中也与人免疫球蛋白有交叉。双PAP法染色中的交叉反应在高于适宜桥抗体浓度时仍可发生。在桥抗体溶液中加入5％的正常人血清可以有效地消除桥抗体所致的交叉染色。本文讨论了这种交叉反应的机理并提出了克服的方法。     

Cross-reaction of antibody against Helicobacter pylori urease B with platelet glycoprotein IIIa and its significance in the pathogenesis of immune thrombocytopenic purpura

Many clinical investigations have suggested that Helicobacter pylori (H. pylori) infection might be associated with immune thrombocytopenic purpura (ITP), but its role in the pathogenesis of ITP is unsettled. In this study, we cultured H. pylori, produced recombinant H. pylori urease (ure) B, and then prepared monoclonal antibody (MoAb) against ureB, 1F11, both 1F11 and MoAb against human platelet glycoprotein (GP) IIIa, SZ21, could bind to the band of GP IIIa of normal platelet lysate, but not to that from a patient with Glanzmann thrombasthenia (GT) whose GP IIb–IIIa complex was absent. Flow cytometry showed that normal platelets were reacted with 1F11 and SZ21, while GT platelets were not. In immuno-radiometric assay, the binding of ¹²⁵I-labeled 1F11 to GP IIIa was higher than that to GP Ib, GP IIb, GP VI, and P-selectin. 1F11 could partly compete with SZ21 in a binding to platelet surface. In addition, 1F11 inhibited platelet aggregation induced by adenosine diphosphate, but had no effect on platelet P-selectin expression or Thromboxane B₂ production of platelets. These results indicate that H. pylori ureB antibody could cross-react with human platelet GP IIIa and partly inhibit platelet aggregation. UreB may be a crucial component of H. pylori involved in the pathogenesis of a subset of ITP. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cross Hypersensitivity Syndrome between Phenytoin and Carbamazepine

Objective: To evaluate the incidence of cross anticonvulsant hypersensitivity syndrome (AHS) between phenytoin (PHT) and carbamazepine (CBZ) in hospitalized patients.Method: Retrospective chart review about the cross AHS was retrieved from pharmacy adverse drug reaction program from 1998 to 2002 in a 450-bed teaching hospital.Main outcome measures: AHS was defined as the appearance of at least two symptoms with the first anticonvulsant drug (ACD). Cross AHS was considered if after withdrawal of a first ACD because of hypersensitivity symptoms, a new episode with similar or new symptoms appeared after exposure to a second ACD. The following symptoms were considered– rash, fever, hepatotoxicity, lymphadenopathies or hematological disturbances.Results: Cross AHS between PHT and CBZ was observed in nine cases (45). After the cross-reaction event, four of them were treated with valproic acid, two with vigabatrin, two with phenobarbital and one with no treatment without developing further AHS.Conclusions: AHS is a severe complication of aromatic ACD that can compromise the future choice of therapy. Because of the high incidence of clinical cross-reaction between these two drugs, non-aromatic ACD alternatives, must be considered.     

Immune response in mouse and malaria-exposed humans to peptides derived from Pfl1-1, a highly repetitive megadalton protein of Plasmodium falciparum

We have investigated the immune response against the Plasmodium falciparum gametocyte-specific antigen Pf11-1. This megadalton parasite molecule has been implicated in the process of erythrocyte rupture during gametogenesis. The molecule is composed in great part of degenerated nonapeptide motifs which are tandemly repeated several hundred times. A computer algorithm searching for T sites predicted that the entire repeat region of the Pf11-1 represents potential T cell antigenic major histocompatibility complex class II-binding sites. To test this hypothesis, synthetic peptides corresponding to two nonamer subtype repeats, differing only at two amino acid positions, were used to immunize congenic mouse strains. Both peptides were shown to contain both B and T cell epitopes. The immune response is restricted to the H-2^d andH-2^khaplotypes. The T cell response against the peptides appeared to be highly specific, clearly discriminating between the two similar nonamer repeat sequences, whereas the humoral response produced cross-reacting antibodies. We also investigated the humoral and T cell reactivities of P. falciparum-primed individuals in West Africa against the synthetic Pf11-1 peptides. Among 51 individuals 35 had antibodies to at least one of the two peptides and a majority of them (28) had antibodies reacting with both peptides. The cellular response was analyzed by [³H] thymidine incorporation or interferon-y release. There was considerable variation in the response to the two peptides. Among the human samples 36% responded to one repeat subtype, while only 13% responded to the second subtype. Interestingly, in individual donors the T cell response to both peptides are associated, suggesting that, as shown for mice, the response is restricted by a genetic element. The data obtained on the two subtypes of the nonamer repeat region suggest that the entire Pfll-1 molecule might induce an unusually heterogenous B and T cell response during natural infection in man.