Investigation Of Cross-reactivity Research Articles

Comparative analysis of cysteine proteases reveals gene family evolution of the group 1 allergens in astigmatic mites.

Astigmatic mites contain potent allergens that can trigger IgE-mediated immune responses, leading to allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. In house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae, group 1 allergens (Der p 1 and Der f 1), characterized as papain-like cysteine proteases, have been defined as the major allergens that have high prevalence and potency. Previous studies of mite group 1 allergens mainly focused on identification, comparison of sequence and structure, as well as the investigation of cross-reactivity. To achieve a comprehensive view of mite group 1 allergens, we performed a comparative genomic analysis of all the cysteine proteases in six astigmatic mite species to elucidate the evolutionary relationships of group 1 allergens. Based on the high-quality and annotated genomes, all the cysteine proteases in six astigmatic mite species were identified by sequence homology search. The phylogenetic relationships, gene synteny and expression levels were revealed by bioinformatic tools. The allergenicity of recombinant cysteine proteases was evaluated by enzyme-linked immunosorbent assay. Tandem duplication was revealed as the major feature of cysteine protease gene evolution in astigmatic mites. The high IgE-binding capacity and the significant expression level of the cysteine protease DP_007902.01 suggested its potential as a novel group 1 allergen of D. pteronyssinus. In addition, gene decay events were identified in the skin-burrowing parasitic mite Sarcoptes scabiei. This comprehensive analysis provided insights into the evolution of cysteine proteases, as well as the component-resolved diagnosis of mite allergies.

Investigation of cross-reactivity between phenobarbital and levetiracetam in children with epilepsy: A prospective, observational multicenter study.

Pharmaceutical allergic reactions due to antiseizure medications (ASMs) are one of the major concerns in the management of patients. Finding an alternative ASM which does not cause allergic reactions and has acceptable effectiveness can be difficult. In this regard, the present study attempts to investigate the cross-reactivity between phenobarbital and levetiracetam in children under treatment for seizure control. The present study is a prospective, observational independent assessor study. 30 children with epilepsy who were hypersensitive to phenobarbital therapy were studied. In order to evaluate the cross-reactivity of the drugs, levetiracetam replaced phenobarbital to control seizure. Within 6months, any allergic reactions and seizure recurrences were evaluated in the patients. 53% of the children in this study were female. The mean age of patients was 42.4months. In patients' follow up no cross-reactive responses were observed in any of the patients. Seizure recurrence rate was 30% in the first six months of follow up that with increasing dosage in the second six months of follow-up, decreased to 10%. Based on the results of this study, in children with epilepsy controlled by phenobarbital if allergic reactions to phenobarbital occur, levetiracetam may be used as a suitable alternative medicine.

Investigation of Cross-Reactivity of Anti-Ephrin-B2 Antibody to Other Ephrin-B Members in an Immunohistochemical Study in a Cohort of Oral Squamous Cell Carcinoma

Ephrin-B1,-B2 and -B3 proteins share a high degree of sequence similarity. Investigation of these proteins as putative prognostic markers in human cancers including oral squamous cell carcinoma (OSCC) has been limited by challenges in generating specific antibodies against them. The current study examined the reactivity of a polyclonal anti-human ephrin-B2 antibody (HPA008999) against ephrin-B proteins and investigated the prognostic significance of immunoreactivity of the same antibody at different intra-tumor sites in OSCC specimens. By amino acid sequence comparison, immunocytochemistry and Western blot analysis on cell lysates and precipitates from HEK-293T cells transfected with EFNB1, EFNB2, or EFNB3 expression constructs, we demonstrated that HPA008999 reacted to all ephrin-B proteins. Using immunohistochemistry (IHC) with the HPA008999 antibody in a cohort (n = 131) of OSCC, we showed high immunoreactivity at the tumor center, but not at the tumor invading front, was significantly associated with worse 5-year overall survival probabilities. In conclusion, the HPA008999 antibody reacted to all ephrin-B proteins and the immunoreactivity at the tumor center might be useful as a prognostic marker in OSCC. These data underscore the need for the investigation of antibodies for cross-reactivity to similar protein members for obtaining reliable and meaningful results in IHC based biomarker studies.

Evolutionary Mechanism of Immunological Cross-Reactivity Between Different GII.17 Variants.

Human norovirus is regarded as the leading cause of epidemic acute gastroenteritis with GII.4 being the predominant genotype during the past decades. In the winter of 2014/2015, the GII.17 Kawasaki 2014 emerged as the predominant genotype, surpassing GII.4 in several East Asian countries. Hence, the influence of host immunity response on the continuous evolution of different GII.17 variants needs to be studied in depth. Here, we relate the inferences of evolutionary mechanisms of different GII.17 variants with the investigation of cross-reactivity and cross-protection of their respective antisera using the expression of norovirus P particles in Escherichia coli. The cross-reactivity assay showed that the antisera of previous strains (GII.17 A and GII.17 B) reacted with recent variants (GII.17 C and GII.17 D) at high OD values from 0.8 to 1.16, while recent variant antisera cross-reacting with previous strains were weak with OD values between 0.26 and 0.56. The cross-protection assay indicated that the antisera of previous strains had no inhibitory effect on recent variants. Finally, mutations at amino acids 353–363, 373–384, 394–404, and 444–454 had the greatest impact on cross-reactivity. These data indicate that the recent pandemic variants GII.17 C and GII.17 D avoided the herd immunity effect of previous GII.17 A and GII.17 B strains through antigenic variation.

Acute generalized exanthematous pustulosis due to diltiazem: Investigation of cross-reactivity with other calcium channel blockers

Acute generalized exanthematous pustulosis due to diltiazem: Investigation of cross-reactivity with other calcium channel blockers

Investigation of cross-reactions with Francisella tularensis antibodies to Brucella

Investigation of cross-reactions with Francisella tularensis antibodies to Brucella

Radioimmunoassay for a novel benzodiazepine inverse agonist, S-8510, in human plasma and urine

A radioimmunoassay (RIA) was developed for a new benzodiazepine inverse agonist, 2-(isoxazol-3-yl)-3,6,7,9-tetrahydroimidazo [4,5-d] pyrano [4,3-b] pyridine monophosphate monohydrate (S-8510), in human plasma and urine. For competitive RIA, three amino derivatives of S-8510 were labelled by the Bolton and Hunter method and rabbit antisera were prepared using three immunogens, conjugates of three carboxyl derivatives of S-8510 with bovine serum albumin. All combinations of the labelled antigens and antisera were examined and homologous combinations were selected for the competitive RIA. One of the three homologous combinations had the best selectivity after investigations of cross-reactivity using 12 related compounds and was very sensitive for S-8510. Next, a pretreatment for biological samples was developed using mixed mode solid-phase extraction (SPE) column followed by the RIA (SPE/RIA). The assay recoveries for human plasma and urine were both excellent and the limits of quantitation were extremely low, 80 and 200 pg ml −1, respectively. Human plasma samples and urine samples after administration of this drug were successfully measured by the SPE/RIA. No cross-reactive metabolites were detected in any fractions after RP-HPLC separation of the plasma samples. The RIA using carefully selected antiserum and labelled antigen was highly specific for unchanged S-8510. To simplify the RIA procedure, a scintillation proximity assay (SPA) using the same labelled antigen and antiserum was developed for analyzing S-8510 in human plasma and found to be very promising.

Occupational wood‐dust sensitivity from Euonymus europaeus (spindle tree) and investigation of cross reactivity between E.e. wood and Artemisia vulgaris pollen (mugwort)

A 44-year-old goldsmith suffered from rhinitis and conjunctivitis after having worked with wood dust from Euonymus europaeus (E.e.) for 15 years. The material was used for drying pieces of jewelry. Very strong reactions could be seen after friction test, scratch test and nasal challenge using wood dust of E.e. RAST-class 3 could be measured with the serum of this patient using E.e. wood and Artemisia vulgaris (A.v. pollen) allergen disks. RAST-inhibition, western-blot (WB) and immunoprint (IP) indicated common allergens in extracts of E.e. wood and A.v. pollen of different degree. In addition this study indicated that subjects suffering from A.v. pollen allergy also show sensitization to E.e. wood since in 22 of 37 A.v. pollen allergies A.v. (RAST class 2-4) IgE-antibodies could be seen. The present case probably demonstrates for the first time an IgE-mediated type I allergy to E.e. wood.

A Rapid and Sensitive Test for Diagnosing Heparin-Associated Thrombocytopenia

Heparin-associated thrombocytopenia (HAT) is a severe complication of heparin therapy. Its diagnosis is difficult. Conventional assays employ platelet aggregometry (PAA) and/or 14C-serotonin release (SRA) which are either insensitive (PAA) or require radioactive tracers (SRA). We here describe a newly developed sensitive and rapid assay based on visual evaluation of heparin-induced platelet activation (HIPA) in microtiter wells. Using sera of 34 patients with clinically suspected HAT we found the HIPA assay to be as sensitive as the SRA and superior to PAA. The HIPA assay allows investigation of crossreactivity with different types of heparins, low molecular weight (LMW) heparins and heparinoids. Three patients who required further parenteral anticoagulation and in whom the HIPA assay was negative before treatment with the LMW heparinoid Org 10172, were treated with this new heparinoid without adverse reactions. We conclude that the HIPA assay may be a useful tool for differential diagnosis and therapy in patients with HAT.

Investigation of cross-reactivity between commercially available antibodies directed against human, mouse, and rat lymphocyte surface antigens and surface markers on canine cells

Using automated flow cytometry, 23 commercially available antibodies (all but one of them monoclonal) raised against surface antigens of specific populations of human, rat, and mouse lymphocytes were tested for cross-reactivity to peripheral blood lymphocytes from five clinically healthy adult dogs. Of all the antibodies tested, only the polyclonal anti-asialo GMl directed against mouse NK cells, and the monoclonal antibodies anti-HLA-DR directed against the human class II antigen and anti-B1, a human pan B cell marker, consistently labeled subpopulations of canine lymphocytes.

Immunological cross-reactions between a cat hair and skin scraping extract and a cat serum and investigation of common allergenic molecules of cat 1 with cat serum.

Investigations of cross reactions between a cat hair and skin scraping extract (CHSS) and a cat serum (CS) were done by several biochemical and immunochemical methods. Furthermore, we looked for common allergenic molecules in a cat 1 reference preparation and in a CS. In crossed (radio) immunoelectrophoresis (CIE/CRIE) of CHSS vs. anti CS-serum one to eight allergen bands could be detected depending on the human serum employed. By constructing allergograms from the CRIE patterns of 17 patients' sera one out of eight allergens could be defined as a major allergen. CRIE of the cat 1 reference vs. anti CS-serum, using the sera of two cat allergic patients, displayed four to nine allergen bands, indicating that the cat 1 reference is not a pure preparation but rather an enriched fraction.

Enzyme-Linked Immunosorbent Assay to Detect Antibody Response against Eimeria bovis and Eimeria zurnii in Calves

In the United States, bovine coccidiosis is primarily a disease of young calves, 6 to 9 mo of age (Fitzgerald, 1975, Bovine Practice 10: 2833). Eimeria zurnii and Eimeria bovis are considered to be the 2 most pathogenic species of cattle (Levine and Ivens, 1970, The coccidian parasites [Protozoa, Sporozoa] of ruminants. University of Illinois Press, Urbana, Illinois). Early detection of the clinical coccidiosis before production of the oocysts, by means of serological methods, may be useful in identifying the infected animals, thereby enabling early and effective treatment. The enzyme-immunosorbent assay (ELISA) has been reported to be sensitive and effective in detecting the serum antibodies against chicken coccidiosis (Rose and Mockett, 1983, Parasite Immunology 5:479-489; Saatara Oz et al., 1984, Journal of Parasitology 70: 859-863). This led to the present investigation of cross-reactivity between a soluble antigen of Eimeria tenella and sera from calves infected with E. zurnii and/or E. bovis. Lack of species specificity might allow the use of a purified avian coccidia antigen to identify coccidiosis in cattle. Twenty Holstein calves were purchased at about 1 wk of age and maintained in indoor pens. Fecal samples were collected before and during the experiment. The calves had not produced oocysts of any species prior to oral inoculation. About 5 wk after purchase, they were divided into 4 groups and were inoculated orally with sporulated oocysts of E. bovis and/or E. zurnii as described below. Group A: 4 calves received 150,000 oocysts of E. bovis (95% pure). Group B: 4 calves received 230,000 oocysts of E. zurnii (78% pure). Group C: 6 calves received 300,000 oocysts of E. bovis and 500,000 oocysts of E. zurnii. Group D: 6 calves were kept as uninfected controls. Contaminating oocysts present were Eimeria auburnensis, Eimeria cylindrica, and Eimeria ellipsoidalis. Calves were bled via the jugular vein prior to inoculation and then weekly to 5 wk post-inoculation (4 wk PI for the E. bovis and E. zurnii group). All of the sera collected were stored at 20 C. Sixteen sera were randomly collected from colostrum-deprived calves that were not positive for coccidia on fecal exam (birth to 13 wk of age) and were used as additional uninfected control

EVALUATION OF AN INDIRECT FLUORESCENT ANTIBODY TEST TO DIAGNOSE BABESIA EQUI INFECTION IN HORSES

An indirect fluorescent antibody (IFA) test for the diagnosis of Babesia equi infections was evaluated. Antigen prepared by conventional methods was of high quality in one instance and of lesser quality in a second when possible autofluorescence of the horse blood caused inconvenience in reading tests. Tests on 14 horses shown by parasitological means to be either infected (9) or uninfected (5) produced reactions at dilutions of 1/270 to 1/7290 for infected and at 1/10 to 1/90 for uninfected animals. The accuracy of the test was further demonstrated during investigations of 701 horses in 3 states of Australia. The 30 horses reacting at 1/270 to 1/2430 were from 33 imported to 3 different farms in Australia from a common source. Investigations of crossreactivity between B. equi and B. bovis of cattle suggested that B. bovis would not interfere with the test for B. equi, but that the reverse was possible.