Immunogenicity of Whole / 生物医学与环境科学（英文）

Objectives@#To evaluate the immunogenicity of @*Methods@#Protein extracts from @*Results@#Immunization with @*Conclusion@#This is the advanced study to investigate the immunogenicity of

Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests / Expressão e análise antigênica da proteína RTP36 recombinante da amostra São Paulo de Ehrlichia canis para testes sorológicos

Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.

Resumo Ehrlichia canis é o principal agente etiológico da erliquiose monocítica canina (EMC), uma doença infecciosa canina globalmente dispersa. No Brasil, a EMC é considerada endêmica, e a infecção pode atingir 65% em cães em alguns estados. O diagnóstico de erliquiose é importante para fins de tratamento e epidemiológicos. A proteína TRP36 de E. canis leva a uma resposta humoral com produção de anticorpos em fase aguda, encontrada durante o curso da doença. O objetivo deste estudo foi obter a proteína TRP36 recombinante da amostra São Paulo de E. canis e avaliar seu potencial como ferramenta para o diagnóstico sorológico da CME. O isolado de E. canis São Paulo foi cultivado em células da linhagem DH82 e o DNA genômico foi obtido. O fragmento de DNA bacteriano que codifica toda a ORF de TRP36 foi clonado no vetor pBAD / Thio-TOPO e transformado em células competentes Escherichia coli DH10B, com o plasmídeo portador de trp36 para expressão de proteínas. Para avaliar a antigenicidade da proteína, 16 amostras de soro canino foram previamente analisadas (por PCR e teste sorológico comercial SNAP®4Dx®). Os resultados estavam de acordo com o teste SNAP®4Dx®. Os experimentos que utilizam essa proteína recombinante como antígeno, visando ao desenvolvimento de um teste sorológico baseado no ELISA, são o próximo passo para produzir um teste de diagnóstico confiável, acessível e útil para o diagnóstico da EMC no Brasil.

DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia

Abstract Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

Assessing and screening for T-cell epitopes from Mycobacterium tuberculosis RD2 proteins for the diagnosis of active tuberculosis

ABSTRACT The Region of D eletion 2 (RD2) of Mycobacterium tuberculosis encodes reserved antigens that contribute to bacterial virulence. Among these antigens, Rv1983, Rv1986, Rv1987, and Rv1989c have been shown to be immunodominant in infected cattle; however, their diagnostic utility has not been evaluated in humans.In this study, we screened 87 overlapping synthetic peptides encoded by five RD2 proteins for diagnosing tuberculosis epitopes in 50 active tuberculosis (TB) cases, 31 non-tuberculosis patients and 36 healthy individuals. A pool of promising epitopes was then assessed for their diagnostic value in 233 suspected TB patients using a whole blood IFN-γ release assay.Only 10 peptides were recognized by more than 10% of active tuberculosis patients. The IFN-γ release responses to Rv1986-P9, P15, P16, Rv1988-P4, P11, and Rv1987-P11 were significantly higher in the active TB group than in the control groups (p < 0.05). The whole blood IFN-γ release assay based on these epitopes yielded a sensitivity of 51% and a specificity of 85% in diagnosing active tuberculosis, and the corresponding results using the T-SPOT.TB assay were 76% and 75%, respectively.In conclusion, these results suggest that the six epitopes from the RD2 of M. tuberculosis have potential diagnostic value in TB.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

ELISA-based assay of immunoglobulin G antibodies against mammalian cell entry 1A (Mce1A) protein: a novel diagnostic approach for leprosy

BACKGROUND Leprosy is a chronic infectious disease caused by the obligate intracellular bacillus Mycobacterium leprae. Because leprosy diagnosis is complex and requires professional expertise, new tools and methodologies are needed to detect cases in early stages and prevent transmission. The M. leprae genome contains mce1A, which encodes a putative mammalian cell entry protein (Mce1A). We hypothesised that the presence of Mce1A on the cell surface could be detected by the host's immune system. OBJECTIVE The aim of this study was to evaluate antibody responses against the Mce1A protein in leprosy patients, household contacts of patients, and the general population to present an addition tool for leprosy diagnosis. METHODS A cross-sectional study involving 89 volunteers [55 leprosy cases, 12 household contacts (HHC) and 22 endemic controls (EC)] was conducted at Couto Maia Hospital, in Salvador, Bahia (BA), Brazil. RESULTS The median anti-Mce1A IgA was significantly higher in multibacillary (MB) and paucibacillary (PB) cases than in EC (p < 0.0001). A similar trend was observed in IgM levels, which were significantly higher in both MB (p < 0.0001) and PB (p = 0.0006) groups compared to in EC individuals. The greatest differences were observed for IgG class-specific antibodies against Mce1A. The median levels of MB and PB were significantly higher compared to both controls HHC and EC (MB or PB vs EC, MB vs HHC p < 0.0001; PB vs HHC, p = 0.0013). Among leprosy cases, IgG enzyme-linked immunosorbent assay sensitivity and specificity were 92.7% and 97.1%, respectively. IgG positivity was confirmed in 92.1% and 94.1% of MB and PB patients, respectively. CONCLUSION This novel diagnostic approach presents an easy, non-invasive, and inexpensive method for leprosy screening, which may be applicable in endemic areas.

Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin

Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.

Comparisons of IgA response in saliva and colostrum against oral streptococci species

Abstract The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies’ samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.

Challenging Mycobacterium tuberculosi s dormancy mechanisms and their immunodiagnostic potential

ABSTRACT Mycobacterium tuberculosis is the etiologic agent of tuberculosis, one of the world's greatest cause of morbidity and mortality due to infectious disease. Many evolutionary mechanisms have contributed to its high level of adaptation as a host pathogen. Prior to become dormant, a group of about 50 genes related to metabolic changes are transcribed by the DosR regulon, one of the most complex and important systems of host-pathogen interaction. This genetic mechanism allows the mycobacteria to persist during long time periods, establishing the so-called latent infection. Even in the presence of a competent immune response, the host cannot eliminate the pathogen, only managing to keep it surrounded by an unfavorable microenvironment for its growth. However, conditions such as immunosuppression may reestablish optimal conditions for bacterial growth, culminating in the onset of active disease. The interactions between the pathogen and its host are still not completely elucidated. Nonetheless, many studies are being carried out in order to clarify this complex relationship, thus creating new possibilities for patient approach and laboratory screening.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

Distrofia muscular facioescapulohumeral en Chile: presentación de serie en hospital de referencia terciario / Facioscapulohumeral muscular dystrophy: Report of seven patients

Background: Facioscapulohumeral muscular dystrophy is the third most common muscular dystrophy with an estimated prevalence of 1 per 20.000 and a normal life expectancy in the majority of patients. However, approximately 15% of patients become wheelchair bound in the course of their life. It is a hereditary autosomal dominant disease with high (95%) penetrance by the age of 20, but with variable degree of phenotypic expression even in the same family group. Symptoms frequently start in the second decade of life, with facial and scapular weakness. Aim: To report the clinical features of seven patients with the disease, seen at a public hospital. Material and Methods: Analysis of seven patients with genetic study seen in a public Hospital in Santiago. Results: The age of patients fluctuated from 18 to 61 years and four were females. The mean age at onset of symptoms was 29 years and four had a family history of the disease. The usual presenting complaint was arm or shoulder asymmetric weakness. Four patients had bone pain. Facial involvement was present in four. A genetic study was done in five patients, the other two patients were relatives, confirming the contraction or lower number of repetitions in D4Z4 region. After 12 years of follow up only 2 patients older than 60 years cannot work and one female patients is in a semi dependent state at the age of 30. Conclusions: The clinical workup in the diagnosis and the timely indication of genetic studies are highlighted, to avoid unnecessary and invasive procedures. The variability in the phenotypic expression in a similar genetic defect is discussed and the genetic or epigenetic mechanisms of this muscular dystrophy are described.

Internações sensíveis à atenção primária específicas de mulheres / Sensitive female-specific hospitalization in primary care

Objetivou-se analisar as internações por condições sensíveis à atenção primária (ICSAP) específicas em mulheres e os fatores que determinam ou influenciam a ocorrência dessas internações (fatores socioeconômicos, sociodemográficos e controle de saúde) por meio de um inquérito de morbidade hospitalar realizado com amostra de 429 mulheres internadas em hospitais conveniados ao Sistema Único de Saúde. O percentual de ICSAP foi 49,42% (n = 212), com destaque para as internações específicas do sexo feminino 19,35% (n = 83). Associaram ao risco de internar por CSAP: idade superior a 60 anos, baixa escolaridade, internação prévia, realização de controle regular de saúde, falta de vínculo com a Estratégia Saúde da Família (ESF) e ser gestante. As causas evidentes foram as condições relacionadas à gravidez, ao parto e ao puerpério e às inflamações nos órgãos pélvicos femininos. Os resultados sugerem falhas no atendimento ambulatorial que deveria ser oportuno e resolutivo no contexto da saúde da mulher.

The scope of this paper was to analyze female-specific sensitive hospitalization occurring in primary care conditions and factors that determine or affect the occurrence of such hospitalizations (social, economic and demographic factors; health control). Analysis was performed by surveys on hospital morbidity with a sample of 429 females attended in Unified Health System (SUS) contracted hospitals. The sensitive hospitalizations percentage in primary care reached 49.42% (n = 212), highlighting female-specific hospitalization at 19.35% (n = 83). Hospitalization risks comprised elderly people over sixty, low schooling, previous hospitalizations, normal health control, lack of association with the Family Health Strategy and pregnancy. Evident causes were related to conditions of pregnancy, childbirth, post-partum and inflammations of the female pelvic organs. Results suggested flaws in outpatient attendance that should be adequate and provide solutions in women’s health.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis (Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.

Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species / Comparación de las técnicas de detección de la reactividad de la proteína de unión de la inmunoglobulina frente a la inmunoglobulina producida por diferentes especies aviarias y mamíferas

The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera).These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.

El fundamento de este estudio radica en el uso de varios ensayos inmunológicos para investigar la reactividad de la proteína de unión de la inmunoglobulina (IBP) frente a las inmunoglobulinas de varias especies aviarias y mamíferas. Las proteínas IBP estudiadas fueron la proteína estafilocócica A (SpA), la proteína estreptocócica G (SpG), la proteína peptoestreptocócica L (SpL), y la proteína recombinante LA (SpLA). Las varias técnicas inmunológicas usadas fueron: la inmunodifusión doble (técnica de Ouchterlony) para examinar las reactividades positivas de la proteína alta; el ensayo por inmunoabsorción ligado a enzimas(ELISA), de tipo directo y competitivo, para examinar la capacidad de realizar uniones positivas de proteína moderada y baja, respectivamente, además del ensayo ELISA 'Sándwich', los análisis inmunoblot, yla purificación de IgG, mediante cromatografía de afinidad, los cuales fueron pruebas sensibles y útiles en el tamizaje y las pruebas de confirmación. La técnica de Ouchterlony mostró que - en comparación con otras proteínas - la SpLA tenía el grado más alto de reactividad con los sueros animales y las inmunoglobulinas purificadas, mientras que la SpL fue la menos reactiva. Con el ELISA directo, la SpL reaccionó con los sueros de mapache, la IgG de conejo, así como con la IgY de palomas y gallinas de Bantam, en tanto con el ELISA directo, la SpA reaccionó con sueros de mofeta, coyote, mapache, mula, asno y seres humanos. ELISA "sándwich" reveló una alta reactividad tanto de SpG como de SpLA, con títulos séricos mamíferos que iban desde 1:32 (suero de mapache) hasta 1:1024 (sueros de mula y de asno). Estos resultados sugieren que la proteína de unión IBP puede usarse en la detección de la inmunoglobulina usando varios ensayos inmunológicos, lo cual es importante para el diagnóstico de enfermedades infecciosas en las poblaciones animales y aviarias bajo estudio, así como para la purificación de inmunoglobulinas.

Comparison of the tuberculin skin test and Quanti-FERON-TB Gold In-Tube [QFT-G] test for the diagnosis of latent tuberculosis infection in dialysis patients

Dialysis patients are more likely than the general population to develop active tuberculosis [TB]. In these patients, the availability of a highly sensitive and specific test to diagnose latent TB will ensure earlier treatment and decreased progression to active disease. In the current study, the Quanti-FERON-TB Gold In-Tube [QFT-G] test was compared with the tuberculin skin test [TST] for the diagnosis of latent tuberculosis infection [LTBI] among 200 hemodialysis patients and 15 confirmed TB disease cases in a tertiary care center in Saudi Arabia. Among the LTBI cases, 26 [13%] were TST positive, and 65 [32.5%] were positive by the QTF-G test, with an overall agreement between the 2 tests of 75.5% [k = 0.34] being observed. Among the confirmed tuberculosis disease cases, none were positive by TST, and 10 [66.7%] were positive by the QTF-G test, resulting in an overall agreement of 33.3% [k = 0]. A comparison between the TST and the QTF-G test was performed based on the sensitivity, specificity, and area under the curve [AUC] obtained for the tests. The QTF-G test was more sensitive and less specific than the TST in predicting the confirmed TB disease cases. When we tested the correspondence of the AUC values between the 2 diagnostic modalities, the obtained p-value was 0.0003. In conclusion, the AUCs of the examined diagnostic modalities are significantly different in predicting LTBI and tuberculosis