IgA Enzyme-linked Immunosorbent Assay Research Articles

Standardization of in-house anti-IgG and IgA ELISAs for the detection of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). RT-PCR detection of viral RNA represents the gold standard method for diagnosis of COVID-19. However, multiple diagnostic tests are needed for acute disease diagnosis and assessing immunity during the COVID-19 outbreak. Here, we developed in-house anti-RBD IgG and IgA enzyme-linked immunosorbent assays (ELISAs) using a well-defined serum sample panel for screening and identification of human SARS-CoV-2 infection. We found that our in-house anti-SARS-CoV-2 IgG ELISA displayed a 93.5% sensitivity and 98.8% specificity whereas our in-house anti-SARS-CoV-2 IgA ELISA provided assay sensitivity and specificity at 89.5% and 99.4%, respectively. The agreement kappa values of our in-house anti-SARS-CoV-2 IgG and IgA ELISA assays were deemed to be excellent and fair, respectively, when compared to RT-PCR and excellent for both assays when compared to Euroimmun anti-SARS-CoV-2 IgG and IgA ELISAs. These data indicate that our in-house anti-SARS-CoV-2 IgG and IgA ELISAs are compatible performing assays for the detection of SARS-CoV-2 infection.

Pyruvate: Ferredoxin oxidoreductase is involved in IgA-related microbiota dysbiosis and intestinal inflammation.

Inflammatory bowel diseases (IBDs) are associated with both immune abnormalities and dysbiosis, characterized by a loss of Faecalibacterium prausnitzii (F. prausnitzii). However, the reason for F. prausnitzii deficiency remains unclear. 16S rDNA seque-ncing and IgA enzyme-linked immunosorbent assay (ELISA) were applied to identify bacterial community and IgA changes in ulcerative colitis (UC) patients. Forced immunization with F. prausnitzii in rabbits was conducted. To screen for potential IgA-reactive proteins in F. prausnitzii lysates, we performed western blotting and mass spectrometry analyses. Pyruvate: ferredoxin oxidoreductase (PFOR) was cloned and purified, then the immunoreactivity of PFOR was verified in peripheral blood mononuclear cells (PBMCs) through PCR, ELISpot assay and single-cell sequencing (scRNA-seq). Finally, the UC fecal dysbiosis was re-analyzed in the context of the phylogenetic tree of PFOR. F. prausnitzii was underrepresented in UC patients with elevated F. prausnitzii-reactive IgA in the fecal supernatant. Forced immunization with F. prausnitzii in rabbits led to high interferon-γ (IFN-γ) transcription in the colon, along with beta diversity disturbance and intestinal inflammation. PFOR was identified as an IgA-binding antigen of F. prausnitzii and the immunoreactivity was validated in PBMCs, which showed elevated expression of inflammatory cytokines. The scRNA-seq revealed enhanced signals in both T regulatory cells (Tregs) and monocytes after PFOR incubation. Furthermore, phylogenetic analysis revealed that PFOR was a common but conserved protein among the gut bacteria. Our results collectively suggest that PFOR is a bioactive protein in the immune system and may contribute to host-microbial crosstalk. Conserved but bioactive microbial proteins, such as PFOR, warrant more attention in future host-microbial interaction studies.

Respiratory Mucosal Immunity: Kinetics of Secretory Immunoglobulin A in Sputum and Throat Swabs From COVID-19 Patients and Vaccine Recipients.

While IgM and IgG response to SARS-CoV-2 has been extensively studied, relatively little is known about secretory IgA (sIgA) response in respiratory mucosa. Here we report IgA response to the SARS-CoV-2 in sputum, throat swabs, and serum with nucleocapsid protein (NP) enzyme-linked immunosorbent assays (ELISA) in a cohort of 28 COVID-19 patients and 55 vaccine recipients. The assays showed sIgA in respiratory mucosa could be detected on the first day after illness onset (AIO), and the median conversion time for sIgA in sputum, throat swabs, and serum was 3, 4, and 10 days, respectively. The positive rates of sIgA first week AIO were 100% (24/28) and 85.7% (24/28) in sputum and throat swabs, respectively, and were both 100% during the mid-onset (2–3 weeks AIO). During the recovery period, sIgA positive rates in sputum and throat swabs gradually decreased from 60.7% (17/28) and 57.1% (16/28) 1 month AIO and the sIgA antibodies were all undetectable 6 months AIO. However, serum IgA positive rate was still 100% at 4 months and 53.6% (15/28) at 6 months. Throat swabs obtained from volunteers who received inactivated SARS-CoV-2 vaccines by intramuscular delivery all showed negative results in IgA ELISA. These findings will likely improve our understanding of respiratory mucosal immunity of this emerging disease and help in containing the pandemic and developing vaccines.

Optimization of a human milk\u2013directed quantitative sIgA ELISA method substantiated by mass spectrometry

Immunoglobulins are the primary protective products in human milk and are responsible for transferring maternal pathogen memory to the infant, providing protection by binding to recognized pathogens and inhibiting virulence. To better understand potentially protective/anti-infective compounds in human milk, the establishment of human milk–tailored analytical approaches is crucial, as most contemporary analytical methods have been optimized for plasma or serum. One of the most prominent immunoglobulins in human milk is secretory immunoglobulin A (sIgA), which may be relevant for the protection of breastfed infants from harmful pathogens. Advanced sIgA detection methods can help monitor the immune status and development of the mother-infant dyad. We therefore developed an enzyme-linked immunosorbent assay (ELISA) sIgA method for the quantitative analysis of IgA plus secretory component (SC), validated with sIgA standards and substantiated by mass spectrometry (MS)–based proteomics. A very strong correlation was observed between the MS-detected IgA1 and the human milk–specific sIgA ELISA (r = 0.82). Overall, the MS data indicate that the developed human milk sIgA ELISA does not differentiate between sIgA1 and sIgA2 and is, therefore, a reflection of total sIgA. Furthermore, our MS data and the human milk–derived sIgA ELISA data are better correlated than data derived from a standard serum IgA ELISA kit (relative to MS IgA1 r = 0.82 and r = 0.42, respectively). We therefore propose our human milk–specific sIgA ELISA as an ideal quantitative indicator of total sIgA with advantages over current serum IgA ELISA kits.

Evaluation of IgG, IgA and IgM against Mycobacterial Antigen in Patients with Extrapulmonary Tuberculosis

Tuberculosis (TB) is a specific infectious disease usually caused by the tubercle bacillus, Mycobacterium tuberculosis (MBT). It primarily affects the lungs and causes pulmonary tuberculosis (PTB) and when it affects other organs it is known as extrapulmonary tuberculosis (EPTB). The diagnosis of TB largely depends on case history, clinical examination, radiological finding and subsequent laboratory confirmation by bacteriological examination. But the radiological findings are not specific and the sensitivity of AFB (acid fast bacilli) smear is poor. To overcome this difficulty, various serological tests have been tried. A novel antigen namely A60 antigen was found to be highly immunogenic and this present study was carried out to see the diagnostic significance of the ELISA test for A60 antigen in diagnosing extrapulmonary tuberculosis. The study was designed as a cross-sectional study involving both extrapulmonary TB patients and controls. Their serum immunoglobulins (IgG, IgA and IgM) specific toA60 antigen were measured by enzyme linked immunosorbent assay (ELISA) technique. In this study, IgG, IgA and IgM was significantly elevated among the EPTB patients as compared to the controls and the IgA and IgM ELISA test combinations was most ideal in diagnosing EPTB. Keywords: Tuberculosis, Extrapulmonary tuberculosis, AFB (acid fast bacilli), A60 antigen, ELISA (enzyme linked immunosorbent assay), Immunoglobulin.

EDTA-Anticoagulated Whole Blood for SARS-CoV-2 Antibody Testing by Electrochemiluminescence Immunoassay (ECLIA) and Enzyme-Linked Immunosorbent Assay (ELISA).

While lateral flow test formats can be utilized with whole blood and low sample volumes, their diagnostic characteristics are inferior to immunoassays based on chemiluminescence immunoassay (CLIA) or enzyme-linked immunosorbent assay (ELISA) technology. CLIAs and ELISAs can be automated to a high degree but commonly require larger serum or plasma volumes for sample processing. We addressed the suitability of EDTA-anticoagulated whole blood as an alternative sample material for antibody testing against SARS-CoV-2 by electro-CLIA (ECLIA; Roche, Rotkreuz, Switzerland) and ELISA (IgG and IgA; Euroimmun, Germany). Simultaneously drawn venous serum and EDTA-anticoagulated whole blood samples from 223 individuals were included. Correction of the whole blood results for hematocrit led to a good agreement with the serum results for weakly to moderately positive antibody signals. In receiver-operating characteristic curve analysis, all three assays displayed comparable diagnostic accuracy (area under the curve (AUC)) using corrected whole blood and serum (AUCs: 0.97 for ECLIA and IgG ELISA; 0.84 for IgA ELISA). In conclusion, our results suggest that the investigated assays can reliably detect antibodies against SARS-CoV-2 in hemolyzed whole blood anticoagulated with EDTA. Correction of these results for hematocrit is suggested. This study demonstrates that the automated processing of whole blood for identification of SARS-CoV-2 antibodies with common ECLIA and ELISA methods is accurate and feasible.

Comparison of Serological Biomarkers in Rheumatoid Arthritis and Their Combination to Improve Diagnostic Performance.

The diagnosis of rheumatoid arthritis (RA) is based on a combined approach that includes serological markers such as rheumatoid factor (RF) and anti-citrullinated peptide/protein antibodies (ACPA). The goal of this study was to evaluate the clinical performance of several RF and ACPA immunoassays for the diagnosis of RA, as well as the diagnostic value of a combinatory approach with these markers. The study cohort included 1,655 patients from the Swiss Clinical Quality Management registry with sera from 968 patients with RA and 687 disease controls, including patients with axial spondyloarthritis (n = 450) and psoriatic arthritis (n = 237). ACPA were determined by anti-CCP2 IgG enzyme-linked immunosorbent assay (ELISA), QUANTA Flash® CCP3 IgG [chemiluminescent immunoassay (CIA)], and QUANTA Lite® CCP3 IgG ELISA. RF was determined by ELISA (QUANTA Lite® RF IgM, RF IgA, and RF IgG) and with two research use only CIAs (QUANTA Flash® RF IgM and RF IgA). All three ACPA assays showed good discrimination between RA patients and controls and good clinical performance. Overall, CCP3 performed better than CCP2. More pronounced differences were observed between the RF assays. We observed that CIA platforms for both RF IgM and RF IgA showed better performance than the ELISA platforms. Excellent and good total agreements were found between ELISA and CIA for CCP3 (total agreement 95.3%, kappa = 0.90), and between CCP2 and CCP3 ELISA (total agreement 86.6%, kappa = 0.73), respectively. RF IgM CIA and ELISA had a good qualitative agreement (86.5%, kappa = 0.73); RF IgA CIA and ELISA showed a moderate total agreement (78.5%, kappa = 0.53). When combinatory analyses were performed, the likelihood of RA increased with dual positivity and triple positivity and combining different markers resulted in higher odds ratio than the individual markers in all cases. ACPA and RF showed good clinical performance in this large Swiss cohort of RA patients and controls. Overall, the performance of CCP3 was superior to CCP2. The combination of these biomarkers in an interval model represents a potential tool for the diagnosis of RA patients.

Molecular Evidence of Chlamydia trachomatis Infection and Its Relation to Miscarriage.

BackgroundChlamydia trachomatis (CT) infection is the most common sexually transmitted disease in the world that can persist and also ascend in the genital tract. This intracellular and silent infection is related to some adverse pregnancy outcomes, such as miscarriage. The aims of this study were to explore the best CT screening tests using blood and vaginal samples and to investigate the correlation between CT infection and the incidence of miscarriage.Materials and MethodsThis case-control study was done in October 2013 through June 2014, using purposive sampling from 157 female participants with or without a history of miscarriage. The samples were taken after each participant had signed a letter of consent and had completed a questionnaire. To achieve the objectives of this study, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests were performed on vaginal swabs and blood samples, respectively.ResultsPCR results showed a significantly higher CT infection rate in the miscarriage group compared to the control group (11.3 vs. 0%, P=0.007). Anti-CT IgG and IgA antibodies were found in 4.2 and 2.1% of cases in the miscarriage group, and in 1.7 and 6.7% of cases in the control group, respectively (P>0.05). Despite lower humoral responses in this study, positive samples were detected only by one of the following techniques; PCR, ELISA IgA and ELISA IgG. It also should be noted that PCR worked best in terms of detection.ConclusionBased on the obtained data, there is a strong association between molecular evidence of CT infection and miscarriage. A higher rate of CT detection in molecular tests compared to serological assays suggests that PCR could be used as the first-choice assay for detection of C. trachomatis. However, the importance of serological tests in detecting potential past CT infection or upper genital infection not amenable to sampling is undeniable.

Comparison the diagnostic value of serological and molecular methods for screening and detecting Chlamydia trachomatis in semen of infertile men: A cross-sectional study

Chlamydia trachomatis (CT) with damaging effects on sperm quality parameters can often cause infertility in men. The main objective of this study was to determine the diagnostic value of polymerase chain reaction (PCR) and enzyme linked immuno sorbent assay (ELISA) for screening and detecting CT in semen samples of infertile men. In this cross-sectional study, 465 men referring to the clinical laboratory of Royan Institute were chosen for primary screening and detection of the presence of CT. 93 samples were normozoospermia with normal sperm parameters i.e. sperm number, motility and morphology (Asymptomatic) and 372 had abnormal sperm parameters (Symptomatic) in semen analysis. ELISA test was performed as the screening test. Samples with optical density (OD) >0.200 were selected as the case and asymptomatic samples with OD <0.200 were selected as the control group for the confirmatory test. PCR assay was used to confirm the serological results. In the case groups (n=62), 4 out of 32 symptomatic samples (12.5%), and 1 out of 30 asymptomatic samples (3.3%) revealed positive results in PCR. No PCR positive sample was observed in the control group (n=34). The final results revealed that considering OD >0.400 as the ELISA positive, the diagnostic value of CT-ELISA positive in symptomatic and asymptomatic infertile patients were 0.019 (7 of 372) and 0.021 (2 of 93), respectively. There was no relationship between the presence of CT infection and different sperm abnormalities. The anti-CT IgA ELISA test may be introduced as an appropriate tool for screening purpose in the seminal plasma to select suspicious samples for PCR confirmatory tests.

Influence of structural dimensions of micro-pillar array in reaction field on sensitivity of enzyme-linked immunosorbent assay (ELISA)

ABSTRACTFor high sensitivity and rapid reaction of enzyme-linked immunosorbent assay (ELISA), the film-stack reaction field with micro-pillars array was designed and developed. The film-stack reaction field was fabricated by a nanoimprint process and an automatic punch-press process. The films with different gaps between micro-pillars (5, 10 and 50 μm) were prepared. These reaction fields were evaluated by IgA ELISA using 96-well microtitre plates and the computational simulation analysis of the fluid flow and the particle trajectory. Compared with ELISA using only the microtitre plate, higher detection sensitivity and shorter incubation time were achieved using the film-stack reaction field due to the increased surface area and the circulating flow through the space between films in a well by the rotation of the film-stack reaction field. Furthermore, in the ELISA results obtained using the film-stack reaction fields, the fluorescence intensities in 10-μm and 50-μm pillar gaps were the minimum and maximum values, respectively. This trend was due to the flow rate between micro-pillars, and the number and the diffusion distance of supplied biomolecules to the inertial space in the film-stack reaction field. In simulation results, the trend of the number of adsorbed biomolecule particles with different gaps between micro-pillars was in agreement with the trend in the ELISA results. Hence, these simulation analyses were validated in the quantitative evaluation of this reaction field and could be applied in the design of this reaction field as an effective design tool.

S-1-Propenylcysteine promotes the differentiation of B cells into IgA-producing cells by the induction of Erk1/2-dependent Xbp1 expression in Peyer's patches

ObjectivesS-Allylcysteine (SAC) and S-1-propenylcysteine (S1PC) are the characteristic sulfur-containing amino acids in aged garlic extract. In this study, we investigated the effect of SAC and S1PC on intestinal immunoglobulin (Ig)A production to gain insight into the immunomodulatory effect of aged garlic extract. MethodsIn vitro study: Mouse splenic lymphocytes were treated with S1PC (0.1 and 0.3 mM) or SAC (0.1 and 0.3 mM) for 3 d, and IgA concentration in the culture medium was examined. In vivo study: Mice were orally administrated S1PC (7.5, 15, and 30 mg/kg) for 5 d and the IgA level in the intestinal lavage fluids as well as the population of IgA-producing cells in Peyer's patches were measured using mouse IgA enzyme-linked immunosorbent assay quantification set and flow cytometer, respectively. ResultsS1PC enhanced IgA production in mouse splenic lymphocytes in culture. However, SAC was ineffective. In addition, oral administration of S1PC to mice increased the IgA level and number of IgA-producing cells in Peyer's Patches. Furthermore, S1PC induced the expression of X-box binding protein 1 (Xbp1) mRNA, an inducer of plasma cell differentiation, in Peyer's patches. This induction was accompanied by the degradation of paired box protein 5 and the activation of mitogen activated protein/extracellular signal-regulated kinase signaling pathway. ConclusionThese results suggest that S1PC increases IgA-producing cells via the enhancement of Erk1/2-mediated Xbp1 expression in the intestine.

Serology in chronic Q fever is still surrounded by question marks

Detection of antibodies using immunofluoresence tests (IFAT) is recommended for diagnosis of chronic Q fever, but other commercial antibody assays are also available. We compared an enzyme-linked immunosorbent assay (ELISA) (Virion/Serion) and a complement fixation test (CFT) (Virion/Serion) for the detection of Coxiella burnetii IgG phase I and IgA phase I in early- and follow-up serum samples from patients with chronic Q fever, diagnosed according to an algorithm that involves IFAT. For this, we tested sera of 49 patients, including 30 proven, 14 probable and five possible chronic Q fever cases. Sensitivity of CFT for diagnosis of chronic Q fever was suboptimal (85 %), as eight patients, including five with chronic Q fever, tested negative at time of diagnosis, whereas IgG phase I antibodies were detected in these five patients by ELISA. Sensitivity of ELISA was higher, although three probable patients were missed. No differences in ELISA IgA phase I detection between proven chronic Q fever and probable were observed; instead possible patients were in majority IgA negative (60 %). Serological examination using ELISA and CFT in follow-up sera from 26 patients on treatment was unsatisfactory. Like IFAT, all kinetic options were possible: decreasing, remaining stable or even increase during time. This study demonstrated that the sensitivity of CFT-based phase I antibody detection is low and therefore not recommended for diagnosis of chronic Q fever. Based on our results, serological follow-up to guide treatment decisions was of limited value.

Evaluation of a WHO-validated Serotype-specific Serological Assay for the Diagnosis of Pneumococcal Etiology in Children With Community-acquired Pneumonia

The etiologic diagnosis of community-acquired pneumonia (CAP) remains challenging in children because blood cultures have low sensitivity. Novel approaches are needed to confirm the role of Streptococcus pneumoniae. In this study, pneumococcal etiology was determined by serology using a subset of blood samples collected during a prospective multicentre observational study of children <15 years of age hospitalized in Belgium with radiogram-confirmed CAP. Blood samples were collected at admission and 3-4 weeks later. Pneumococcal (P)-CAP was defined in the presence of a positive blood or pleural fluid culture. Serotyping of S. pneumoniae isolates was done with the Quellung reaction. Serological diagnosis was assessed for 9 serotypes using World Health Organization-validated IgG and IgA serotype-specific enzyme-linked immunosorbent assays (ELISAs). Paired admission/convalescent sera from 163 children were evaluated by ELISA (35 with proven P-CAP and 128 with nonproven P-CAP). ELISA detected pneumococci in 82.8% of patients with proven P-CAP. The serotypes identified were the same as with the Quellung reaction in 82% and 59% of cases by IgG ELISA and IgA ELISA, respectively. Overall, ELISA identified a pneumococcal etiology in 55% of patients with nonproven P-CAP. Serotypes 1 (51.6%), 7F (19%) and 5 (15.7%) were the most frequent according to IgG ELISA. In conclusion, the serological assay allows recognition of pneumococcal origin in 55% of CAP patients with negative culture. This assay should improve the diagnosis of P-CAP in children and could be a useful tool for future epidemiological studies on childhood CAP etiology.

Erken Dönem İnsan Trişinellozunda Özgün Antikor Yanıtlarının Değerlendirilmesi

Objective: Trichinellosis is a zoonotic disease in humans caused by Trichinella sp. Since the signs and symptoms due to Trichinellosis are not pathognomonic, the detection of serum antibodies is useful for the early diagnosis. The present study was undertaken to evaluate the specific antibody responses in confirmed cases of a trichinellosis outbreak, caused by T. britovi in Turkey. Material and Methods: Serum samples from 171 trichinellosis patients, 127 healthy individuals and 32 patients with other parasitosis were examined for IgG, IgA and IgM antibodies by an enzyme linked immunosorbent assay (ELISA) using excretory/secretory (ES) antigen. Results: IgG responses were positive in all patients, whereas IgA and IgM responses were positive in 50.9% and 59.6%, respectively. The specificity of antibody assays was 97.5% for IgA and IgM and 99.4% for IgG antibodies. According to the receiver-operator characteristic (ROC) curve analysis, area under the ROC curve (AUC) values of IgG, IgA and IgM ELISA were 99.7%, 74.2% and 78.6%, respectively. Conclusion: The results of this study suggest that IgG ELISA using ES antigen is the choice for diagnosis and in house IgM ELISA is more sensitive than IgA ELISA in diagnosing trichinellosis about 3-6 weeks after consumption of suspected meat. To the best of our knowledge, this is the first report presenting specific IgA and IgM antibody responses in such a large group of serum samples obtained in a T. britovi outbreak.

External quality assessment of pertussis serology in Germany

The purpose of this investigation was to test the performance of pertussis serology in diagnostic laboratories. The World Health Organization (WHO) Reference Reagent (06/142) and a sample with a low level of antibodies were sent to 200 participants of an external quality assessment (EQA) programme in Germany. The results were reported qualitatively and quantitatively, and were converted into IU/ml when possible. A total of 183 participants reported results. IgG, IgA and IgM enzyme-linked immunosorbent assays (ELISAs) with mixed antigens were used by 111, 110 and 113 participants, respectively, and 69 and 44 participants used IgG and IgA ELISAs with purified pertussis toxin (PT), respectively. IgG, IgA and IgM immunoblots were employed by 62, 63 and 11 participants, respectively. Most tests could distinguish between the positive and negative samples, but quantitative results were reported partly in non-comparable units. Only 37 % of participants used ELISAs that gave results comparable to the expected values in IU/ml and that could be interpreted according to published recommendations.

Serologic host response to Helicobacter pylori and Campylobacter jejuni in socially housed Rhesus macaques (Macaca mulatta)

BackgroundHelicobacter pylori are successful colonizers of the human gastric mucosa. Colonization increases the risk of peptic ulcer disease and adenocarcinoma. However, potential benefits of H. pylori colonization include protection against early-onset asthma and against gastrointestinal infections. Campylobacter jejuni are a leading cause of bacterial diarrhea and complications include Guillain-Barré syndrome. Here, we describe the development of reliable serological assays to detect antibodies against those two bacteria in Rhesus macaques and investigated their distribution within a social group of monkeys.MethodsTwo cohorts of monkeys were analyzed. The first cohort consisted of 30 monkeys and was used to establish an enzyme-linked immunosorbent assay (ELISA) for H. pylori antibodies detection. To evaluate colonization of those macaques, stomach biopsies were collected and analyzed for the presence of H. pylori by histology and culture. C. jejuni ELISAs were established using human serum with known C. jejuni antibody status. Next, plasma samples of the 89 macaques (Cohort 2) were assayed for antibodies and then statistically analyzed.ResultsAn H. pylori IgG ELISA, which was 100% specific and 93% sensitive, was established. In contrast, the IgA ELISA was only 82% specific and 61% sensitive. The CagA IgG assay was 100% sensitive and 61% of the macaques were positive. In cohort 2, 62% macaques were H. pylori sero-positive and 52% were CagA positive. The prevalence of H. pylori IgG and CagA IgG increased with monkey age as described for humans. Of the 89 macaques 52% showed IgG against C. jejuni but in contrast to H. pylori, the sero-prevalence was not associated with increasing age. However, there was a drop in the IgG (but not in IgA) mean values between infant and juvenile macaques, similar to trends described in humans.ConclusionsRhesus macaques have widespread exposure to H. pylori and C. jejuni, reflecting their social conditions and implying that Rhesus macaques might provide a model to study effects of these two important human mucosal bacteria on a population.

Assessment of the IgA Immunoassay Diagnostic Potential of the Mycobacterium tuberculosis MT10.3-MPT64 Fusion Protein in Tuberculous Pleural Fluid

Pleural tuberculosis (PL-TB) remains difficult to diagnose. An enzyme-linked immunosorbent assay (ELISA) was developed based on a construction containing the fusion of the Rv3019c (MT10.3) and Rv1980c (MPT64) gene sequences, and its performance was evaluated in an area where TB is endemic. A total of 92 pleural fluid (PF) samples at serial dilutions of 1:50 to 1:800 were included in the ELISA IgA MT10.3-MPT64 evaluation: 70 from TB patients and 22 from patients with other pleurisies. Confirmation of the expression and subsequent purification of the protein was made by SDS-PAGE and Western blot assays, resulting in a 36-kDa protein. ELISA IgA MT10.3-MPT64 showed sensitivities of 61.4%, 58.6%, 62.9%, 67.1%, and 70% at each PF dilution, respectively. The cumulative results of all dilutions increased sensitivity to 81.4% without jeopardizing specificity. Similar results were also obtained at the combined dilutions of 1:50, 1:200, and 1:800 or 1:50 plus 1:800 dilutions (80%). The overall sensitivity of the reference test, i.e., histopathological examination, was 74%. But, via the ELISA IgA MT10.3-MPT64 test, sensitivity was high for specimens with a negative culture (23/27; 85.2%) or nonspecific histopathology (17/18; 94.4%). Our findings demonstrated the promising use of this test as an adjunct in PL-TB diagnoses, particularly in cases with lower bacterial loads and false-negative results in the reference tests, since the new test includes such important features as quick and easy application, high sensitivity and, perhaps most importantly, affordability, which is so crucial for its widespread use in developing countries.

Calcium phosphate coupled Newcastle disease vaccine elicits humoral and cell mediated immune responses in chickens

Calcium phosphate (CaP) particles were coupled with inactivated Newcastle disease virus (NDV) vaccine. The surface morphology of CaP particles coupled to NDV was found to be spherical, smooth and with a tendency to agglomerate. The mean (± SE) size of CaP particles was found 557.44 ± 18.62 nm. The mean percent encapsulation efficiency of CaP particles coupled to NDV assessed based on total protein content and haemagglutination (HA) activity in eluate was found to be 10.72 ± 0.89 and 12.50 ± 2.09, respectively. The humoral and cell mediated immune responses induced by CaP coupled NDV vaccine were assessed in comparison to a commercial live vaccine (RDV ‘F’). CaP coupled NDV vaccine elicited prolonged haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titres in the serum even at fourth and fifth week post-vaccination (PV), unlike RDV ‘F’ inoculated chickens whose titres declined to insignificant levels by this time. CaP coupled NDV vaccine could stimulate HI antibodies in tracheal washings and tears from second and first week PV, respectively. IgA ELISA antibodies were also seen in tracheal washings of these birds from third week PV and in tears from second week PV. CaP coupled NDV vaccine elicited cell mediated immune responses (CMI) from two to four weeks PV. The stimulation indices obtained after stimulation with specific antigen was not significantly different between CaP coupled antigen and live NDV virus except on first week PV. However, CaP coupled antigen did not cause suppression of lympo proliferation as indicated by statistically similar responses to mitogen, concanavalin A between the two groups. Overall, CaP coupled NDV vaccine elicited stronger and prolonged immune responses in comparison to the commercial live vaccine. No increase in the serum calcium and phosphorous levels were seen in CaP coupled NDV vaccine inoculated chickens.

Development of a Bacillus subtilis -Based Rotavirus Vaccine

Bacillus subtilis vaccine strains engineered to express either group A bovine or murine rotavirus VP6 were tested in adult mice for their ability to induce immune responses and provide protection against rotavirus challenge. Mice were inoculated intranasally with spores or vegetative cells of the recombinant strains of B. subtilis. To enhance mucosal immunity, whole cholera toxin (CT) or a mutant form (R192G) of Escherichia coli heat-labile toxin (mLT) were included as adjuvants. To evaluate vaccine efficacy, the immunized mice were challenged orally with EDIM EW murine rotavirus and monitored daily for 7 days for virus shedding in feces. Mice immunized with either VP6 spore or VP6 vegetative cell vaccines raised serum anti-VP6 IgG enzyme-linked immunosorbent assay (ELISA) titers, whereas only the VP6 spore vaccines generated fecal anti-VP6 IgA ELISA titers. Mice in groups that were immunized with VP6 spore vaccines plus CT or mLT showed significant reductions in virus shedding, whereas the groups of mice immunized with VP6 vegetative cell vaccines showed no difference in virus shedding compared with mice immunized with control spores or cells. These results demonstrate that intranasal inoculation with B. subtilis spore-based rotavirus vaccines is effective in generating protective immunity against rotavirus challenge in mice.

Identifying Infections with Respiratory Syncytial Virus by Using Specific Immunoglobulin G (IgG) and IgA Enzyme-Linked Immunosorbent Assays with Oral-Fluid Samples

Currently, respiratory syncytial virus (RSV) infection is identified in epidemiological studies by virus antigen or nucleic acid detection in combination with serology. Oral-fluid specimens may provide a noninvasive alternative to blood, and oral fluid is more suitable for sampling outside of the clinic setting. We evaluated an indirect enzyme-linked immunosorbent assay for the detection of RSV-specific immunoglobulin G (IgG) and IgA by using oral-fluid samples collected from individuals with RSV infections confirmed by an immunofluorescent antibody test. For five children sampled repeatedly from birth, antibody profiles in oral fluid quite consistently tracked those in paired sera, and RSV infections were detected by rising titers of antibodies of at least one Ig class. Specific IgG responses were generally more reliable than IgA responses, except in early infancy, where the reverse was sometimes true. For a further five young children from whom oral fluid was collected weekly following RSV infection, boosted antibody responses, frequently of a transient nature, lasting a few weeks, were observed; specific IgG responses were of longer duration and more pronounced than specific IgA responses. Our data show significant promise for the use of oral fluid alone in RSV infection surveillance. The observed rapid dynamics of the antibody responses are informative in defining study sampling intervals.