Qualitative and quantitative analysis of antibody response in childhood tuberculosis against antigens of Mycobacterium tuberculosis

Purpose: Serodiagnosis of tuberculosis in children, using available crude antigens, has been difficult. The tests lack sufficient sensitivity and/or specificity. In this study, western blot analysis of M. tuberculosis H37Rv culture filtrate antigen (CFA) was carried out, to identify diagnostically useful antigens. In addition, the CFA was also used in enzyme linked immunosorbent assay (ELISA), to measure antibodies of multiple isotypes. Methods: Specific IgG, IgA and IgM antibodies were estimated in the sera from 26 clinically/ bacteriologically diagnosed cases of childhood tuberculosis (CTB) and 61 normal children (CNHS), using culture filtrate antigen. Western blot analysis with culture filtrate antigen was carried out to qualitatively compare the antibody profile among the CTB, with childhood normal controls and adult TB. Results: IgG positivity was only 7.6% with culture filtrate antigen in the CTB group, while 3.2% among the controls were also positive. However, the results of IgA and IgM isotypes were better. By combination of all the three isotypes an increased sensitivity of 57.7% with a specificity of 93.5%, was obtained. Immunoblot analysis revealed marked difference among antibodies in the region of 16, 19, 38 and 45kDa between CTB and CNHS. Conclusions: Our findings point to a limited sensitivity of 57.7% in ELISA with culture filtrate antigen. However, antibodies around 16, 19,38 and 45kDa region may be useful in differentiating the CTB patients from CNHS by immunoblot assay

Isolation and Evaluation of Diagnostic Value of Two Major Secreted Proteins of Mycobacterium Tuberculosis

Two secreted antigens of Mycobacterium tuberculosis, namely the antigen 85 complex (30/31) and 38kDa antigens, were purified from the whole culture filtrate by using two dimensional preparative electrophoresis and anion exchange chromatography, respectively. Individual components of the antigen 85 complex namely, antigen 85A, 85B and 85C, were separated using hydrophobic interaction chromatography. The humoral antibody activity to these antigens in sputum positive cases of active pulmonary tuberculosis and normal healthy volunteers was determined by enzyme linked immunosorbent assay (ELISA) and immunoblot. Recombinant 38kDa and antigen 6 were used as reference antigens for the assay. None of the healthy volunteers reacted with the 38kDa antigen, while 52% of the TB sera reacted with it. Of the three components of the antigen 85 complex, 85B gave the highest positivity of 40 per cent. The results of combination of 38kDa with antigen 6 offered better results with 76% positivity

Production & characterization of monoclonal antibodies to Mycobacterium tuberculosis

Background & objectives: Monoclonal antibodies (MAbs) against Mycobacterium tuberculosis H37Rv culture filtrate (CF) were raised by immunizing BALB/c mice and characterization was done. Attempts have been directed towards identifying mycobacterial antigens in biological fluids by employing polyclonal and monoclonal antibodies specific for M. tuberculosis. Immunohistologic studies, using MAbs for the localization of whole or fragmented bacilli in the biopsy specimens were also carried out. Methods: Intrasplenic IS and intraperitoneal IP routes of immunization, were compared. The MAbs were characterized for their isotype, binding specificity, nature of binding epitope, reactivity in immunoassays etc. Results: IS and IF’ routes of immunization, were compared and IP was found superior. Ten MAbs designated TRC l-10 were produced. Of these, 7 MAbs, TRC 1-7 reacted with the 30/31 kDa doublet (antigen 85 complex), TRC 8 with 12 kDa in addition to 30/31 kDa and TRC 9 and 10 with the 24 and 12 kDa antigens respectively. Six MAbs were classified as broadly cross reactive and 2 showed limited cross reactivity. TRC 8 and 10 showed species specificity. Employing TRC 8 in sandwich ELISA, antigen was detected in sera from 17 of 25 pulmonary tuberculosis patients and 3 of 20 controls. TRC 8 was found to be useful in detecting antigens specifically in M. tuberculosis and M. leprae infected tissues, by immunoperoxidase staining. Interpretation & conclusion: TRC 8 was found to be restricted in its reactivity to M. tuberculosis complex and M, leprae. TRC 8 may prove useful in immuno-diagnosis of tuberculosis

HLA-DR phenotypes and IgG, IgA and IgM antibody responses to Mycobacterium tuberculosis culture filtrate and 30 kDa antigens in pulmonary tuberculosis

The role of HLA-DR genetic make-up on the IgG, IgA and IgM antibody response to Mycobacterium tuberculosis culture filtrate and 30 kDa antigens was studied in pulmonary tuberculosis. The study was carried out in HLA-DR typed active pulmonary tuberculosis (ATB) patients (n = 37), inactive (cured) pulmonary tuberculosis (ITB) patients (n = 79) and normal healthy subjects (NHS; n = 46). In ATB and ITB (cured) patients, IgG antibody (optical density at 490 nm for 1 : 3200 dilution) as measured by enzyme-linked immunosorbent assay was the predominant one than IgA and IgM antibodies. Increased IgG antibody titre to culture filtrate (P = 0.03) and decreased titre to 30 kDa antigen were observed with HLA-DR1-positive ATB patients than non-DR1 (ATB) patients. Moreover, HLA-DR4- and HLA-DR6-positive ATB patients showed trends toward an increased IgG antibody response to 30 kDa antigen than HLA-DR4- and HLADR6- negative (ATB) patients respectively. Significantly increased IgA antibody to 30 kDa antigen was observed with HLA-DR1-positive ATB patients than non-DR1 patients (P = 0.03). The study suggests that multiple HLA-DR molecules may regulate the IgG and IgA antibody responses to various proteins of M. tuberculosis. Moreover, HLA-DR phenotypes and increased IgG and IgA antibody titres may be useful to differentiate M. tuberculosis-infected subjects from normal subjects and cured patients with the same HLA-DR phenotypes or genetic make-up

Ofloxacin resistance in Mycobacterium tuberculosis: An increasing concern

Multidrug resistance tuberculosis (MDR-TB) associated with the development of resistance to fluoroquinolones (FQs) especially ofloxacin is a matter of concern, as they had been earlier recommended drugs for usage in the MDR-TB treatment regimens, and moxifloxacin and other quinolones are still on the list. Mycobacterium tuberculosis acquires resistance to FQs mainly through mutations in the quinolone resistance determining regions (QRDRs) of the gyrA gene and less frequently in the gyrB gene. A literature search on the geographical distribution of ofloxacin resistance in TB shows that there is a mild surge in reporting of the resistance to ofloxacin in tuberculosis patients. Molecular tests demonstrating mutations in gyrA and gyrB genes is widely used to detect ofloxacin resistance and the broadly available commercial assay for the rapid detection of second-line-drug resistance, including FQ resistance, the GenoType MTBDRsl assay (Hain Life science, Nehren, Germany), detects the most common mutations found in the QRDR of gyrA while its new version 2.0 detects mutations in the gyrB as well. It has been shown that on reviewing the frequency and geographic distribution of gyrA and gyr B mutations associated with FQ resistance, there do exist geographic differences in the frequencies within and across countries. Cross-resistance to FQs is an area of concern, although some studies show that concordance in resistance among the FQ agents, lower level of cross-resistance has also been reported. The presence of ofloxacin resistance is an alarm signal while Moxifloxacin and other FQs are still the recommended drugs for the resistant TB cases. The WHO recommendation that ofloxacin be phased out from MDR-TB regimens is well justified. It is important that rationale usage of ofloxacin is needed for preventing ofloxacin resistance, to aid in the management of tuberculosis

Aflatoxins B1 in different grades of chillies (Capsicum annum L.) in India as determined by indirect competitive-ELISA

Samples of the three grades of chilli pod (grades 1 to 3) were collected during surveys in 1998 and 1999 from the principal market yards and cold storage facilities of the major chilli-growing areas of Andhra Pradesh (AP), India. Chilli powders were collected from different supermarkets in Hyderabad, AP. They were analysed for aflatoxin B1 (AFB1) content by an indirect competitive ELISA. To avoid the influence of interfering substances present in chilli extracts, it was necessary to prepare the aflatoxin standards in methanol extracts of chillies free from aflatoxins. For nine representative samples there was good agreement between ELISA and HPLC estimations of AFB1 and the results suggested that the ELISA procedure adopted was dependable. Of the 182 chilli samples tested, 59% of the samples were contaminated with AFB1 and 18% contained the toxin at non-permissible levels. The highest AFB1 concentration of 969 µg/kg was found in one sample representing grade 3. Overall the maximum percentage of chilli pods showing AFB1 levels higher than 30 μg/kg (non-permissible levels) was in grade 3. Chilli pods stored in refrigerated rooms showed the lowest proportion of samples containing aflatoxin. Nearly 9% of the chilli powders sold in supermarkets contained non-permissible aflatoxin levels. This report highlights the importance of using grade 1 chilli pods to minimize aflatoxin contamination

Prime-Boost Vaccination With Covaxin/BBV152 Induces Heightened Systemic Cytokine and Chemokine Responses

Covaxin/BBV152 is a whole virion inactivated SARS-CoV-2 vaccine. The effect of prime-boost vaccination with Covaxin on systemic immune responses is not known. We investigated the effect of Covaxin on the plasma levels of a wide panel of cytokines and chemokines at baseline (M0) and at months 1 (M1), 2 (M2) and 3 (M3) following prime-boost vaccination in healthy volunteers. Our results demonstrate that Covaxin induces enhanced plasma levels of Type 1 cytokines (IFNγ, IL-2, TNFα), Type 2/regulatory cytokines (IL-4, IL-5, IL-10 and IL-13), Type 17 cytokine (IL-17A), other pro-inflammatory cytokines (IL-6, IL-12, IL-1α, IL-1β) and other cytokines (IL-3 and IL-7) but diminished plasma levels of IL-25, IL-33, GM-CSF and Type 1 IFNs. Covaxin also induced enhanced plasma levels of CC chemokine (CCL4) and CXC chemokines (CXCL1, CXCL2 and CX3CL1) but diminished levels of CXCL10. Covaxin vaccination induces enhanced cytokine and chemokine responses as early as month 1, following prime-boost vaccination, indicating robust activation of innate and adaptive immune responses in vaccine recipients

Inactivated COVID-19 vaccines: durability of Covaxin/BBV152 induced immunity against variants of concern

BACKGROUND: Covaxin/BBV152 is one of the most widely used vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and one of the few vaccines used extensively in low- and middle-income countries (LMIC). METHODS: We investigated the effect of Covaxin on the SARS-CoV-2 specific IgG and IgA and neutralizing antibody (NAb) levels at baseline (M0) and at Months 1 (M1), 2 (M2), 3 (M3), 4 (M4), 6 (M6) and 12 (M12) following vaccination in healthcare workers. In addition, we also examined the NAb levels against variant lineages of B.1.617.2 (Delta, India), B.1.617.2.1 (Delta Plus, India), B.1.351 (Beta, SA), B.1.1.7 (Alpha, UK) and B.1.1.529 (Omicron). RESULTS: Covaxin induces enhanced SARS-CoV-2 binding antibodies of IgG and IgA responses against both spike (S) and nucleocapsid (N) antigens at M1, M2, M3, M4, M6 and M12 in comparison with M0. Our data also reveal that NAb levels against the ancestral strain (Wuhan, wild type) are elevated and sustained at M1, M2, M3, M4, M6 and M12 in comparison with M0 and against variant lineages of B.1.617.2 (Delta, India), B.1.617.2.1 (Delta Plus, India), B.1.351 (Beta, SA) and B.1.1.7 (Alpha, UK) are elevated at M3, M6 and M12 in comparison with M0. However, NAb levels against B.1.1.529 (Omicron) was consistently below the limit of detection except at M12. CONCLUSION: Thus, Covaxin induces an enhanced humoral immune response, with persistence till at least 12 months post-vaccination against most SARS-CoV-2 variants

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