Determination of optimum levels of binding antibody units (BAU) of new quantitative chemiluminescent immuno-assay (CLIA) in COVID-19 vaccinated volunteer blood donors.

BACKGROUND: For assessment of COVID-19 vaccine efficacy, neutralization activity of anti-SARS-CoV-2 antibody is measured. This study was undertaken to determine optimum levels of binding antibody units (BAU/ml) in new quantitative chemiluminescent assay (CLIA) that corresponded to neutralizing potential (30% inhibition) of sVNT assay. METHODS: Ninety-one blood samples were analyzed by CLIA and sVNT assays. Test samples (n = 75) were collected from blood donors post-2nd vaccination dose, while control samples (n = 16) were archived pre-COVID donor samples. Correlation between CLIA and sVNT was calculated and receiver operating characteristic (ROC) curve was drawn and analyzed. RESULTS: Results indicated excellent correlation between 57.5 BAU/ml on CLIA and 30%inhibition on sVNT assay. ROC curve analysis revealed that the area under the curve (AUC) was 0.971. DISCUSSION: The present study determined that 57.5 BAU/ml on CLIA corresponded to 30% inhibition on sVNT assay. Periodic quantitative analysis.

Analytical and clinical performance evaluation of enhanced chemiluminescence-based fourth-generation HIV combo assay: Report from tertiary health-care setup in North India.

INTRODUCTION: HIV fourth-generation assay, designed for the detection of HIV p24 antigen along with anti-HIV antibodies of both immunoglobulin M and immunoglobulin G type against HIV 1 and HIV 2 viral antigens, have helped in the early detection of HIV infection and supports in minimizing the transmission risk in the acute phase of infection. The objective of this study was to evaluate the analytical and clinical performance of HIV fourth-generation assay based on enhanced chemiluminescence technology. MATERIALS AND METHODS: The analytical performance of the assay was evaluated in terms of accuracy, precision, limit of detection, type of sample (serum vs. plasma), cross-reactivity (with other transfusion transmissible infections markers), and interference (with endogenous substances). Proficiency control material included kit-controls, archived known positive donor samples, third-party controls, and World Health Organization (WHO)/National Institute for Biological Standards and Controls (NIBSC, MHRA, UK) controls. The clinical performance was evaluated using routine donor and patient samples received during the study period. RESULTS: HIV fourth-generation assay showed reliable and reproducible results measured in terms of coefficient of variation % with kit-controls, archived known positive donor samples, third-party controls, and WHO international standards for anti-HIV 1 and 2 antibodies, HIV1 p24 antigens and HIV2 p26 antigen controls. The analytical sensitivity of the HIV fourth-generation assay was found to be 0.1 IU/mL of HIV1 p24 antigen control and there was no cross-reactivity or interference observed. In the clinical performance of the assay, HIV fourth-generation assay showed reliable performance in both donor and patient samples. CONCLUSION: HIV fourth-generation assay meets the requirements for its use as a screening assay for HIV infection based on the analytical and clinical performance of the assay.

Humoral immune response to infective larval antigen in Brugia malayi infected Mastomys natalensis.

The sequential changes in the humoral immune response against infective larval antigens during the course of Brugia malayi infection in Mastomys natalensis have been studied using enzyme linked immunosorbent assay. IgM antibody against B. malayi infective larval excretory secretory (ES) antigen was detected in the peripheral circulation within a week of infection, whereas IgM antibody against B. malayi infective larval somatic antigen and IgG antibody against both somatic and ES antigens were detected on day 20 post-inoculation. Thereafter, the antibody levels showed a steady increase until day 150. A gradual decrease of IgM antibody level was observed upto day 360, whereas IgG antibody level was decreased upto day 250 and then maintained almost the same level upto day 360. Wuchereria bancrofti cross reactive antigen as well as B. malayi infective larval ES antigen were detected in blood circulation on day 20, the level increased upto day 150 and then remained almost the same upto day 360 with slight variations. Studies of antigen and antibody levels in microfilaraemic and amicrofilaraemic animals show that there is no significant difference in antibody level whereas elevated antigen titre was observed in active infection with microfilaraemia.

Detection of filarial antigen using antibodies raised against Wuchereria bancrofti microfilarial SDS soluble antigen.

Polyclonal antibodies raised in mouse ascitic fluid against Wuchereria bancrofti microfilarial antigens (Wb Mf SDS S Ag) were studied for their diagnostic use in bancroftian filariasis using a dip stick, enzyme-linked immunosorbent assay. In sandwich ELISA, 100% of microfilaremic sera (30 out of 30) 53% of acute filarial sera (7/13), 40% of subacute filarial sera (6 out of 15), 13% of chronic filarial sera (2/15) and 20% of endemic area normal sera (3/15) showed the presence of filarial antigen. Determination of filarial antigen titer in microfilaremic sera showed an apparent positive correlation between microfilarial density and antigen titer. The antibody raised against Wb Mf SDS S Ag was found to be cross reactive with phosphorylcholine epitopes. The filarial antigen detected by anti Wb Mf SDS S Ag antibodies in sandwich ELISA is possibly associated with the active stage (microfilaremia) of infection.

Antigenic analysis of excretory-secretory products of Wuchereria bancrofti and Brugia malayi infective larval forms by SDS-PAGE.

Excretory-secretory (ES) products of W. bancrofti and the closely related B. malayi infective larval forms were analysed for their antigenic activity by SDS-PAGE followed by Western blotting as well as by gel elution-sandwich ELISA using filarial serum immunoglobulin-G (FSIgG) as a capture antibody. In W. bancrofti infective larval ES products, the protein molecules of 66, 46, 35, 33, 30 and 14 kDa molecular wt. showed antigenic activity by immuno blotting technique. In sandwich ELISA technique eventhough all SDS-PAGE fractions except ESA 6 (55-47 kDa) showed antigenic positivity, the fractions ESA 8 (37-31 kDa) and ESA 9 (31-25 kDa) showed high reciprocal antigen titre of 262144 and 32768 respectively. In B. malayi infective larval ES products, the protein molecules of 109, 102, 97 and 77 kDa molecular wt. showed reactivity with FSIgG by blotting technique, where as in sandwich ELISA except ESA 7 (47-37kDa), all fractions showed antigenic positivity. However, these fractions failed to show high antigen titre similar to W. bancrofti ES products with FSIgG.

Diagnostic use of filarial antibody and antigen isolated from hydrocele fluid for human filariasis.

Paired samples of serum and hydrocele fluid of filarial patients associated with hydrocele were analysed for filarial antibody and antigen. Sera samples showed higher titers of filarial antibody and antigen compared to their corresponding hydrocele fluid samples. HFIgG isolated from hydrocele fluid was equally useful as FSIgG isolated from serum and detected filarial antigen in 23 out of 26 microfilaraemic sera, 7 out of 10 chronic sera and 3 out of 18 endemic normal sera by inhibition ELISA. Filarial antigen was isolated from hydrocele fluid. In inhibition ELISA antigen fraction, HFA-9 (20-25 kDa) isolated by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis showed high reciprocal antigen titer of 2048. This active antigen fraction was evaluated for its diagnostic utility in comparison with Wuchereria bancrofti microfilariae excretory-secretory antigen (Wb mf ES Ag) in inhibition ELISA. Both antigen preparations detected filarial antigen in about 80% of microfilaraemic sera, 60% chronic sera and 20-30% of endemic normal sera. This study showed that antibody and antigen isolated from hydrocele fluid were equally sensitive as FSIgG and Wb mf ES Ag in the detection of filarial antigen by inhibition ELISA. Thus hydrocele fluid may be used as an alternative source for the isolation of antibody and antigen of immunodiagnostic importance.

Penicillinase ELISA for detection of tubercular antigen in tuberculosis.

Stick sandwich enzyme linked immunosorbent assay (ELISA) using rabbit anti PPD-RT 23 immunoglobulins and enzyme penicillinase has been explored for detection of tubercular antigen in sera and CSF samples of pulmonary tuberculosis and tubercular meningitis (TBM) respectively. The analysis of sera showed 73.3% of pulmonary tuberculosis cases, 16.2% of healthy controls and 44.4% of Hansen's disease positive for tubercular antigen. The accuracies of positive and negative predictive values were 69% and 82% respectively. The analysis of CSF samples showed the presence of tubercular antigen in 76.4% of TBM, 16.6% of pyogenic meningitis cases, 19.4% of neurological diseases other than meningitis and 16.1% non-neurological disease controls. The accuracies of positive and negative predictive values were 48% and 94% respectively. Hence this simple test using economical and indigenous reagents can be applied for the diagnosis of pulmonary and extra-pulmonary tuberculosis.

Diagnostic use of polyclonal antibodies raised in mouse ascitic fluid in bancroftian filariasis.

Polyclonal antibodies were produced against Brugia malayi adult antigens (BmA (PBS) SAg and BmA (SDS) SAg) in mouse ascitic fluid by immunising Balb/c mice intraperitoneally with high ratio of adjuvant to immunogen. The diagnostic use of these antibodies in detecting circulating filarial antigen in bancroftian filariasis was studied by sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using stick assay system. Both antibodies raised against PBS and SDS soluble antigens were found to be equally sensitive and relatively specific in detection of circulating filarial antigen. When anti BmA (PBS) SAg antibody was used in sandwich ELISA, 90% of microfilaraemic sera, 30-40% of acute and sub acute filarial sera, 20% of chronic filarial sera, 7% of endemic normal sera and none of 15 non-endemic normal sera were positive for filarial antigen. Using anti BmA (SDS) Sag antibody, 93% of microfilarial sera, 40% of acute and sub acute filarial sera, 20% of chronic filarial sera and none of 15 endemic and non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detection using anti BmA S Ag antibodies produced in mouse ascitic fluid in sandwich ELISA may be useful in detection of active stage (microfilaraemia) of infection.

Evaluation of fractionated circulating filarial antigen in diagnosis of bancroftian filariasis.

Circulating filarial antigen (CFA) isolated from the plasma of microfilaraemic patients was fractionated on an Ultrogel ACA 34 column. The second protein peak (CFA2) showing filarial antigen was further fractionated by DEAE-cellulose column chromatography into two fractions (CFA2 DE1 and DE2). CFA2 DE1 fraction, showing antigenic activity, was further evaluated in an ELISA for its diagnostic use in bancroftian filariasis. Studies with CFA2 DE1 and anti-CFA2 DE1 antibody showed that they were highly active in the detection of filarial antibody and antigen in asymptomatic microfilaraemia sera and thus obviate the need for the tedious night blood collection and examination. Fractionated filarial plasma can be another candidate antigen for immunodiagnosis of bancroftian filariasis.

Analysis and diagnostic use of Brugia malayi adult antigen in bancroftian filariasis.

Detergent-soluble antigens of Brugia malayi adult worms (BmA SDS S Ag) were analysed for their antigenic activity and potential use in diagnosis of bancroftian filariasis. Analysis of SDS-PAGE fractions of BmA SDS S Ag against antifilarial antibodies, that is, human filarial serum immunoglobulin G and anti BmA SDS S Ag antibody, revealed two active antigen fractions: BmA-6 and BmA-9. Antibodies raised to BmA-6 and BmA-9 were tested with antigens isolated from infected human body fluids such as circulating filarial antigen (CFA2), urinary filarial antigen (UFAC2) and hydrocele fluid antigen (HFA). Both antibodies showed high reactivity with CFA2-1, 6 and 9 as well as UFAC2-5, 6 and 9 antigenic fractions. In immunoblotting studies, anti BmA-6 antibody detected specific antigens of high microfilaraemic reactivity such as 120, 54, 26 and 22 kDa. In inhibition ELISA using anti BmA SDS S Ag antibody and antigen fraction BmA-6, filarial antigen was detected in 85% of microfilaraemic, 35% of clinical filarial and 20% of endemic normal sera samples. When anti BmA SDS S Ag antibody and BmA-9 were used, 80% of microfilaraemic, 35% of clinical filarial and 25% of endemic normal sera showed positive reaction for filarial antigen. The analysis of urine samples showed the presence of filarial antigen in 76 and 72% of microfilaraemic cases using BmA-6 and BmA-9 fractions respectively while only 20% of endemic normals were positive using both the antigen fractions.(ABSTRACT TRUNCATED AT 250 WORDS)