Antigen-antibody Assay Research Articles

Potential for high dynamic range Sedia Limiting Antigen antibody assay to support viral load monitoring during antiretroviral therapy

Potential for high dynamic range Sedia Limiting Antigen antibody assay to support viral load monitoring during antiretroviral therapy

Resistance levels to non-nucleoside reverse transcriptase inhibitors among pregnant women with recent HIV infection in Malawi.

Background:Information on HIV drug resistance (HIVDR) prevalence in people newly diagnosed with HIV is limited. We implemented a cross-sectional study to estimate HIVDR prevalence among pregnant women recently infected with HIV in Malawi.Methods:The HIVDR study was nested within a routine antenatal clinic (ANC) sentinel surveillance survey. Dried blood spot samples were tested for recent infection using a limiting antigen antibody assay together with HIV viral load testing. HIV-1 protease and reverse transcriptase were sequenced using Sanger sequencing. Drug susceptibility was predicted using Stanford HIVdb algorithm (version 8.9). Weighted analysis was performed in Stata 15.1.Results:Of the 21,642 pregnant women enrolled in the ANC survey, 8.4% (1826/21,642) tested HIV positive. Of these, 5.0% (92/1826) had recent HIV infection, and 90.2% (83/92) were tested by PCR. The amplification and sequencing success rate was 57.8% (48/83). The prevalence of any HIVDR was 14.6% (5/45) (95% CI: 4.7–36.8%), all of which indicated HIVDR to nonnucleoside reverse transcriptase inhibitors (NNRTIs). HIVDR to nucleoside reverse transcriptase inhibitors was 7.9% (2/45) (95% CI: 1.4–34.6%). Resistance to protease inhibitors currently in use in Malawi was not observed.Conclusions:Despite the low number of cases with presumed TDR, our study hints that resistance to NNRTIs was high, above the 10% target for regimen change. Further investigation is needed to establish the exact magnitude of presumed TDR among women recently infected with HIV. These findings support the transition to an integrase inhibitor-based first-line regimen for patients initiating or on ART.

Piezoelectric point-of-care biosensor for the detection of SARS-COV-2 (COVID-19) antibodies

It is always challenging to diagnose a disease using a biosensor reliably, and quickly with high sensitivity and selectivity, simultaneosuly. Recently the world experienced a global pandemic caused by a novel coronavirus (COVID-19). Although the vaccines are available, COVID-19 resulted a huge threat to the entire world with high mortality rates. Irrespective of a specific disease, there is a constant need for a cheaper and faster in-vitro, lab-on-a-chip sensor with high sensitivity and selectivity. Such sensors will not only facilitate the disease detection but will expedite and vaccine development process through detection of its corresponding antibodies when developed. In this article, we present an ultrasonic guided wave sensor using 128° YX lithium niobate piezoelectric wafer, specially designed in a shape of a multi-threaded comb with cantilever beams which is equally selective and sensitive for the detection of corresponding antigen-antibody assays. As a proof of concept in this article, the diagnostic sensor is created and tested for detection of SARS-COV-2 antibodies. Sensors were functionalized with SARS-COV-2 antigens and target antibody for the same was detected. Unique and judicially tuned acoustic features are analyzed for successful detection of the right antibodies. The proposed lab-on-a-chip device utilizes a wide range of diagnostic frequencies resulting into a highly sensitive platform for the diagnostics even to the slightest biophysical changes. The proposed sensor is also believed to extend to the detection of various other antigens/antibodies of different diseases in the future.

Laboratory Evaluation of Hepatitis C Virus Infection in Patients Undergoing Hemodialysis from North East India

Subjects undergoing hemodialysis have enhanced vulnerability to hepatitis C virus (HCV) infection due to invasive procedures and poor infection control practices. Early detection and treatment are essential to prevent cross-infection and mortality/morbidity. However, common use anti-HCV antibody tests lack the necessary accuracy, and alternative tests (e.g. core antigen detection kits) which are available need to be examined as a viable alternative. A total of 270 continuous serum samples were collected from patients undergoing dialysis within 15 months of study period. Sequentially, multiple tests were performed - immunochromatography-based rapid test, third-generation ELISA i.e. (anti-HCV antibody detection), fourth-generation ELISA (HCV antigen-antibody combined detection assay), and HCV RNA quantitative real time polymerase chain reaction (qPCR) assay. Diagnostic parameters of serological kits were compared in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, and so on. Statistical Package for the Social Sciences was used. HCV-combined core antigen-antibody assays performed better than other serological assays in reference to the gold standard HCV RNA. This fourth-generation assay yielded a Kappa value of 0.947 compared with the value of 0.747 and 0.619 for anti-HCV ELISAand rapid detection test. Other parameters such as sensitivity, specificity, PPV, NPV, and so on were also better for fourth-generation ELISA compared with third-generation ELISA and other serological assays. HCV RNA was negative in 7.3% of anti-HCV-positive patients and was detected in 11.4% of anti-HCV ELISA-negative patients. In about 1.6% of HCV RNA-positive cases, fourth-generation ELISA was negative and had low HCV viral load (650 IU/ml and below). Fourth generation ELISA detected additional 7.4% HCV positive cases (compared to third generation kits) and upon cost effective analyis, additional cost to be bear for the better detection (by fourth generation kit) was found to be only INR 27 per 1% increased case detection. In resource scant setup, screening and follow-up of patients undergoing hemodialysis can be performed by fourth-generation HCV ELISA (antigen-antibody combined assay) instead of the current practice of anti-HCV antibody ELISA. Better yield in detection rate will compensate for slight addition to costs.

Rapid HIV Antigen-Antibody Assays and Detection of Acute HIV Infection in Sub-Saharan Africa.

Detection of acute HIV infection is a unique problem that fourth-generation HIV assays were expected to alleviate. In this commentary, we draw attention to the limitations and challenges with use of currently available rapid antigen-antibody (Ag/Ab) combination tests for detection of acute HIV infection in sub-Saharan Africa. Laboratory-based HIV-1 Ag/Ab immunoassays are complex, requiring specialized equipment and handling that are currently not affordable in many settings in Africa. The point-of-care Ag/Ab platform on the other hand is easier to deploy and potentially more accessible in resource-limited settings. However, available fourth-generation HIV-1 rapid diagnostic tests have demonstrated poor performance characteristics in field studies where non-B subtypes of HIV-1 dominate. The potential for point-of-care HIV-1 Ag/Ab diagnostics to significantly improve detection of acute HIV infection remains yet to be realized in sub-Saharan Africa. Assay platforms need to be optimized to identify local circulating subtypes, and optimal algorithms need to be determined.

Asthma and lung function in adulthood after a viral wheezing episode in early childhood.

Viral aetiology of infection has a significant role in the long-term outcome of early-childhood wheezing. This study examines asthma and lung function in adulthood after early-childhood wheezing induced by respiratory syncytial virus (RSV) and rhinovirus (RV). A total of 100 children were hospitalized for a wheezing episode at less than 24 months of age from 1992 to 1993 in Kuopio University Hospital (Finland). Adenovirus, influenza A and B virus, parainfluenza (1-3) virus, and RSV were tested on admission using antigen detection and antibody assays, and RSV and RV were tested by polymerase chain reaction (PCR). In 2010, 49 cases and 60 population controls attended a follow-up study, which included spirometry with bronchodilation test and fractionally exhaled nitric oxide (FENO ) measurements. Current asthma was present in 64% of the cases with RV-induced wheezing (OR 17.0 [95%CI 3.9-75.3] vs controls), in 43% of the cases with RSV-induced wheezing episode (6.1 [1.5-24.9] vs controls), and in 12% of the controls. The RV group showed significantly higher mean FENO values than the RSV group and controls. RV-positive cases had lower MEF50 before bronchodilation and higher MEF50, FEV1, and FEV1/FVC bronchodilation responses than controls. RSV-positive cases had lower FVC than controls before bronchodilation. Cases with RV- and RSV-induced early-childhood wheezing had increased risk for asthma in adulthood, and RV-positive cases had significantly higher FENO values than RSV-positive cases and controls. Compared to controls, RV-positive cases showed more bronchial reactivity, and RSV-positive cases showed lower FVC before bronchodilation in lung function testing. Children with RV- or RSV-induced wheezing in early childhood have an increased risk for asthma and lung function abnormalities in adulthood.

EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

Estimation of the True Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Autoantibody Concentration in the Sera of Normal Subjects

According to our previous studies, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) autoantibodies (GMAbs) are normally present in healthy people. To date, antigen-antibody capture assay has measured the concentrations. However, nonspecific IgG other than GMAbs and immune complex formation with intrinsic GM-CSF prevents us from measuring the accurate concentrations. To solve these problems, we developed a liquid phase high-sensitivity assay using biotinylated GM-CSF (bGM-CSF) and streptavidin coated plate, which could measure the concentration of GMAbs over 0.02 ng/ml. Using this method, the true GMAbs concentration in 30 normal subjects was estimated to be 52.6 ng/ml for the median value, ranging 10.6 to 799.5 ng/ml. Thus, we propose a novel method that enables us to estimate the true concentration of GMAbs in the sera of healthy subjects.

Evaluation the performance of three HIV antigen-antibody combination assays in the detection of HIV in the window period

Objective To evaluate the detectability of HIV antigen-antibody in the window period of acute infection by three HIV antigen-antibody assays. Methods Twenty-two samples of HIV seroconversion serum panels and thirty-seven HIV acute infected plasm samples from our laboratory collected from cohort study of men who have sex with men between 2009 and 2011,were assayed by ECLIA,CLIA and ELISA methods.All assays were evaluated for the ability to detect HIV in the window period,and the sensitivity of each assay for acute samples was analyzed.Chi square test was used for statistical analysis. Results The ability of detecting HIV in the window period of each assay was different.For HIV seroconversion serum panels,the results of ECLIA and CLIA assays were consistent,and the window period was shortened at least 1 to 5 days compared with ELISA assay.For HIV acute samples,all were HIV positive by ECLIA or CLIA assay,but for ELISA assay,94.6% was positive.For samples before seroconversion,ECLIA and CLIA assay had the same sensitivity(93.5%),which is superior to ELISA assay(71.0%)(χ2=5.14,P<0.05). Conclusion The ability of detecting HIV in the window period was different for each assay.The results of ECLIA and CLIA assay are consistent,superior to ELISA assay.（Chin J Lab Med,2014,37:613-616） Key words: HIV antigens; HIV antibodies; Electrochemistry; Luminescent measurements; Enzyme-linked immunosorbent assay; Immunoassay

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity, cost-effectiveness and their suitability for high-throughput testing. Enzyme immunoassay (EIAs) for hepatitis C virus (HCV) antibodies were the initial screening tests introduced. The "first generation" antibody EIAs detected seroconversion after unduly long infectious window period. Improved HCV antibody assays still had an infectious window period around 66 d. HCV core antigen EIAs shortened the window period considerably, but high costs did not lead to widespread acceptance. A fourth-generation HCV antigen and antibody assay (combination EIA) is more convenient as two infectious markers of HCV are detected in the same assay. Molecular testing for HCV-RNA utilizing nucleic acid amplification technology (NAT) is the most sensitive assay and shortens the window period to only 4 d. Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now < 1 per million donations. However, HCV serology still continues to be retained as some donations are serology positive but NAT negative. In resource constrained countries HCV screening is highly variable, depending upon infrastructure, trained manpower and financial resource. Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres. The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays. Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.

Detecting HIV antibodies in oral fluid: validation of a commercial antigen-antibody assay

Detecting HIV antibodies in oral fluid: validation of a commercial antigen-antibody assay

Real-life evaluation of a human immunodeficiency virus screening algorithm using a single combined p24 antigen–antibody assay

Since May 2010, human immunodeficiency virus (HIV) screening in France has been performed using a single combined fourth-generation assay. One of our major concerns is to verify that this screening strategy is able to diagnose HIV primary infection as soon as possible. Thus, the sensitivity and specificity of this strategy were evaluated on 49,623 serum samples, including 29 primary infections, received for routine HIV testing between September 2010 and November 2011. Specimens were screened using the Enzygnost HIV Integral II enzyme-linked immunosorbent assay (ELISA) kit. All positive sera, according to the manufacturer's recommendations [signal-to-cutoff ratio (S/CO) ≥ 1] were retested using the Architect HIV Ag/Ab Combo. Moreover, we defined a grey zone (0.5 < S/CO < 1) and sera within this grey zone were retested using the VIDAS HIV DUO Ultra test and HIV-1 RNA was checked by the Abbott RealTime PCR kit. Screening tests were positive for all primary infections. All samples within the grey zone proved VIDAS HIV DUO Ultra and HIV-1 RNA negative. Overall, the ELISA test sensitivity and specificity were 100 and 99.79 %, respectively. The false-positive rate was higher when S/CO was in the low range (1 to 5). Adding a second screening test for positive sera reduced the false-positive rate from 0.20 to 0.02 %. HIV screening with a single combined assay did not miss any documented primary infection during this evaluation period, even without extending the positivity zone.

A phenotype–genotype correlation of ADAMTS13 mutations in congenital thrombotic thrombocytopenic purpura patients treated in the United Kingdom

ADAMTS13 mutations play a role in thrombotic thrombocytopenic purpura (TTP) pathogenesis. To establish a phenotype-genotype correlation in a cohort of congenital TTP patients. Clinical history and ADAMTS13 activity, antigen and anti-ADAMTS13 antibody assays were used to diagnose congenital TTP, and DNA sequencing and in vitro expression were performed to identify the functional effects of the ADAMTS13 mutations responsible. Seventeen (11 novel) ADAMTS13 mutations were identified in 17 congenital TTP patients. All had severely reduced ADAMTS13 activity and antigen levels at presentation. Six patients with pregnancy-associated TTP and six patients with childhood TTP were homozygous or compound heterozygous for ADAMTS13 mutations located in the metalloprotease (MP), cysteine-rich, spacer and/or distal thrombospondin type 1 domains. The adults had TTP precipitated by pregnancy, and had overall higher antigen levels (median, 30 ng mL(-1) ; range, < 10-57 ng mL(-1) ) than the children (median, 14 ng mL(-1) ; range, < 10-40 ng mL(-1)). Presentation in the neonatal period was associated with more intensive treatment requirements. The two neonates with the most severe phenotype had mutations in the first thrombospondin type 1 motif of ADAMTS13 (p.R398C, p.R409W, and p.Q436H). Using transfected HEK293T cells, we have shown that p.R398C and p.R409W block ADAMTS13 secretion, whereas p.Q436H allows secretion at reduced levels. This study confirms the heterogeneity of ADAMTS13 defects and an association between ADAMTS13 genotypes and TTP phenotype.

Performance of an antigen–antibody combined assay for hepatitis C virus testing without venipuncture

BackgroundHepatitis C virus (HCV) is underdiagnosed and therefore increasing the opportunities for HCV testing without venipuncture may be useful. ObjectivesWe evaluated the analytical performance of a modified, commercially available, combined HCV antigen–antibody assay (cEIA) (Monolisa® HCV-Ag-Ab-ULTRA) and a commercially available point-of-care (POC) device (OraQuick® HCV) on fingerstick blood (FSB) and oral mucosal transudate (OMT). Study designFSB, OMT and serum samples were collected from 113 cases of HCV-antibody-positive patients and 88 HCV-antibody-negative controls. The HCV-antibody-positive group included 63 patients with quantifiable HCV-RNA (56%) and 17 HIV/HCV co-infected patients (15%). FSB and OMT specimens were collected as dried blood spots (DBSs) or with the OraSure collection system, before testing with cEIA. ResultsWith FSB specimens, the cEIA and the POC device exhibited 100% specificity and 98.2% and 97.4% sensitivity, respectively. The specificity of the cEIA in FSB sharply decreased if stored 3days at room temperature. With OMT specimens, the cEIA sensitivity (71.7%) and specificity (94.3%) were significantly lower than the performance of OraQuick® HCV (sensitivity, 94.6%; specificity, 100%). The optical densities obtained with the cEIA in FSB and OMT were lower in HIV/HCV co-infected patients compared with HCV monoinfected patients. ConclusionThe cEIA using FSB specimens collected on DBSs preserved in appropriate storage conditions was a reliable alternative, equivalent to the POC assay, for HCV testing without venipuncture. The cEIA was not adapted for HCV testing on OMT.

Performance evaluation of a new fourth-generation HIV combination antigen–antibody assay

Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys® HIV combi PT assay is a fourth-generation antigen–antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay’s specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys® assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3–7.1 days observed with comparators. The analytical sensitivity of the Elecsys® HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys® assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys® assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys® HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.

237-P Establishing a reference range for control beads in single antigen antibody assays

237-P Establishing a reference range for control beads in single antigen antibody assays

Fever and Rash in an 8-month-old Girl

Fever and Rash in an 8-month-old Girl

Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.

Hepatitis C virus among non-injecting cocaine users (NICUs) in South America: can injectors be a bridge?

To investigate the factors associated with hepatitis C virus (HCV) infection among non-injecting cocaine users (NICUs) and to compare practices associated with HCV and HIV infection. An intercountry cross-sectional study. Setting Buenos Aires and Montevideo metropolitan areas. A total of 871 NICUs. NICUs were interviewed and their blood was drawn and used for HCV, HIV, HBV surface antigen (HbsAg), HB-anticore and Venereal Disease Research Laboratory (VRDL) antibody assays. Bivariate and multivariate logistic regression analyses included comparisons of HCV and HIV mono-infected participants with HCV-HIV seronegatives. Prevalence rates were 8.8 [95% confidence interval (CI): 6.9-10.8) for HCV and 7.9 (95% CI: 6.1-9.7) for HIV. HCV-infected NICUs were twice as likely as HCV-HIV seronegatives to have shared straws for cocaine snorting or sniffing, even when adjusted for other variables. HCV prevalence rates ranged from 3.6% among NICUs who denied sharing straws and having had an injection drug user (IDU) or an HIV-positive sexual partner to 12.6% among participants who reported ever having shared straws or having had either an IDU- or HIV-positive sexual partner (χ(2) (trend) = 6.56, P = 0.01). Non-injecting cocaine users from South America are vulnerable to multiple infections and HCV infection appears to occur through the sharing of straws. HCV infection is associated with intimate relationships with IDUs or HIV-seropositive partners, supporting the hypothesis that HCV risk may be due primarily to risk-taking behaviour associated with drugs in this population.

56-P: Factors Affecting the Relationship Between Single Antigen Antibody Assays and Crossmatch Results

56-P: Factors Affecting the Relationship Between Single Antigen Antibody Assays and Crossmatch Results