Publications by authors named "Sheikh M Talha"

18 Publications

  • Page 1 of 1

Double-Antigen Lateral Flow Immunoassay for the Detection of Anti-HIV-1 and -2 Antibodies Using Upconverting Nanoparticle Reporters.

Sensors (Basel) 2021 Jan 6;21(2). Epub 2021 Jan 6.

Department of Biotechnology, University of Turku, 20520 Turku, Finland.

Rapid diagnostic tests (RDTs) are often used for the detection of anti-human immunodeficiency virus (HIV) antibodies in remote locations in low- and middle-income countries (LMIC) with low or limited access to central laboratories. The typical format of an RDT is a lateral flow assay (LFA) with visual interpretation prone to subjectivity. This risk of misinterpretation can be overcome with luminescent upconverting nanoparticle reporters (UCNPs) measured with a miniaturized easy-to-use reader instrument. An LFA with UCNPs for anti-HIV-1/2 antibodies was developed and the assay performance was evaluated extensively with challenging patient sample panels. Sensitivity ( = 145) of the UCNP-LFA was 96.6% (95% CI: 92.1-98.8%) and specificity ( = 309) was 98.7% (95% CI: 96.7-99.7%). Another set of samples ( = 200) was used for a comparison between the UCNP-LFA and a conventional visual RDT. In this comparison, the sensitivities for HIV-1 were 96.4% (95% CI: 89.8-99.3%) and 97.6% (95% CI: 91.6-99.7%), for the UCNP-LFA and conventional RDT, respectively. The specificity was 100% (95% CI: 96.4-100%) for both assays. The developed UCNP-LFA demonstrates the applicability of UCNPs for the detection of anti-HIV antibodies. The signal measurement is done by a reader instrument, which may facilitate automated result interpretation, archiving and transfer of data from de-centralized locations.
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http://dx.doi.org/10.3390/s21020330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825344PMC
January 2021

Upconverting nanoparticle reporter-based highly sensitive rapid lateral flow immunoassay for hepatitis B virus surface antigen.

Anal Bioanal Chem 2021 Feb 23;413(4):967-978. Epub 2020 Nov 23.

Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, 121001, India.

Detection of hepatitis B Virus surface antigen (HBsAg) is an established method for diagnosing both acute and chronic hepatitis B virus (HBV) infection. In addition to enzyme immunoassays (EIAs), rapid diagnostic tests (RDTs) are available for the detection of HBsAg in resource-poor settings. However, the available RDTs have inadequate sensitivity and therefore are not suitable for diagnosis of patients with low levels of HBsAg and for blood screening. To provide a high-sensitivity RDT, we developed a lateral flow immunoassay (LFIA) for HBsAg utilizing upconverting nanoparticle (UCNP) reporter. The UCNP-LFIA can use whole blood, serum, or plasma and the results can be read in 30 min using a reader device. When compared with a commercial conventional visually read LFIA, the developed UCNP-LFIA had a Limit of Detection (LoD) of 0.1 IU HBsAg/ml in spiked serum, whereas the LoD of the conventional LFIA was 3.2 IU HBsAg/ml. The developed UCNP-LFIA fulfills the WHO criterion for blood screening (LoD ≤ 0.13 IU HBsAg/ml) in terms of LoD. The UCNP-LFIA and conventional LFIA were evaluated with well-characterized sample panels. The UCNP-LFIA detected 20/24 HBsAg-positive samples within the HBsAg Performance Panel and 8/10 samples within the Mixed Titer Performance Panel, whereas the conventional LFIA detected 8/24 and 4/10 samples in these panels, respectively. The performance of the assays was further evaluated with HBsAg-positive (n = 108) and HBsAg-negative (n = 315) patient samples. In comparison with a central laboratory test, UCNP-LFIA showed 95.4% (95% CI: 89.5-98.5%) sensitivity whereas sensitivity of the conventional LFIA was 87.7% (95%CI: 79.9-93.3%).
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http://dx.doi.org/10.1007/s00216-020-03055-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813740PMC
February 2021

Ultrasensitive and Robust Point-of-Care Immunoassay for the Detection of Malaria.

Anal Chem 2020 12 23;92(24):15766-15772. Epub 2020 Nov 23.

Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad 121001, Haryana, India.

malaria is widespread in the tropical and subtropical regions of the world. There is ongoing effort to eliminate malaria from endemic regions, and sensitive point-of-care (POC) diagnostic tests are required to support this effort. However, current POC tests are not sufficiently sensitive to detect in asymptomatic individuals. After extensive optimization, we have developed a highly sensitive and robust POC test for the detection of infection. The test is based on upconverting nanophosphor-based lateral flow (UCNP-LF) immunoassay. The developed UCNP-LF test was validated using whole blood reference panels containing samples at different parasite densities covering eight strains of from different geographical areas. The limit of detection was compared to a WHO-prequalified rapid diagnostic test (RDT). The UCNP-LF achieved a detection limit of 0.2-2 parasites/μL, depending on the strain, which is 50- to 250-fold improvement in analytical sensitivity over the conventional RDTs. The developed UCNP-LF is highly stable even at 40 °C for at least 5 months. The extensively optimized UCNP-LF assay is as simple as the conventional malaria RDTs and requires 5 μL of whole blood as sample. Results can be read after 20 min from sample addition, with a simple photoluminescence reader. In the absence of a reader device at the testing site, the strips after running the test can be transported and read at a central location with access to a reader. We have found that the test and control line signals are stable for at least 10 months after running the test. The UCNP-LF has potential for diagnostic testing of both symptomatic and asymptomatic individuals.
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http://dx.doi.org/10.1021/acs.analchem.0c02748DOI Listing
December 2020

Glycovariant-based lateral flow immunoassay to detect ovarian cancer-associated serum CA125.

Commun Biol 2020 Aug 21;3(1):460. Epub 2020 Aug 21.

Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.

Cancer antigen 125 (CA125) is a widely used biomarker in monitoring of epithelial ovarian cancer (EOC). Due to insufficient cancer specificity of CA125, its diagnostic use is severely compromised. Abnormal glycosylation of CA125 is a unique feature of ovarian cancer cells and could improve differential diagnosis of the disease. Here we describe the development of a quantitative lateral flow immunoassay (LFIA) of aberrantly glycosylated CA125 which is widely superior to the conventional CA125 immunoassay (CA125IA). With a 30 min read-out time, the LFIA showed 72% sensitivity, at 98% specificity using diagnostically challenging samples with marginally elevated CA125 (35-200 U/mL), in comparison to 16% sensitivity with the CA125IA. We envision the clinical use of the developed LFIA to be based on the substantially enhanced disease specificity against the many benign conditions confounding the diagnostic evaluation and against other cancers.
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http://dx.doi.org/10.1038/s42003-020-01191-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442799PMC
August 2020

High-sensitivity lateral flow immunoassay with a fluorescent lanthanide nanoparticle label.

J Immunol Methods 2019 02 4;465:39-44. Epub 2018 Dec 4.

Department of Biotechnology, University of Turku, Turku, Finland.

Lateral flow (LF) immunoassays are commonly used for point-of-care testing and typically incorporate visually read reporters, such as gold particles. To improve sensitivity and develop quantitative LF immunoassays, visual reporters can be replaced by fluorescent reporters detected by an instrument. In this study, we used fluorescent europium(III) chelate doped nanoparticle (Eu-np) reporters to develop a quantitative high-sensitivity LF immunoassay for free prostate specific antigen (fPSA). Furthermore, we tested different simplified formats of the assay and the effect of different modifiable parameters on the detection limit of the assay: dynamic range, assay duration and number of assay steps. The molar detection limits of the different assay formats were compared with published detection limits of LF immunoassays with different reporters. The cutoff was calculated from 11 female serum samples. The detection limit of the sensitivity optimized fPSA assay with fPSA spiked into pooled female serum was 0.01 ng/ml, which is approximately 100-fold lower than the most sensitive gold particle LF assays and 10-fold lower than other Eu-np and carbon nanoparticle based LF immunoassays. Thus, Eu-np reporters can be used to develop highly sensitive and quantitative LF immunoassays.
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http://dx.doi.org/10.1016/j.jim.2018.12.001DOI Listing
February 2019

Microparticle-based platform for point-of-care immunoassays.

Anal Biochem 2018 05 24;548:66-68. Epub 2018 Feb 24.

Department of Biotechnology, University of Turku, Turku, Finland.

There is a need for quantitative and sensitive, yet simple point-of-care immunoassays. We have developed a point-of-care microparticle-based immunoassay platform which combines the performance of a microtiter well-based assay with the usability of a rapid assay. The platform contained a separate reaction and detection chambers and microparticles for the solid-phase. Photoluminescent up-converting nanoparticles (UCNPs) were used as labels. The platform was tested with a cardiac troponin I assay, and a limit of detection of 19.7 ng/L was obtained. This study demonstrates the feasibility of developing point-of-care assays on the new platform for various analytes of interests.
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http://dx.doi.org/10.1016/j.ab.2018.02.021DOI Listing
May 2018

Lateral flow immunoassay with upconverting nanoparticle-based detection for indirect measurement of interferon response by the level of MxA.

J Med Virol 2017 04 27;89(4):598-605. Epub 2016 Sep 27.

Department of Virology, University of Turku, Turku, Finland.

Myxovirus resistance protein A (MxA) is a biomarker of interferon-induced gene expression state involved in many viral infections and some autoimmune disorders. It has a variety of potential utilities in clinical diagnostics, including distinguishing between bacterial and viral infections. Currently, MxA-assays are used for monitoring of IFN-β therapy in multiple sclerosis (MS) patients. As a proof-of-concept for rapid quantitative measurement of interferon response, a lateral flow immunoassay (LFIA) with upconverting nanoparticle (UCNP) reporters was developed and evaluated with clinical whole blood samples to assess the potential for a rapid and user-friendly quantitative assay for MxA, since the currently available rapid test for MxA (FebriDX) produces only qualitative result. The high detection sensitivity enabled by the UCNP reporter technology allowed the sample pre-treatment with dilution of whole blood into lysis buffer at a detectable analyte concentration. The assay can be done within 2 hr and the results correlate with the reference MxA-ELISA, which requires an overnight incubation. With 36 samples, R for linear regression was 0.86. The assay detected 96% of the IFN-β responders with 89% specificity using a cut-off level of 100 μg/L for an elevated MxA-concentration. J. Med. Virol. 89:598-605, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jmv.24689DOI Listing
April 2017

Europium nanoparticle-based simple to perform dry-reagent immunoassay for the detection of hepatitis B surface antigen.

J Virol Methods 2016 Mar 4;229:66-9. Epub 2016 Jan 4.

Department of Biotechnology, University of Turku, Turku, Finland.

Hepatitis B infection, caused by hepatitis B virus (HBV), presents a huge global health burden. Serological diagnosis of HBV mainly relies on the detection of hepatitis B surface antigen (HBsAg). Although there are high sensitivity commercial HBsAg enzyme immunoassays (EIAs) available, many low-resource laboratories lacking trained technicians continue to use rapid point-of-care assays with low sensitivities for HBsAg detection, due to their simplicity to operate. We developed a time-resolved fluorometric dry-reagent HBsAg immunoassay which meets the detection limit of high sensitivity EIAs but is simple to operate. To develop the assay, anti-HBsAg monoclonal antibody coated on europium nanoparticles was dried atop of biotinylated anti-HBsAg polyclonal antibody immobilized on streptavidin-coated microtiter wells. To test a sample in dry-reagent assay, serum sample and assay buffer were added to the wells, incubated, washed and europium signals were measured. The assay showed a detection limit of 0.25 ng/ml using HBsAg spiked in serum sample. When evaluated with 24 HBV positive and 37 negative serum samples, assay showed 100% sensitivity and specificity. Assay wells are stable for at least 26 weeks when stored at 4°C, and can tolerate elevated temperatures of up to 35°C for two weeks. The developed assay has high potential to be used in low-resource laboratories.
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http://dx.doi.org/10.1016/j.jviromet.2016.01.001DOI Listing
March 2016

Array-in-well platform-based multiplex assay for the simultaneous detection of anti-HIV- and treponemal-antibodies, and Hepatitis B surface antigen.

J Immunol Methods 2016 Feb 19;429:21-7. Epub 2015 Dec 19.

Department of Biotechnology, University of Turku, Turku, Finland.

Multiplex assays detecting sets of related clinical analytes simultaneously can save considerable amount of time and resources. Array-in-well (AIW) is a powerful platform for the multiplex detection of different analytes where microarrays can be printed at the bottom of microtiter wells, thus combining the potential of microarrays with the ease of handling microtiter wells. We have developed a single-step AIW assay for the simultaneous screening of HIV, Treponema pallidum subspecies pallidum (causing syphilis) and Hepatitis B virus infections targeting the specific detection of anti-HIV- and treponemal-antibodies and Hepatitis B surface antigen (HBsAg), respectively, using two different fluorescent label technologies i.e. DyLight 633 and europium nanoparticle. Double-antigen assay formats were used for anti-HIV- and treponemal-antibody detection that can simultaneously detect both IgG and IgM, and thus reduce the window period of detection. AIW assay was evaluated with well characterized serum/plasma samples (n=111), and the qualitative results were in near complete agreement with those of the reference assays. The AIW assay exhibited 100% sensitivities for all three analytes, and 100% specificities for anti-HIV antibodies and HBsAg, and 98.6% specificity for treponemal antibodies. The limit of detection of HBsAg in AIW assay was 0.18 ng/ml. This high performing AIW assay has the potential to be used as a multiplex screening test for these three infections.
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http://dx.doi.org/10.1016/j.jim.2015.12.007DOI Listing
February 2016

Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.

J Virol Methods 2016 Feb 23;228:67-73. Epub 2015 Nov 23.

Department of Biotechnology, University of Turku, Turku, Finland.

There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.
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http://dx.doi.org/10.1016/j.jviromet.2015.11.015DOI Listing
February 2016

All-in-one dry-reagent time-resolved immunofluorometric assay for the rapid detection of HIV-1 and -2 infections.

J Virol Methods 2015 Dec 22;226:52-9. Epub 2015 Oct 22.

Department of Biotechnology, University of Turku, Turku, Finland.

An all-in-one (AIO) dry-reagent time-resolved fluorometric immunoassay that requires minimal liquid handling was developed for the detection of anti-HIV-1 and -2 antibodies. To prepare the AIO wells, in vivo biotinylated capture antigens (r-Bio-HIV-1env and r-Bio-HIV-2env) were immobilized on streptavidin-coated microtitration wells and Eu(III) chelate labelled non-biotinylated tracer antigens [r-HIV-1env-Eu(III) and r-HIV-2env-Eu(III)] were dried in stable form in the same wells. The HIV AIO assay was evaluated with serum/plasma samples (n=148) from in-house and commercial panels at two different incubation times of 15 min and 1h. The overall sensitivity of the AIO assay was 98.6% and specificity was 100% for both the incubation times. The AIO assay can accept whole blood matrix. This assay is envisioned to fill the gap between the rapid point-of-care assays and traditional enzyme immunoassays (EIA) in terms of complexity and turnaround time, without compromising the performance.
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http://dx.doi.org/10.1016/j.jviromet.2015.10.004DOI Listing
December 2015

Effects of blood sample anticoagulants on lateral flow assays using luminescent photon-upconverting and Eu(III) nanoparticle reporters.

Anal Biochem 2016 Jan 25;492:13-20. Epub 2015 Sep 25.

Department of Biotechnology, University of Turku, 20520 Turku, Finland.

Many quantitative and semiquantitative lateral flow (LF) assays have been introduced for clinical analytes such as biomarkers for cancer or acute myocardial infarction (AMI). Various detection technologies involving quantitative analyzing devices have been reported to have sufficient analytical sensitivity and quantification capability for clinical point-of-care tests. Fluorescence-based detection technologies such as quantum dots, Eu(III) nanoparticles, and photon-upconverting nanoparticles (UCNPs) have been introduced as promising solutions for point-of-care devices because of their high detectability by optical sensors. Lateral flow assays can be used for various sample types, e.g., urine, saliva, cerebrospinal fluid, and blood. This study focuses on the properties of serum and plasma because of their relevance in cancer and AMI diagnostics. The limit of detection was compared in LF assays having Eu(III) nanoparticles or UCNPs as reporters and the antibody configurations for two different analytes (prostate-specific antigen and cardiac troponin I (cTnI)). The results indicate a significant effect of anticoagulants in venipuncture tubes. The samples in K3EDTA tubes resulted in significant interference by decreased reporter particle mobility, and thus the limit of detection was up to eightfold less sensitive compared to serum samples. Despite the matrix interference in the cTnI assay with UCNP reporters, limits of detection of 41 ng/L with serum and 66 ng/L with the Li-heparin sample were obtained.
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http://dx.doi.org/10.1016/j.ab.2015.09.009DOI Listing
January 2016

Europium nanoparticle-based high performing immunoassay for the screening of treponemal antibodies.

PLoS One 2013 26;8(12):e84050. Epub 2013 Dec 26.

Department of Biotechnology, University of Turku, Turku, Finland.

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p=0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084050PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873409PMC
August 2014

Fed-batch cultivation of Escherichia coli expressed designer hepatitis C virus diagnostic intermediate and its evaluation.

Biotechnol Appl Biochem 2012 Nov-Dec;59(6):437-44

Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

The present study aimed for an enhanced induction strategy combined with high-level production of a capture antigen of hepatitis C virus (HCV) for use in diagnosis of HCV infection. We have expressed the synthetic gene encoding for HCV multiepitope protein in pET-28a(+) vector and investigated its production in Escherichia coli BL21(DE3) cells using high-cell-density fed-batch cultivation. A maximum cell dry mass of 30 g/L was obtained, and the culture was induced with 1, 5, and 10 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for ∼4 H at 30°C; a maximum protein production of 1.5 g/L was observed in the case of induction with 10 mM IPTG. The enhanced induction strategy resulted in a ∼15-fold increase as compared to 1 mM IPTG. The protein was purified using a simple immobilized metal affinity chromatography procedure, yielding 16.6 mg/g dry cell weight of pure protein with more than 99% purity. Further, the protein was evaluated for its diagnostic potential by using the commercially available HCV Seroconversion Panel, Worldwide HCV Performance Panel, and Viral Coinfection Panel. The protein showed high sensitivity and specificity, which was comparable to the best performing commercially available enzyme immunoassay (EIA) kits.
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http://dx.doi.org/10.1002/bab.1044DOI Listing
March 2014

Escherichia coli-expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen.

BMC Infect Dis 2012 Nov 27;12:325. Epub 2012 Nov 27.

Department of Biotechnology, University of Turku, 20520 Turku, Finland.

Background: The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies.

Methods: A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II)-affinity chromatography. Biotinylated and europium(III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131), that included an in-house panel and four commercially procured panels.

Results: In-frame deletion of three hydrophobic regions, spanning amino acid residues 1-43, 519-538 and 676-706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses.

Conclusions: This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test.
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http://dx.doi.org/10.1186/1471-2334-12-325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3519745PMC
November 2012

A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2.

J Virol Methods 2011 Apr 11;173(1):24-30. Epub 2011 Jan 11.

International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.

This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1env, incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu(3+) chelate or with nanoparticles doped with the Eu(3+) chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu(3+) chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios.
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http://dx.doi.org/10.1016/j.jviromet.2011.01.001DOI Listing
April 2011

Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay.

J Nanobiotechnology 2010 Nov 26;8:27. Epub 2010 Nov 26.

Department of Biotechnology, University of Turku, Turku, Finland.

A highly specific and novel dual-label time-resolved immunofluorometric assay was developed exploiting the unique emission wavelengths of the intrinsically fluorescent terbium (Tb3+) and europium (Eu3+) tracers for the simultaneous detection of human immunodeficiency virus 1 (HIV-1) and hepatitis B virus (HBV) infections, respectively. HIV-1 infection was detected using a double antigen sandwich format wherein anti-HIV-1 antibodies were captured using an in vivo biotinylated version of a chimeric HIV-1 antigen and revealed using the same antigen labeled with Tb3+ chelate. Hepatitis B surface antigen (HBsAg), which served as the marker of HBV infection, was detected in a double antibody sandwich using two monoclonal antibodies (mAbs), one chemically biotinylated to capture, and the other labeled with Eu3+ nanoparticles, to reveal. The performance of the assay was evaluated using a collection (n = 60) of in-house and commercially available human sera panels. This evaluation showed the dual-label assay to possess high degrees of specificity and sensitivity, comparable to those of commercially available, single analyte-specific kits for the detection of HBsAg antigen and anti-HIV antibodies. This work demonstrates the feasibility of developing a potentially time- and resource-saving multiplex assay for screening serum samples for multiple infections in a blood bank setting.
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http://dx.doi.org/10.1186/1477-3155-8-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001693PMC
November 2010

Inexpensive designer antigen for anti-HIV antibody detection with high sensitivity and specificity.

Clin Vaccine Immunol 2010 Mar 20;17(3):335-41. Epub 2010 Jan 20.

ecombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.

A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.
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http://dx.doi.org/10.1128/CVI.00283-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837966PMC
March 2010