Antigen detection in primary HIV infection

Serial blood samples were obtained from 21 homosexuals who had developed symptomatic primary infection with human immunodeficiency virus (HIV) after a median incubation time of 14 days. During the first two weeks after the onset of illness HIV antigen (p24) was detected in the blood by enzyme linked immunosorbent assay (ELISA). During the second and third weeks after the onset of illness p24 antibody was detected by Western blot assay and antigen concentrations rapidly decreased to undetectable values. Dissociation of antigen-antibody complexes showed complexed antigen during the phase of declining concentrations of free antigen. Neither free nor complexed antigen was detected in any serum samples for several months thereafter, which suggested that failure to detect HIV antigen reflected low or absent synthesis of viral protein rather than masking of antigen by antibodies. Reappearance of HIV antigen with a fall in p24 antibody concentration was observed in a few patients six months or more after the onset of disease.The combined use of antigen and antibody assays made it possible to obtain evidence of infection with HIV in all of the 95 serum samples tested, illustrating the usefulness of these assays for diagnosing infection with HIV in its early stages.     

Characterization of monoclonal antibodies directed against distinct conserved epitopes of human immunodeficiency virus type 1 core proteins

Summary Two mouse hybridoma cell lines secreting antibodies to the Human Immunodeficiency Virus (HIV) p25 major core protein and its precursors p55 and p41, were developed after immunization with the highly cytopathic Zaïrian HIV-1 isolate, NDK. These monoclonal antibodies also react with the gag gene products from HIV-1-BRU prototype and present cross reaction with HIV-2-ROD, and SIV-AGM. They map into topographically distinct areas of p25 and define epitopic regions topographically separated from those recognized by four other anti-p25 mAb suggesting the existence of at least 6 spatially distinct epitopic regions on HIV-1-p25 core protein.Abbreviations HIV Human Immunodeficiency Virus - SIV Simian Immunodeficiency Virus - HTLVI Human T cell Leukaemia Virus - AIDS Acquired Immune Deficiency Syndrome - mAb Monoclonal Antibody - ELISA Enzyme Linked Immunosorbent Assay - PBS Phosphate Buffered Saline     

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10^-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10^-18 moles of the p24/assay corresponds to ca. 10³ copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10² copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.     

Expression of an immunogenic region of HIV by a filamentous bacteriophage vector.

Vectors derived from the Escherichia coli filamentous phage, fd-tet, expressing parts of the human immunodeficiency virus (HIV) gag genes were constructed and analyzed. The immunoreactive domain of HIV Gag antigens was produced in the form of a fusion protein, with a phage minor coat protein, called protein III, playing an important role in phage infectivity. A micropanning procedure, utilizing the strong affinity of biotinylated antibody to streptavidin, was applied for the selection of clones. A simple preparation procedure consisting of polyethyleneglycol precipitation of the recombinant phage from the E. coli supernatant allowed us to detect HIV antigens by enzyme-linked immunosorbent assay (ELISA). Cloned FUSE-gag, as isolated using anti-Gag RL4.72.1 monoclonal antibody (mAb), contained a nucleotide sequence coding for 91 amino acids of HIV Gag p24. It specifically reacted with the mAb in the ELISA. Construction of the mAb-selectable phages permitted localization of epitopes for mAb. Infectivity of the phage clone was specifically neutralized by the anti-HIV mAb. Immunoelectroblotting analysis of recombinant phages revealed the presence of an about 65-kDa band reacting with anti-HIV mAb. This Mr corresponded to the size of the fused form of the FUSE 1 protein III. Human sera from HIV-infected and uninfected individuals reacted with recombinant protein III, as well as the original form of protein III.     

HIV antigen and antibody detection: variable responses to infection in the Edinburgh haemophiliac cohort

Sequential serum samples from 18 haemophiliac patients exposed simultaneously to human immunodeficiency virus type 1 (HIV 1) in early 1984 were tested retrospectively for serological markers of infection. Assay for total antibodies to HIV established that the time to seroconversion might be as long as 110 days after exposure to contaminated factor VIII; serum samples were also tested by Western blotting, by enzyme linked immunosorbent assay (ELISA) for specific antibodies to envelope and core proteins, and for p24 antigen by two assay systems during the two years after infection. The studies showed that five of the 12 patients for whom serum samples obtained between exposure and seroconversion were available had transient p24 antigenaemia. Although amounts of total antibody to HIV and of antibodies to envelope proteins rose continuously during the two years of the study, amounts of antibody to the core protein were variable and tended to decline in patients who became symptomatic. Two patients had persistent p24 antigenaemia that began four months after seroconversion; these patients remained asymptomatic. One patient who developed the acquired immune deficiency syndrome (AIDS) had transient antigenaemia at the time of seroconversion but failed to show any antigen for the rest of the study; progression to AIDS was accompanied by an increase in antibodies to envelope proteins.Much of the variability in the course of infection with HIV must represent the differences in the susceptibility of the patients to infection.     

人免疫缺陷病毒早期蛋白Nef的表达、纯化及免疫原性研究

目的：通过原核细胞表达人免疫缺陷病毒（HIV）Nef抗原,制备特异抗血清,为Nef抗原检测提供技术方法。方法：以HIVBotswana毒株基因组为模板,用PCR法获得Nef蛋白编码基因,将其克隆到pET30a载体中,在大肠杆菌中表达Nef融合蛋白;用纯化的融合蛋白免疫BALB／c小鼠获得抗血清,用真核表达的Nef抗原对其特异性进行分析。结果：构建的Nef融合基因在大肠杆菌中获得表达,相对分子质量约为36x103,免疫BALB／c小鼠获得针对融合蛋白的高效价抗血清,ELISA抗体滴度为1：6400;免疫荧光和Westemblot检测表明,该抗血清能特异地与重组痘苗病毒表达的Nef抗原反应。结论：在大肠杆菌中表达了HIVNef融合蛋白,制备了Nef融合蛋白的高效价小鼠免疫血清,该血清能特异性识别HIVNef抗原,为HlVNef抗原检测提供了技术方法。     

A new capture test using conjugated peptides for the detection of HIV antibodies

Abstract A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591–611) YLKDQQLLGIWGCSGKLICTT, the gp120 sequence (314–329) IRIQRGPGRAFVTIGK and the p27 sequence (182–198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N -hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indirect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies.     

Detection of HIV1 RNA and p24 antigen in HIV1-infected human liver

To investigate the possibility of HIV1 infection of liver cells, liver samples from 17 infected patients were studied. A monoclonal antibody directed against the p24 gag HIV1 protein was used in an immunoperoxidase assay and yielded positive results in 7/17 samples. Staining by anti-p24 antibody was of three types: diffuse in Kupffer cells of most samples, inside granuloma and in some sinusoidal cells the origin of which was difficult to ascertain. In 5 futher liver specimens, HIV1 RNA were identified by in situ hybridization in inflammatory mononuclear cells and/or in scattered sinusoidal cells. Attempts to locate the CD4 membrane antigen showed that it was mainly present on endothelial sinusoidal cells. These results indicate that liver cells, including Kupffer cells, might be infected by HIV1.     

A new capture test using conjugated peptides for the detection of HIV antibodies.

A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591-611) YLKDQQLLGIWGCSGKLICTT, the gp120 sequence (314-329) IRIQRGPGRAFVTIGK and the p27 sequence (182-198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N-hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indirect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies.     

Screening for antiviral inhibitors of the HIV integrase-LEDGF/p75 interaction using the AlphaScreen luminescent proximity assay

Small-molecule inhibitors of HIV integrase (HIV IN) have emerged as a promising new class of antivirals for the treatment of HIV/AIDS. The compounds currently approved or in clinical development specifically target HIV DNA integration and were identified using strand-transfer assays targeting the HIV IN/viral DNA complex. The authors have developed a second biochemical assay for identification of HIV integrase inhibitors, targeting the interaction between HIV IN and the cellular cofactor LEDGF/p75. They developed a luminescent proximity assay (AlphaScreen) designed to measure the association of the 80-amino-acid integrase binding domain of LEDGF/p75 with the 163-amino-acid catalytic core domain of HIV IN. This assay proved to be quite robust (with a Z' factor of 0.84 in screening libraries arrayed as orthogonal mixtures) and successfully identified several compounds specific for this protein-protein interaction.     

HIV-1 Gag p17-p24在痘苗病毒中的表达及性质研究

研究了重组痘苗病毒表达的HIV1核心蛋白(Gag)p17p24蛋白的一些生物学及免疫学特点。间接免疫荧光、DotELISA及Westernblot结果表明,构建的两株重组病毒分别表达了HIV1Gagp24及p17p24融合蛋白。电镜观察证实,Gagp24及p1724重组蛋白均可形成病毒样粒子。重组病毒可诱导小鼠产生抗HIV1Gagp24抗体。重组病毒感染BHK21细胞后,可见由于细胞凋亡而致的染色体DNA断裂“梯子”电泳图。     

Development of Ss-NIE-1 Recombinant Antigen Based Assays for Immunodiagnosis of Strongyloidiasis

Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG₄ specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG₄. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG₄ ELISA were 95% and 93%, respectively. For the IgG₄ Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG₄ ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.     

Development Of A Nucleic Acid Lateral Flow Strip For Detection Of Hepatitis C Virus (Hcv) Core Antigen

The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10⁴ copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills.     

Enzyme-Linked Immunosorbent Assays for the Specific and Sensitive Quantification of Methanosarcina mazei and Methanobacterium bryantii

Three microtitration plate enzyme-linked immunosorbent assays (ELISAs) have been developed: a competitive ELISA and a two-site (or indirect sandwich) ELISA for Methanosarcina mazei S6 and a two-site ELISA for Methanobacterium bryantii FR-2. The assays were sensitive, with limits of cell protein detection of 3 ng ml⁻¹, 5 ng ml⁻¹, and 50 ng ml⁻¹, respectively, and showed good precision. The M. mazei assays used monoclonal antibodies and were entirely species specific, showing no cross-reaction with methanogens of other genera or with other species of the same genus. The Methanobacterium bryantii assay, which used two polyclonal antisera, showed only a slight cross-reaction with one other Methanobacterium species but no cross-reaction with methanogens of other genera. The use of the ELISAs for quantitative analysis of mixed cultures and of sewage sludge samples was investigated. Sludge diluted at 1:10³ or more caused no significant interference in any of the three ELISAs. Various cultures of bacteria, methanogens, and nonmethanogens at a protein concentration of 50 μg ml⁻¹ showed no significant interference in the M. mazei competitive assay and the Methanobacterium bryantii two-site assay, although they did cause falsely low results in the M. mazei two-site assay.     

R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens.

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.     

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

ABSTRACT: BACKGROUND: HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. METHOD: A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. RESULTS: The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. CONCLUSIONS: When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3--4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.     

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.