Journal: The Journal of Experimental Medicine

Article Title: Determinants of Human Immunodeficiency Virus Type 1 Escape from the Primary CD8 + Cytotoxic T Lymphocyte Response

doi: 10.1084/jem.20040511

Figure Lengend Snippet: Epitopes/Epitope-containing Regions Identified as Being Recognized by the CD8 + T Cell Response during Acute/Early HIV-1 Infection in Subjects BORI, WEAU, and SUMA

Article Snippet: , gp160 , 828- EGT D R IV I EI -836 , gp160 EI9 , ND.

Techniques: Infection

Journal: The Journal of Experimental Medicine

Article Title: Determinants of Human Immunodeficiency Virus Type 1 Escape from the Primary CD8 + Cytotoxic T Lymphocyte Response

doi: 10.1084/jem.20040511

Figure Lengend Snippet: Relative immunodominance and functional avidity of T cell responses to different viral epitopes during acute, subacute, and early HIV-1 infection in subjects BORI, WEAU, and SUMA. The T cell response to each of the gp160, Gag, and Tat epitopes recognized by subjects BORI, WEAU, and SUMA was measured at time points during acute HIV-1 infection (9–16 DFOSx and before detection of HIV-1 antibodies by ELISA or Western immunoblot), subacute infection (3–4 wk FOSx and before full immunoblot seroconversion), and early infection (between 5 and 10 wk FOSx) using IFN-γ ELISPOT assays. The relative immunodominance of the responses to different epitope peptides at each time point is shown, calculated by expressing the number of epitope–peptide-specific IFN-γ–producing cells as a percentage of the overall response detected to all the peptides tested at that time point. The magnitude of individual epitope-specific responses is shown in Table S2. The functional avidity of patient T cell responses to each of the epitopes they recognized was also compared by assessing the ability of polyclonal patient CTL or epitope-specific CTL lines to mediate lysis of autologous EBV-B-LCL target cells pulsed with different concentrations of the epitope peptide in 51 Cr release assays. The values shown represent the peptide concentration required to sensitize target cells for half-maximal CTL lysis.

Article Snippet: , gp160 , 828- EGT D R IV I EI -836 , gp160 EI9 , ND.

Techniques: Functional Assay, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Enzyme-linked Immunospot, Expressing, Lysis, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Determinants of Human Immunodeficiency Virus Type 1 Escape from the Primary CD8 + Cytotoxic T Lymphocyte Response

doi: 10.1084/jem.20040511

Figure Lengend Snippet: Summary of sequence changes detected over time in each subject's plasma virus quasispecies within regions recognized by the CD8 + T cell response. Sequence changes occurring within epitope-containing regions in the in vivo viral quasispecies over time in (a) BORI, (b) WEAU, and (c) SUMA are shown. A panel of molecular clones spanning each epitope-containing region was derived from plasma viral RNA (cDNA) at serial time points in infection (DFOSx), and the sequence of independent clones was determined. Each panel shows the deduced amino acid sequence of clones from a single epitope/epitope-containing region. Where overlapping CD8 T cell epitopes were identified within one region, their relative location is indicated. For each epitope/epitope-containing region, the deduced amino acid sequence of the predominant viral species present at the earliest time point tested (the index sequence) is shown in full. Any variant sequences present at this time point, plus all sequences determined at subsequent time points were compared with the index sequence; variant amino acids are indicated, with a dash (-) denoting amino acid identity. The slash (/) denotes an amino acid deletion, and insertions are indicated by vertical pipes. At each time point, the proportion of clones analyzed that had a given sequence is shown, and the total percentage of clones analyzed with variant sequences is also indicated. Sequence analysis of the WEAU gp160 AY9 epitope at time points from D16 to D136 was reported previously (reference ).

Article Snippet: , gp160 , 828- EGT D R IV I EI -836 , gp160 EI9 , ND.

Techniques: Sequencing, Clinical Proteomics, Virus, In Vivo, Clone Assay, Derivative Assay, Infection, Variant Assay

Journal: The Journal of Experimental Medicine

Article Title: Determinants of Human Immunodeficiency Virus Type 1 Escape from the Primary CD8 + Cytotoxic T Lymphocyte Response

doi: 10.1084/jem.20040511

Figure Lengend Snippet: Extent and kinetics of escape-conferring mutation in epitope-containing regions in Env, Gag, and Tat in subjects BORI, WEAU, and SUMA. Panels a and e; b; and c illustrate (for subjects BORI, WEAU, and SUMA, respectively) the extent and kinetics of accumulation of mutations (with the exception of those shown not to confer CTL escape in ) within epitope-containing regions in gp160, Gag, and Tat (% mutant) at time points analyzed during the first 400 DFOSx. In BORI, the five mutating regions shown constituted five out of seven epitope-containing regions identified in this patient; in WEAU, the 4 mutating epitope-containing regions constituted 4 of 14 regions to which responses were identified, and in SUMA the single mutating epitope-containing region (which contained the Tat FK10/VI10/MY9 epitopes) constituted 1 of 21 regions to which responses were identified. Panels d and f show the relative immunodominance (calculated as described in the legend to ) of the response to the gp160 EL9 epitope in subjects BORI and SUMA respectively. Panels e and g illustrate the kinetics of accumulation of escape-conferring mutations (% mutant) in this epitope within the viral quasispecies in subjects BORI and SUMA, respectively.

Article Snippet: , gp160 , 828- EGT D R IV I EI -836 , gp160 EI9 , ND.

Techniques: Mutagenesis

Journal:

Article Title: The Simian Immunodeficiency Virus Envelope Glycoprotein Contains Two Epitopes Presented by the Mamu-A*01 Class I Molecule

doi:

Figure Lengend Snippet: Identification of gp160 epitopes. PBMC restimulated with gp160 peptide pool 2 (A) and pool 6 (C) were tested for lysis of autologous targets sensitized with three peptide subpools. Individual peptides in each positive subpool were then tested for recognition by pool 2-specific (B) and pool 6-specific (D) CTL. Sequences of the individual peptides are shown in single-letter code.

Article Snippet: Sets of overlapping SIV gp160 and Gag peptides (20 amino acids offset by 10 residues) were synthesized by Chiron Mimotopes (Clayton, Australia).

Techniques: Lysis

Journal:

Article Title: The Simian Immunodeficiency Virus Envelope Glycoprotein Contains Two Epitopes Presented by the Mamu-A*01 Class I Molecule

doi:

Figure Lengend Snippet: Class I MHC restriction of gp160 peptide 54-specific CTL. (A) DNA extracted from B-LCL lines of the indicated macaque was amplified with PCR primers specific for Mamu-A*01 and a conserved region of the class II Mamu-DRB gene. (B) B-LCL from these animals were sensitized with peptide 54 and tested for lysis by an antigen-specific CTL line from animal VW6. The E:T ratio was 40:1 in a 4-h assay.

Article Snippet: Sets of overlapping SIV gp160 and Gag peptides (20 amino acids offset by 10 residues) were synthesized by Chiron Mimotopes (Clayton, Australia).

Techniques: Amplification, Lysis

Journal:

Article Title: The Simian Immunodeficiency Virus Envelope Glycoprotein Contains Two Epitopes Presented by the Mamu-A*01 Class I Molecule

doi:

Figure Lengend Snippet: Lysis of target cells expressing SIV gp160. Peptide 54-specific (A) and peptide 15-specific (B) CTL were tested for lysis of Mamu-A*01-transfected 721.221 cells that were sensitized with the relevant peptide or infected with VVgp160 or VVgag.

Article Snippet: Sets of overlapping SIV gp160 and Gag peptides (20 amino acids offset by 10 residues) were synthesized by Chiron Mimotopes (Clayton, Australia).

Techniques: Lysis, Expressing, Transfection, Infection

Journal:

Article Title: HIV-gp160 modulates differentially the production in vitro of IgG, IgA and cytokines by blood and tonsil B lymphocytes from HIV-negative individuals

doi: 10.1046/j.1365-2249.2003.02152.x

Figure Lengend Snippet: Baseline levels culture conditions of in vitro production of IgA (a) and IgG (b) by unseparated peripheral blood mononuclear cells or tonsil mononuclear cells obtained from HIV − and HIV + individuals. Mononuclear cells B lymphocytes were induced to terminal differentiation by exogenous IL-2 and IL-10 in the presence of gp160 (but in the absence of CD40L signalling as the cells were unseparated)

Article Snippet: The recombinant soluble gp160 used was a chimeric protein composed of gp120 and gp41 derived from MN and LAI strains, respectively (Aventis Pasteur, Marcy l’Etoile, France).

Techniques: In Vitro

Journal:

Article Title: HIV-gp160 modulates differentially the production in vitro of IgG, IgA and cytokines by blood and tonsil B lymphocytes from HIV-negative individuals

doi: 10.1046/j.1365-2249.2003.02152.x

Figure Lengend Snippet: Purified CD19+ B cells from blood (a) and tonsils (b) from HIV−, non-exposed individuals were stimulated with anti-µ antibodies, IL-2, IL-10 and CD40 signalling in the presence (w) or the absence (w/o) of recombinant glycosylated (top two panels) and non-glycosylated (bottom two panels) gp160. Shown is the differential production of IgG and IgA. IgG and IgA production is expressed as log10 protein level (ng/ml) deduced from the background control. The asterisk (*) represents statistical difference (P < 0·05). (a, b) Top: ▪, w/o gp160; □, w gp160. (a, b) Bottom: ▪, w/o gp160 E. coli; □, w gp160 E. coli.

Article Snippet: The recombinant soluble gp160 used was a chimeric protein composed of gp120 and gp41 derived from MN and LAI strains, respectively (Aventis Pasteur, Marcy l’Etoile, France).

Techniques: Purification, Recombinant, Control

Journal:

Article Title: HIV-gp160 modulates differentially the production in vitro of IgG, IgA and cytokines by blood and tonsil B lymphocytes from HIV-negative individuals

doi: 10.1046/j.1365-2249.2003.02152.x

Figure Lengend Snippet: Purified CD19+ B cells from blood (left panels) and tonsils (right panels) from HIV−, non-exposed individuals were stimulated with anti-µ antibodies, IL-2, IL-10, and CD40 signalling in the presence (w) or the absence (w/o) of recombinant gp160. B cells were then labelled with FITC-conjugated anti-sIg antibodies (and counterlabelled with a RPE-Cy5-conjugated anti-CD20 MoAb for gating). Shown is the result of flow cytometry analyses of the percentage of sIg+ B cells before and after exposure of starting B cell populations after 45 min incubation with gp160. HIV1-gp160 was the Aventis-Pasteur glycosylated vaccine candidate. ▪, w/o gp160; □, w gp160.

Article Snippet: The recombinant soluble gp160 used was a chimeric protein composed of gp120 and gp41 derived from MN and LAI strains, respectively (Aventis Pasteur, Marcy l’Etoile, France).

Techniques: Purification, Recombinant, Flow Cytometry, Incubation

Journal:

Article Title: HIV-gp160 modulates differentially the production in vitro of IgG, IgA and cytokines by blood and tonsil B lymphocytes from HIV-negative individuals

doi: 10.1046/j.1365-2249.2003.02152.x

Figure Lengend Snippet: Purified CD19+ B cells from blood (a) and tonsils (b) from HIV−, non-exposed individuals were stimulated with anti-µ antibodies, IL-2, IL-10 and CD40 signalling in the presence (w) or the absence (w/o) of recombinant gp160. Shown is the differential production of various cytokine mRNA. mRNA was semiquantified by means of RT-PCR (lane 1: anti-µ + IL-2 + IL-10 + sCD40L; lane 2: anti-µ + IL-2 + IL-10 + sCD40L + gp160; lane 3: negative control; lane 4: positive control).

Article Snippet: The recombinant soluble gp160 used was a chimeric protein composed of gp120 and gp41 derived from MN and LAI strains, respectively (Aventis Pasteur, Marcy l’Etoile, France).

Techniques: Purification, Recombinant, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control

Journal:

Article Title: Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization

doi: 10.1128/JVI.76.11.5357-5368.2002

Figure Lengend Snippet: Schematic representation of functional domains and mutations in HIV-1 Env glycoproteins. Full-length envelope polyprotein, gp160, with the indicated features based on the amino acid residues of HXB2 is shown (top). Functional domains include the gp120/gp41 cleavage site (residues 510 and 511), the fusion domain (residues 512 to 527), the two heptad repeats (residues 546 to 579 and residues 628 to 655), the transmembrane domain (residues 684 to 705), and the cytoplasmic domain (residues 706 to 856). The mutant forms of the envelope proteins are shown below the structure of gp160. COOH deletions were introduced that terminate the envelope protein at positions 752, 704, or 680 to produce gp150, gp145, or gp140, respectively. Two internal deletions that removed the cleavage site, the fusion domain, and the region between the two heptad repeats were introduced into gp160, gp150, gp145, and gp140. A further deletion in the COOH-terminal region at position 592 removed the second heptad repeat and the transmembrane domain to produce gp128ΔCFI. To disrupt potential glycosylation sites, asparagine (N) residues at 11 positions (88, 156, 160, 197, 230, 234, 241, 262, 276, 289, and 295) were replaced with aspartic acid (D) residues in both gp160 and gp150. Versions of both gp160 and gp150 were created with a total of 17 mutated glycosylation sites by including six additional N-to-D substitutions at positions 332, 339, 356, 386, 392, and 448.

Article Snippet: Competitive inhibition of binding with sCD4 was assessed by serial dilution of sCD4 (1:3, 1:9, 1:27) from 500 ng/well compared to no sCD4 in a 96-well plate that was previously coated with CD4, followed by incubation with either gp140ΔCFI or r-gp160 (Intracel).

Techniques: Functional Assay, Mutagenesis

Journal:

Article Title: Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization

doi: 10.1128/JVI.76.11.5357-5368.2002

Figure Lengend Snippet: Comparison of the expression of the HIV-1 gp160 with codon-optimized gp160. (A and B) Expression of plasmids encoding Rev-dependent and Rev-independent codon-modified gp160 (lanes 1 and 2). (A) The upper panel shows expression of Rev-dependent viral gp160 (left) and codon-modified gp160 (right) in transfected 293 cells. The lower panel shows comparable expression of β-actin in these transfected cells. (B) Processing of gp160 was detected with a monoclonal antibody to gp41with viral or codon-altered gp160, as indicated (lanes 3 to 9). (C) Expression of mutant CXCR4-tropic HIV Env glycoproteins with COOH-terminal truncations is shown. (D and E) CXCR4-tropic envelope proteins containing mutant glycosylation sites and mutant functional domains are shown. The indicated proteins were detected by immunoblotting as described above. Cell lysates produced by transfection with vector containing no insert were used as controls (vector, first lane in each panel).

Article Snippet: Competitive inhibition of binding with sCD4 was assessed by serial dilution of sCD4 (1:3, 1:9, 1:27) from 500 ng/well compared to no sCD4 in a 96-well plate that was previously coated with CD4, followed by incubation with either gp140ΔCFI or r-gp160 (Intracel).

Techniques: Expressing, Modification, Transfection, Mutagenesis, Functional Assay, Western Blot, Produced, Plasmid Preparation

Journal:

Article Title: Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization

doi: 10.1128/JVI.76.11.5357-5368.2002

Figure Lengend Snippet: Cytotoxicity of full-length gp160 is eliminated by deletion of the COOH-terminal cytoplasmic domain. Cell rounding and detachment were not observed in control-transfected 293 cells (A), in contrast to full-length gp160 (B), and were observed to a lesser extent in cells transfected with gp150 (C), in contrast to gp145 (D) or gp140 (E).

Article Snippet: Competitive inhibition of binding with sCD4 was assessed by serial dilution of sCD4 (1:3, 1:9, 1:27) from 500 ng/well compared to no sCD4 in a 96-well plate that was previously coated with CD4, followed by incubation with either gp140ΔCFI or r-gp160 (Intracel).

Techniques: Transfection

Journal:

Article Title: Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization

doi: 10.1128/JVI.76.11.5357-5368.2002

Figure Lengend Snippet: Interaction of gp140ΔCFI with defined monoclonal antibodies or CD4 and biochemical analysis for oligomerization of gp140ΔCFI. (A) Analysis of the antigenic structure of soluble gp140ΔCFI with monoclonal antibodies. The envelope glycoproteins from the supernatants of the 293 cells transfected with the vector expressing gp140ΔCFI were immunoprecipitated with either 5 μg of monoclonal antibodies 2F5, 2G12, F105, and b12 or with 5 μg of HIV-1 Ig. The proteins were analyzed by SDS-PAGE and detected by Western blotting using the polyclonal antibodies against gp160. The arrow indicates the position of gp140ΔCFI. A nonspecific (ns) band that cross-reacted with the antibody is indicated. (B) Quantification of 2F5 and 2G12 binding to gp140ΔCFI using the indicated concentrations of each antibody as shown compared to a nonreactive Ig isotype (control). The intensity of the gp140ΔCFI band was determined by quantitative phosphorimaging. The arrow indicates the position of gp140ΔCFI. (C) Interaction of soluble gp140ΔCFI protein with CD4. Binding of gp140ΔCFI and gp160, compared to that of controls transfected with vector alone, in an ELISA with CD4 is shown (left panel). The values represent the mean and standard deviation (error bars) for each point. The ability of sCD4 to compete with binding to these envelopes is shown (right panel). (D) Biochemical analysis of soluble gp140ΔCFI oligomerization. Western blot analysis to detect the gp140ΔCFI in different fractions, fractions 1 to 10, after sucrose density gradient. Fraction 1 represents the greatest density, and fraction 10 represents the least density. Equal volumes of the samples from each fraction were analyzed in an SDS-polyacrylamide gel under nonreducing conditions, except the molecular weight marker. The proteins were detected by Western blotting using polyclonal antibody against gp160. The positions of dimer, trimer, and aggregates are shown. The lower panel shows the presence of monomer in these fractions run under reducing conditions. (E) Molecular-exclusion chromatography of soluble of gp140ΔCFI. Membrane-free supernatant containing gp140ΔCFI was analyzed on a Superdex 200 column and compared with a mixture of molecular weight standards; the position of each marker is indicated by arrows. Fractions were analyzed for gp140ΔCFI by immunoprecipitation followed by Western blotting and were quantitated by densitometry.

Article Snippet: Competitive inhibition of binding with sCD4 was assessed by serial dilution of sCD4 (1:3, 1:9, 1:27) from 500 ng/well compared to no sCD4 in a 96-well plate that was previously coated with CD4, followed by incubation with either gp140ΔCFI or r-gp160 (Intracel).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Molecular Weight, Marker, Chromatography

Journal:

Article Title: Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization

doi: 10.1128/JVI.76.11.5357-5368.2002

Figure Lengend Snippet: Antibody response against HIV-1 envelope proteins in DNA immunized mice. (A) Comparison of the antibody response in mice immunized with gp140ΔCFI or other Env plasmid expression vectors. Sera were collected 2 weeks after the last immunization and used to immunoprecipitate codon-altered gp160 from lysates of transfected 293 cells as described before. The quantitation of the immunoprecipitated gp160 was done as described for panel B. The average of the normalized data has been presented as a bar diagram. Error bars are indicated. (B) Antibody responses in mice immunized with gp140ΔCFI or gp128ΔCFI relative to a V3-specific monoclonal antibody standard (monoclonal antibody 1727). Antisera from immunized mice were diluted in immunoprecipitation buffer, and 1 μl of each diluted serum was used to immunoprecipitate codon-altered HIV-1 gp160 from lysates of transfected 293 cells as described in the legend to Fig. ​Fig.3A.3A. The gels were scanned, and the intensity of the gp160 band was determined by densitometry using the program ImageQuant and presented relative to the intensity of gp160 immunoprecipitated with positive control sera (rabbit anti-gp160), which was used to normalize data between experiments. These data are presented graphically to facilitate comparison among groups. Monoclonal antibody 1727 interacts with the V3 loop of HIV IIIb and was kindly provided by the NIH AIDS Research and Reference Reagent Program, from Jon Laman. (C) Antibody responses in mice immunized with gp140 or gp140ΔCFI were determined by immunoprecipitation and Western blotting. Animals received two booster doses (100 μg) of the same plasmid, 2 weeks apart. Sera (1 μl) collected 2 weeks after the last immunization were used to immunoprecipitate codon-optimized HIV-1 gp160 from lysates of transfected 293 cells containing 400 μg of total protein. Each lane corresponds to the serum from an animal immunized with either the control vector (lanes 1 and 2), CXCR4-tropic gp140 (lanes 3 to 6), or plasmid that expresses gp140 with the indicated mutant functional domains (lanes 7 to 10). A mouse monoclonal antibody to gp160 (HIV-1 V3 monoclonal [IIIB-V3-13]; NIH AIDS Research and Reference Reagent Program) was used as a positive control (lane 11).

Article Snippet: Competitive inhibition of binding with sCD4 was assessed by serial dilution of sCD4 (1:3, 1:9, 1:27) from 500 ng/well compared to no sCD4 in a 96-well plate that was previously coated with CD4, followed by incubation with either gp140ΔCFI or r-gp160 (Intracel).

Techniques: Plasmid Preparation, Expressing, Transfection, Quantitation Assay, Immunoprecipitation, Positive Control, Western Blot, Mutagenesis, Functional Assay

Journal:

Article Title: Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization

doi: 10.1128/JVI.76.11.5357-5368.2002

Figure Lengend Snippet: CTL response against HIV-1 envelope proteins in DNA-immunized mice and generation of a neutralizing antibody response in guinea pigs. (A) The CTL response to CXCR4-tropic Env and indicated deletion mutants is shown. (B and C) The CTL responses to CXCR4-tropic envelope with glycosylation site and ΔCFI mutations are shown, respectively. Spleen cells were isolated from immunized mice 2 weeks after the final immunization and stimulated in vitro with irradiated cells expressing gp160 with addition of human interleukin 2 (5 U/ml) at day 4. The cytolytic activity of the restimulated spleen cells was tested after 7 days against V3 peptide-pulsed BC10ME cells. Similar findings were observed with target cells that stably express full-length Env (data not shown). (D) Preimmune sera were collected from four guinea pigs prior to immunization or after DNA priming and ADV boosting with gp140ΔCFI as described in the text. Both preimmune sera and postimmune sera were diluted, and neutralizing activity was measured by reduction of HIV-IIIB virus compared to the untreated control. Neutralizing antibody titers were analyzed as previously described (26). The data represent the dilutions at which the sera can neutralize the virus in the MT2 assay. Standard deviations are indicated.

Article Snippet: Competitive inhibition of binding with sCD4 was assessed by serial dilution of sCD4 (1:3, 1:9, 1:27) from 500 ng/well compared to no sCD4 in a 96-well plate that was previously coated with CD4, followed by incubation with either gp140ΔCFI or r-gp160 (Intracel).

Techniques: Isolation, In Vitro, Irradiation, Expressing, Activity Assay, Stable Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antibody detection by agglutination–PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis

doi: 10.1073/pnas.1711004115

Figure Lengend Snippet: Principle scheme of antibody detection by ADAP for HIV diagnosis. (A) HIV virus contains many immunogenic proteins, including viral capsid protein p24 (blue) and envelope glycoprotein gp160, which can be cleaved into gp41 (brown) and gp120 (green). (B) Recombinant viral proteins are activated by installation of maleimides onto lysine residues via the small-molecule cross-linking agent sulfo-SMCC. Thiol-functionalized DNA covalently ligates to these maleimides by Michael addition to form protein–DNA conjugates. (C) Upon incubation with antibody-containing samples, antibodies and conjugates form immune complexes, allowing nearby DNA to be ligated into a full-length amplicon upon addition of a universal bridge oligonucleotide and DNA ligase. Each amplicon bears unique primer binding sites for independent amplification and quantification by real-time qPCR. Critically, DNA conjugates alone without ligation bear only one primer binding site and are therefore PCR-incompetent. Only successful ligation into a full-length amplicon enables exponential amplification by PCR. This “turn-on” mechanism allows ADAP to leverage PCR’s analytical sensitivity while preserving assay specificity.

Article Snippet: To demonstrate that viral antigen–DNA conjugates were capable of detecting their cognate antibodies, we obtained a panel of highly purified human antibodies against p24, gp41, and gp160 derived from HIV patients (Immunodx).

Techniques: Recombinant, Incubation, Amplification, Binding Assay, Ligation, Preserving

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antibody detection by agglutination–PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis

doi: 10.1073/pnas.1711004115

Figure Lengend Snippet: Singleplex ADAP analysis of OF samples. (A) OF samples from HIV-negative (n = 22) and HIV-positive (n = 22) patients were analyzed by ADAP. Using cutoff values established from HIV-negative samples, HIV-positive samples showed 91% positivity for p24 and 100% for gp41, gp120, and gp160. (B) Cumulative signal from all anti-HIV antibodies. All 22 HIV-positive samples showed a higher signal than 22 HIV-negative samples. By defining positivity as the presence of two or more HIV antibodies, singleplex ADAP analysis yields 100% clinical sensitivity and specificity in comparison with the clinical gold-standard EIA. (C) The cumulative signal intensities of ADAP correlated well with the signal intensities of EIA (R = 0.80, P < 0.05).

Article Snippet: To demonstrate that viral antigen–DNA conjugates were capable of detecting their cognate antibodies, we obtained a panel of highly purified human antibodies against p24, gp41, and gp160 derived from HIV patients (Immunodx).

Techniques: