Detection of antibodies to Epstein-Barr virus antigens by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) is described for the serologic analysis of antibodies to Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA), viral capsid antigen (VCA), and early antigen (EA). The specificity of each of the ELISAs was demonstrated by the use of well-characterized human sera shown by immunofluorescence assay to be variously reactive for antibodies to one or more of the three viral antigens studied. The ELISA for EBNA was four to 256 times more sensitive than immunofluorescence assays with all 33 EBNA-positive sera tested. The ELISAs for VCA and EA were also more sensitive than immunofluorescence assays: approximately 50% of the sera tested showed higher antibody titers. Sera that were negative for all three antigens by immunofluorescence assay were also negative by ELISA for each antigen. These ELISAs for EBV are rapid, sensitive, and objective and thus provide new and valuable methods for the detection of antibodies to EBV-related antigens.     

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1.

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.     

Evaluation of a near-patient test and 2 enzyme-linked immunosorbent assay-based assays for detecting anti-herpes simplex virus type-2 antibodies.

Several type-specific serologic assays for herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), based on glycoprotein G1 (gG1) and gG2, have recently been developed. These include immunodot (POCkit HSV-2) and enzyme-linked immunosorbent assay (ELISA). The diagnostic value of POCkit HSV-2, a near-patient test, and of 2 immunoenzymatic, type-specific assays was evaluated on 122 patients attending an STD clinic. Western blot was used as the reference test. The sensitivity of POCkit HSV-2 was good but the specificity was poor, so that in a population with low seroprevalence, a positive result is likely to be a false positive. Analysis of 2 currently available HSV type-specific ELISAs yielded results suggesting that the sensitivity of these tests may also be suboptimal.     

Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41.

The human immunodeficiency virus transmembrane glycoprotein gp41 has at its amino terminus a strongly hydrophobic stretch of 28 amino acids flanked by a highly conserved series of polar amino acids. To investigate the role in syncytium formation of the hydrophobic amino terminus of gp41 and the polar border of this hydrophobic region, we introduced eight single-amino acid substitutions and one double-amino acid substitution in the amino-terminal 31 amino acids of gp41. The mutant envelope glycoproteins were expressed from two distinct human immunodeficiency virus type 1 envelope glycoprotein expression vectors; the effects of the mutations on syncytium formation, envelope glycoprotein transport, secretion, and CD4 receptor-binding were analyzed. Results showed that polar substitutions throughout the hydrophobic amino terminus of gp41 greatly reduced or blocked syncytium formation mediated by the human immunodeficiency virus type 1 envelope glycoproteins, as did nonconservative mutations in the polar border of the hydrophobic amino terminus. Mutations at gp41 amino acids 15, 26, and 29 also significantly increased the extent of gp120 secretion into the extracellular medium. None of the mutations detectably affected envelope glycoprotein processing or envelope glycoprotein binding to CD4.     

Immunofluorescence, enzyme-linked immunosorbent assay, particle agglutination and western blot for the detection of antibody to human immunodeficiency virus type 1

Immunofluorescence assay (IFA) has been applied for detection of antibody to human immunodeficiency virus type 1 (HIV-1). To compare the IFA with an enzyme-linked immunosorbent assay (ELISA) and particle agglutination (PA), we examined the antibody response to HIV-1 in 475 sera from AIDS, PGL and ARC patients as well as several risk groups and healthy persons by three methods. The positive results by any methods were confirmed by western blot (WB). The results by all methods were well correlated on the sera from 45 asymptomatic male homosexuals and 70 female prostitutes. There were some false positive results by ELISA in the sera from prisoners and healthy persons. Four sera from drug abusers were positive only by PA and IFA and were negative by ELISA. All were WB-inconclusive. Particle agglutination and IFA results were compared with western blot analysis on 208 ELISA-positive sera. All IFA-strongly positive sera (84%) were positive by western blot. The sera with weakly positive, negative and inconclusive results by IFA (16%) were possibly any of positive, inconclusive or negative by western blot. By PA, 200 of 208 (97%) sera were PA-positive and 1% of these sera were WB-inconclusive while the PA-negative sera were either negative or inconclusive by western blot. These results suggested that PA is a simple and sensitive method for screening of HIV-1 antibody while IFA could be a primary confirmatory test and western blot would then be used for confirming any IFA-negative or inconclusive results.     

False positivity of enzyme-linked immunosorbent assay for measurement of secretory IgA antibodies directed at HIV type 1 antigens

We have determined that polymeric IgA in saliva of HIV-1-uninfected individuals binds in varying degrees to components of culture supernatants containing HIV-1 recombinant proteins when ELISA is used for the determination. This finding did not extend to salivary IgG antibodies. Further, such problems were not encountered in Western blot. Binding did not appear to be mediated by salivary proteins known to bind to IgA, including secretory component, amylase, lactoferrin, lysozyme, galactosyl transferase, or secretory leukocyte protease inhibitor, and was not influenced by blocking reagents or by changes in secondary anti-IgA antibodies. Although these findings will not likely impact on the use of saliva as a diagnostic fluid for HIV-1 infection (the HIV-1 response in saliva is mostly of the IgG isotype), they indicate that assessments of this secretion as an indicator of IgA mucosal immune responses to HIV-1 vaccines should be undertaken with caution.     

Correlation of enzyme-linked immunosorbent assays for serum human immunodeficiency virus antigen and antibodies to recombinant viral proteins with subsequent clinical outcomes in a cohort of asymptomatic homosexual men

A cohort of asymptomatic homosexual men at a Boston community health center was screened for the presence of human immunodeficiency virus (HIV) serum antigen and antibodies to recombinant proteins containing portions of the envelope and the gag (core) gene products. Of 196 asymptomatic men screened, 149 were antigen-negative/antibody-negative, 41 were antigen-negative/antibody-positive, and six were antigen-positive/antibody-positive. All three men in whom the acquired immune deficiency syndrome (AIDS) developed over the next year were antigen-positive at enrollment. Although a larger portion of the men who were antigen-positive and did not demonstrate progression to AIDS after one year had thrush, zoster, or generalized lymphadenopathy, the associations were not statistically significant. Whereas all of the seropositive men had antibody to viral envelope antigens, about a quarter did not have detectable antibodies to recombinant core antigens. However, all of these men had detectable antibody to core antigens by Western blot. Titers to recombinant core and envelope antigens tended to be lower in the men with AIDS. HIV-infected persons who are more likely to have enhanced immuno-compromise may be identified by these newer tests, but further longitudinal studies will be necessary to fully understand their prognostic value.     

Antibody epitopes sensitive to the state of human immunodeficiency virus type 1 gp41 oligomerization map to a putative alpha-helical region.

Two antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic peptides in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gp160 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gp160 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic alpha-helix.     

Serial enzyme-linked immunosorbent assays for antibody to Candida antigens during induction chemotherapy for acute leukemia

Two or more sera were collected from 68 adult patients who received a total of 100 courses of induction chemotherapy for acute leukemia. Sera were tested by ELISAs for antibody to candidal mannan and a major cytoplasmic antigen. Twenty episodes of induction chemotherapy were accompanied by invasive candidiasis of skin, esophagus, or deeper organs. Levels of antibody to mannan rose more than 2.75 times normal activity in nine of these episodes and were already above this level in all sera in two episodes. Levels of antibody to mannan exceeded 2.75 times normal in only two of the remaining episodes in which invasive candidiasis was not observed. An eightfold increase in antibody to mannan occurred at some point in five of the 20 episodes complicated by invasive candidiasis but in only one of the remaining 80 episodes. Antibody to the major cytoplasmic antigen was detected infrequently in sera from episodes complicated by invasive candidiasis. Further studies of precise serial measurements of antibody to mannan therefore appear warranted.     

Comparative performances of serologic and molecular assays for detecting human T lymphotropic virus type 1 and type 2 (HTLV-1 and HTLV-2) in patients infected with human immunodeficiency virus type 1 (HIV-1)

The present study evaluated several techniques currently available (commercial kits and in-house assays) for diagnosing human T lymphotropic viruses types 1 and 2 in two groups of patients enrolled at HIV/AIDS specialized care services in São Paulo: Group 1 (G1), n = 1608, 1237 male/371 female, median age 44.3 years old, majority using highly active antiretroviral therapy (HAART); G2, n = 1383, 930 male/453 female, median age of 35.6 years old, majority HAART naïve. Enzyme immunoassays [(EIA) Murex and Gold ELISA] were employed for human T lymphotropic viruses types 1 and 2 screening; Western blotting (WB), INNO-LIA (LIA), real-time PCR pol (qPCR), and nested-PCR-RFLP (tax) were used to confirm infection. Samples were considered human T lymphotropic viruses types 1 and 2 positive when there was reactivity using at least one of the four confirmatory assays. By serological screening, 127/2991 samples were positive or borderline, and human T lymphotropic virus infection was confirmed in 108 samples (three EIA-borderline): 56 human T lymphotropic virus type 1 [G1 (27) + G2 (29)]; 45 human T lymphotropic virus type 2 [G1 (21) + G2 (24)]; one human T lymphotropic virus type 1 + human T lymphotropic virus type 2 (G2); six human T lymphotropic virus [G1 (2) + G2 (4)]. Although there were differences in group characteristics, human T lymphotropic viruses types 1 and 2 prevalence was similar [3.1% (G1) and 4.2% (G2), p = 0.113]. The overall sensitivities of LIA, WB, qPCR, and PCR-RFLP were 97.2%, 82.4%, 68.9%, and 68.4%, respectively, with some differences among groups, likely due to the stage of human T lymphotropic virus infection and/or HAART duration. Indeterminate immunoblotting results were detected in G2, possibly due to the seroconversion period. Negative results in molecular assays could be explained by the use of HAART, the occurrence of defective provirus and/or the low circulating proviral load. In conclusion, when determining the human T lymphotropic virus infection, the findings highlight that there is a need to consider the blood samples with borderline results in screening assays. Of all the tested assays, LIA was the assay of choice for detecting human T lymphotropic virus type 1 and human T lymphotropic virus type 2 in human immunodeficiency virus type 1-infected patients.     

International collaborative study to compare assays for antibodies that neutralize human immunodeficiency virus

A variety of techniques are currently in use to measure antibodies that neutralize human immunodeficiency virus (HIV), and standardization of these assays is needed. Eleven laboratories participated in this comparison study of 14 methods for detection of HIV neutralizing antibodies (NA). A panel of 10 coded sera and a positive and a negative control serum were tested in each assay. The 10 coded samples included aliquots of the same sera that were distributed as positive and negative control sera, an aliquot of the WHO reference human anti-HIV-1 serum, and seven other sera from people with HIV-1 infection. Each laboratory reported features of its test methods and results. The results demonstrated excellent within laboratory and between laboratory consistency. The most important variable appeared to be the virus strain used. Cell line, conditions of neutralization or culture, method of endpoint determination, or use of VSV pseudotypes did not appear to be important variables. The results indicate that standardization of HIV-1 NA assays should be readily achievable.     

A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1.

We have established a panel of human monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1). The antibodies 2F5 and 2G12 have been identified to be two of the most potently in vitro neutralizing antibodies against HIV-1. Here we report on a further antibody, 4E10, of similar in vitro neutralizing potency. 4E10 binds to a novel epitope C terminal of the ELDKWA sequence recognized by 2F5, which has been so far the only described broadly neutralizing anti-gp41 antibody. Both 4E10 and 2F5 bind only weakly to infected cells compared with gp120-specific 2G12 and polyclonal anti-HIV-1 immunoglobulin (HIVIG), but show potent in vitro neutralizing properties. 4E10 neutralizes potently not only tissue culture-adapted strains but also primary isolates of different clades, including A, B, C, D, and E. Viruses that were found to be resistant to 2F5 were neutralized by 4E10 and vice versa; none of the tested isolates was resistant to both anti-gp41 antibodies. This confirms that the region recognized by 2F5 and 4E10 is essential for viral infectivity and may be important for vaccine design. Moreover, our results suggest that 4E10 should be further investigated for passive anti-HIV immunotherapy.     

HIV抗体诊断试剂的临床质量评估

目的掌握国内市售艾滋病病毒（HIV）抗体诊断试剂的实际质量情况,为选用质量优良的HIV抗体诊断试剂提供依据。方法用5种酶联免疫吸附试验（ELISA）（双抗原夹心法）（A、B、C、D、E）试剂和3种快速试剂（胶体金法、乳胶层析法）（F、G、H）,分别对各自的血清盘（包括：阳性样品、阴性样品和现场样品）检测抗体,并对各参评试剂的性能指标进行分析。结果国家参考品评估结果：ELISA试剂的功效率为89.58%～100.00%,试剂D的敏感性只有85.71%,试剂E的功效率为100.00%。快速试剂的功效率为93.33%～95.56%。自备样品评估结果：ELISA试剂的功效率为98.19%～99.55%,其中试剂D敏感性和功效率最低,分别为96.83%和98.19%。快速试剂的功效率为92.44%～97.33%。结论除了试剂E没出现假阴性、试剂H没出现假阳性结果外,其他试剂均不同程度地出现了假阳性和假阴性共同存在的现象。试剂质量参差不齐,假阳性或假阴性会导致资源浪费或HIV的传播,国产试剂质量有待进一步提高。     

A new solid-phase enzyme-linked immunosorbent assay for specific antibodies to measles virus

A convenient and flexible enzyme-linked immunosorbent assay system for the detection of specific antibodies to measles virus has been developed. In this system infected cells are desiccated on 96-well microtiter plates and stored at room temperature. After incubation of samples to be tested in the cell-coated plates and subsequent washing, bound antibodies are detected with a peroxidase-conjugated staphylococcal protein A probe. After another washing and the addition of the appropriate substrate, the amount of bound probe is estimated by colorimetric analysis. This technique offers several advantages. The need for a purified viral antigen source is obviated. The plates are easily prepared and can be stored for months at room temperature. Major viral epitopes, including surface glycoproteins as well as cytoplasmic viral antigens, are preserved despite desiccation. The method is more sensitive than the conventional means of virus-specific antibody detection.     

Sequence similarities between human immunodeficiency virus gp41 and paramyxovirus fusion proteins

Cell fusion is a characteristic cytopathic effect induced by the human immunodeficiency virus (HIV) that leads to the formation of syncytia between infected lymphocytes. Although this process has been shown to occur following the specific binding of the 110-120 kD externalized envelope molecule of the virus with the CD4 glycoprotein, the region of the HIV envelope that directly mediates cell fusion is unknown. In an attempt to identify this fusion domain, we compared the amino acid sequences from the envelope molecules of several HIV isolates to the fusion proteins of paramyxoviruses. We found that the amino terminal region of the HIV transmembrane protein gp41 had a striking degree of similarity with the fusion domain of the respiratory syncytial virus. Moreover, similar sequences were noted in the fusion proteins of other paramyxoviruses and the transmembrane envelope proteins of a variety of lentiviruses suggesting that a functional relationship exists between these glycoproteins. This finding indicates that the amino terminal region of the HIV gp41 molecule may mediate cell fusion for this virus, and could be an important target in the design of immunologic strategies for the prevention of HIV infection in vivo.     

A mutation in the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 dominantly interferes with fusion and infectivity.

Several domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been identified that are involved in HIV-1-mediated membrane fusion. One domain that is involved in membrane fusion is the hydrophobic amino terminus of the HIV-1 transmembrane glycoprotein gp41. Here we show that a polar substitution at gp41 amino acid 2 (the 41.2 mutation) results in an envelope glycoprotein that dominantly interferes with both syncytium formation and infection mediated by the wild-type HIV-1 envelope glycoprotein. The interference by the 41.2 mutant is not a result of aberrant envelope glycoprotein synthesis, processing, or transport. The 41.2 mutant elicits a dominant interfering effect even in the presence of excess wild-type glycoprotein, suggesting that a higher-order envelope glycoprotein complex is involved in membrane fusion. These results shed light on the process by which the HIV-1 envelope glycoproteins induce membrane fusion reactions and present a possible approach to anti-HIV therapy.     

Herpes simplex virus type 1 and human immunodeficiency virus type 1 antigens in platelets from a hemophilia B patient with human immunodeficiency virus type 1-related thrombocytopenia.

We analyzed platelet-associated antigens from a hemophilia B patient with human immunodeficiency virus type 1 (HIV-1)-related thrombocytopenia. Two bands appeared at 31,000 and 37,000 daltons in the platelet lysate after reaction with autologous serum in SDS-PAGE and Western blots. The band at 37,000 daltons was obtained using anti-herpes simplex type 1 (HSV-1) rabbit antiserum. Doublet bands at 36,000 and 37,000 daltons also appeared after reaction with HSV-1 seropositive human serum. The band at 31,000 daltons appeared after reaction with anti-HIV-1 rabbit serum. These results suggest that the platelet-associated antigens in this patient are components of both HSV-1 and HIV-1 antigens. In addition, acyclovir decreased his PAIgG level and increased his platelet count, and zidovudine increased his platelet count. Thus, we concluded that each of the platelet-associated antigens is partially responsible for the thrombocytopenia by causing deposition of immune complexes in this patient.