Comparison of the avidity index method and the serologic testing algorithm for recent human immunodeficiency virus (HIV) seroconversion, two methods using a single serum sample for identification of recent HIV infections

A study was designed to compare an avidity index method to the serologic testing algorithm for recent human immunodeficiency virus (HIV) seroconversion (STARHS) for the detection of recent HIV infection. One hundred sixty HIV-positive sera were tested. Both techniques performed similarly in identifying recent infections, although STARHS tended to misclassify more individuals that had long-standing infection as being recently infected.     

Evaluation of immunoglobulin M and G capture enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue infections.

BACKGROUND: Serological assays are widely used to confirm dengue virus infections and to differentiate between a primary and a secondary infection. OBJECTIVE: Two commercial dengue diagnostic kits, Panbio Dengue IgM Capture and Dengue IgG Capture ELISA (Brisbane, Australia) were evaluated. STUDY DESIGN: Three hundred and seventy-three serum samples were tested. Panel sera included samples from dengue confirmed cases (representing both primary and secondary infections), from non-dengue infectious diseases, and from healthy individuals. The MAC-ELISA/Dengue IPK was used for the detection of anti-dengue virus IgM antibody in the sera and the ELISA inhibition method (EIM/Dengue IPK) was used to differentiate between primary and secondary infections. Both these reference assays, which were previously developed in the Arbovirus Laboratory at the "Pedro Kouri" Tropical Medicine Institute, were employed as the gold standard. RESULTS: High sensitivity (96.8%) and specificity (99.4%) were found with the commercial diagnostics when compared to the reference methods. Furthermore, high concordance 95.5% in classifying dengue infection types (primary or secondary infections) was observed. CONCLUSIONS: The Panbio Dengue IgM and IgG assays offer a good alternative for dengue diagnosis. They are easy to perform and results can be obtained in less than 3h.     

Detection of nephropathia epidemica (Puumala virus)-specific immunoglobulin M by enzyme-linked immunosorbent assay.

Nephropathia epidemica (NE), a less severe form of hemorrhagic fever with renal syndrome, is caused by Puumala virus (PUU). This communication reports the development of a mu-capture enzyme-linked immunosorbent assay (ELISA) for detection of specific immunoglobulin M (IgM) antibodies to PUU virus in human sera. Acute- and early-convalescent-phase sera (collected 1 to 41 days after disease onset) from 29 Swedish patients with clinical NE were tested for PUU virus-specific IgG and IgM antibodies by the indirect immunofluorescence test and ELISA, respectively. Late-convalescent-phase serum was also collected from 18 of these patients 3 to 24 months postinfection and assayed. The IgM ELISA values were strongly positive in sera collected during the first 2 months; at 3 to 9 months, they were negative or in the lower range of significance, and at 24 months all sera were negative. Paired sera from NE patients often fail to show seroconversion or a significant titer rise when tested by indirect immunofluorescence. Since all acute- and early-convalescent-phase sera were positive by IgM ELISA, this test could become an important tool for early diagnosis of acute human NE infections.     

Development of quality control procedures for the human immunodeficiency virus type 1 antibody enzyme-linked immunosorbent assay.

A standardized pool of human sera that was positive for human immunodeficiency virus type 1 (HIV-1) antibody was developed. This positive control serum was used to analyze test differences among eight laboratories, among the HIV-1 antibody test kits of three different manufacturers, among different lots of the same test kit, and among pipetting devices and techniques. The standardized pool of human sera was tested 327 times by the different laboratories. In terms of positive tests, a reproducibility of 99.69% was achieved; however, significant test variance among laboratories, among test kit lots, and among pipetting devices and techniques could be demonstrated if the tests were compared on the basis of the net positive optical density (OD) value. This value was calculated by subtracting the cutoff OD value (i.e., the value below which an OD value was considered negative for HIV-1 antibody) from the observed OD value of the standardized pool of human sera. The results obtained suggest that this strategy can be used for proficiency testing, for monitoring the quality of HIV-1 antibody enzyme-linked immunosorbent assay reagents, and for evaluating pipetting devices and techniques.     

Evaluation of a West Nile virus immunoglobulin A capture enzyme-linked immunosorbent assay

An in-house-developed enzyme-linked immunosorbent assay detected West Nile virus (WNV) immunoglobulin A (IgA) in 65 of 68 sera from WNV-infected patients; 40 of 63 WNV IgM-positive, IgG-negative serum or plasma specimens; 65 of 67 WNV IgM-positive, IgG-positive specimens; 0 of 70 WNV IgM-negative, IgG-negative specimens; and 0 of 64 archived blood donation sera. WNV IgA is thus highly prevalent among WNV-infected patients and typically appears after WNV IgM but before WNV IgG.     

Evaluation of viral-lysate enzyme-linked immunosorbent assay kits for detecting human immunodeficiency virus (type 1) infections using human sera standardized by quantitative western blotting.

The first generation of proprietary reagents for detecting antibodies to the Human Immunodeficiency Virus Type 1 (HIV-1) by enzyme-linked immunosorbent assay (ELISA) used as antigen partially purified virus from cell culture lysates. These tests, which are still in use, may vary in their antibody measurement capabilities if different proportions of the viral polypeptides are present in the viral lysate mixtures. We determined the quantities of antibodies in the serum of persons infected with HIV-1 by dilution analysis using 3 ELISA kits: Abbott [A], Du Pont [D], Genetic Systems [G]. The proportionate antibody titres of each serum to p24gag and gp160env/120env were established by quantitative Western blotting. Serum antibody titres were high, frequently over 1:10,000, a result observed both by ELISA and Western blot. For Kit D, sera with high proportions of antibody to p24gag produced antibody titration curves with steep slopes whereas shallower slopes were found in sera with high proportions of antibody to gp160env. In contrast, Kit A gave steeper slopes with sera enriched for gp160env antibodies. Kit G gave results with slopes intermediate between Kits A and D. Serum antibody titres differed between kits depending upon the proportion and concentration of antibodies in a given serum to gp160env and p24gag. The findings that both the concentration and proportion of antibodies to specific viral polypeptides in human sera markedly affect the signal intensity produced by proprietary ELISAs suggest the need for several control sera which reflect the diversity of human serum responses. Standardization of human reference sera by quantitative Western blotting will assist in evaluation and quality control of ELISA tests.     

Development of immunoglobulin M capture enzyme-linked immunosorbent assay to differentiate human flavivirus infections occurring in Australia

We report the development of a flavivirus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MAC-ELISA) which improves the determination of an infecting flavivirus serotype over that by current serological methods. A panel of 165 IgM-positive sera from flavivirus patients with specific diagnostic results was tested by the flavivirus MAC-ELISA using a panel of 10 antigens. For 134 of these sera (81.2%), the highest reactivity was demonstrated against the infecting virus, which was consistent with the original diagnostic result. Specific antibody reactions inconsistent with the original diagnosis were found for six sera (3.6%). In our experience, the flavivirus-serotyping ELISA provides a rapid and accurate alternative to other serological tests, such as hemagglutination inhibition, for the specific diagnosis of flavivirus infections.     

Use of four dengue virus antigens for determination of dengue immune status by enzyme-linked immunosorbent assay of immunoglobulin G avidity

We used an enzyme-linked immunosorbent assay (ELISA) of immunoglobulin G avidity to determine the dengue immune status of 105 pairs of serum samples from patients infected with dengue virus. This study shows that a simple avidity test, for which only one acute-phase serum sample is required, is potentially more useful than the hemagglutination inhibition test for the discrimination of primary from secondary dengue virus infection, whatever the type of dengue antigen used.     

Immunoglobulin G antibody capture enzyme-linked immunosorbent assay: a versatile assay for detection of anti-human immunodeficiency virus type 1 and 2 antibodies in body fluids.

In tests on specimens of dried blood, saliva, and urine from 55 human immunodeficiency virus (HIV)-seropositive and 55 HIV-seronegative patients, an immunoglobulin G capture enzyme immunoassay for the detection of antibodies to HIV types 1 and 2, GACELISA, gave 109 of 110, 109 of 109, and 109 of 110 correct results, respectively. This performance, achieved in a laboratory previously unfamiliar with the assay, suggests that GACELISA is a useful new epidemiological tool for the study of HIV infection, equally applicable to all three kinds of specimen.     

Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-arboviral immunoglobulin G (IgG ELISAs) were developed for a comprehensive array of medically important arboviruses from the Alphavirus, Flavivirus, and Bunyavirus genera. Tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. MAbs were screened for suitability as capture vehicles for antigens from the three genera. The final test configuration utilized group-reactive MAbs eastern equine encephalitis virus 1A4B-6, dengue 2 virus 4G2, and La Crosse encephalitis virus 10G5.4 to capture the specific inactivated viral antigens. Serum IgG was detected by using alkaline phosphatase-conjugated anti-human IgG (Fc portion). A dilution of 1:400 was chosen as the universal screening serum dilution, with endpoint titrations of serum samples testing positive eliminating occasional false-positive results. IgG ELISA results correlated with those of the standard plaque-reduction neutralization assays. As expected, some test cross-reactivity was encountered within the individual genera, and tests were interpreted within the context of these reactions. The tests were standardized for laboratory diagnosis of arboviral infections, with the intent that they be used in tandem with the corresponding IgM antibody-capture ELISAs.     

Detection of immunoglobulin G antibodies to cytomegalovirus antigens by antibody capture enzyme-linked immunosorbent assay

An antibody capture enzyme-linked immunosorbent assay (ELISA) that uses horseradish-peroxidase-labeled antigen for the detection of immunoglobulin G (IgG) antibodies to cytomegalovirus (CMV) is described. A microtiter plate was coated with anti-human IgG and consecutively incubated with serum specimens, enzyme-labeled CMV antigen made from CMV-infected cell nuclei, and substrate. The CMV IgG antibody content was determined spectrophotometrically and expressed as absorbance. Furthermore, to reveal any nonspecific reactions, all sera were tested against an enzyme-labeled control antigen made from uninfected cell nuclei. The problem with nonspecific reactions was small and was circumvented by the addition of unlabeled control antigen to the conjugates. For epidemiological studies the test was not as sensitive as other serological tests. On the other hand, the IgG antibody capture ELISA was highly sensitive for detecting the serological antibody response in patients with primary and recurrent CMV infections. Thus, one positive serum remained positive at a serum dilution of 1:10(7). The specificity of the test was shown by a blocking experiment and by testing 126 complement fixation-positive sera, of which 97% were positive. There was a rather good correlation between the complement fixation test and the IgG antibody capture ELISA (rs = 0.79, P less than 0.001). The test is especially useful when tests for CMV antibodies of the IgM, IgA, and IgE classes are run by similar antibody capture ELISAs, since the same procedure and conjugate are used.     

Synthetic-peptide-based enzyme-linked immunosorbent assay for screening human serum or plasma for antibodies to human immunodeficiency virus type 1 and type 2.

A synthetic-peptide-based enzyme-linked immunosorbent assay (EIA) capable of screening for antibodies to both human immunodeficiency virus type 1 (HIV-1) and HIV-2 has been developed for use in blood banks and diagnostic laboratories. Microtiter wells are coated with two synthetic peptides, one corresponding to the highly conserved envelope region of HIV-1 and another corresponding to the conserved envelope region of HIV-2. Overall, sensitivity was 100% in 303 individuals diagnosed with AIDS and 96 individuals diagnosed with AIDS-related complex, 14.8% in a study of 500 high-risk group members, 99.9% in 600 EIA repeatedly reactive (RR)-HIV-1 Western blot (WB)-positive repository specimens, and 100% for 222 geographically diverse HIV-1 specimens and 216 confirmed HIV-2-positive specimens evaluated. The specificity was determined to be 99.72% for a total of 13,004 serum and plasma samples from random volunteer donors evaluated across five blood banks. Forty donors who were found to be EIA RR-WB indeterminate but nonreactive on the United Biomedical, Inc., test (UBI HIV 1/2 EIA) were prospectively followed as an additional measure of specificity. None of the 40 low-risk cases evolved into a positive WB pattern at follow-up. The sensitivity and specificity of this new assay are comparable to those of other Food and Drug Administration-licensed HIV-1 and HIV-1-HIV-2 assays that are currently available in the United States. The UBI HIV 1/2 EIA affords laboratories another choice in the detection of antibodies for HIV-1 and HIV-2 with a test based on an alternative antigen format.     

Sensitive chemiluminescent enzyme-linked immunosorbent assay for quantification of human immunoglobulin G and detection of herpes simplex virus

A chemiluminescent enzyme-linked immunosorbent assay (CELISA) was developed for detecting human immunoglobulin G and herpes simplex viral antigen. A comparison of CELISA with a conventional absorptiometric detection system showed that CELISA was 100 times more sensitive than absorptiometry for the measurement of human immunglobulin G. Similarly, CELISA detected as few as 40 plaque-forming units of herpes simplex virus in contrast to 2,500 plaque-forming units detected by absorptiometry. Of 18 specimens which were positive for herpes simplex virus type 1 by isolation in tissue culture, 15 (83%) were detected by CELISA within a few hours; in certain cases, several days were necessary for detection of virus by isolation techniques.     

Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections

We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.     

Salivary human immunodeficiency virus (HIV)-1-specific immunoglobulin A in HIV-1-exposed infants in Kenya

Humoral immunity, and specifically immunoglobulin A (IgA) that is directed against human immunodeficiency virus (HIV)-1, may contribute to protection against HIV-1 acquisition at mucosal surfaces. HIV-1-specific IgA has been detected in genital tract secretions of HIV-1-uninfected commercial sex workers with HIV-1 exposure, and may be produced in parotid saliva by infants exposed orally to HIV-1 during delivery and breastfeeding. To explore this hypothesis, we collected saliva from 145 infants aged < or = 6 months enrolled in a perinatal HIV-1 transmission study in Nairobi and from 55 control infants without HIV-1 exposure who were born to HIV-1-seronegative mothers. Among the 145 infants, 115 (79%) remained uninfected during the 12-month study period and 30 (21%) became HIV-1-infected during follow-up. Nine (8%) of the 115 HIV-1-exposed, uninfected infants had detectable levels of HIV-1 gp160-specific IgA compared with four (13%) of 30 infected infants and none of 55 control infants (P = 0.47 and P = 0.03 respectively). Among the nine HIV-1-exposed, uninfected infants with positive assays, median age was 1 month and none acquired HIV-1 during follow-up. We conclude that HIV-1-specific salivary IgA responses may be generated by very young infants exposed perinatally to maternal HIV-1. Mucosal responses would be an appropriate target for paediatric vaccines against breast milk HIV-1 transmission.     

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes

An antigen-capture enzyme-linked immunosorbent assay (C-ELISA) was developed for detection and identification of infectious bronchitis virus (IBV) serotypes Arkansas, Connecticut, and Massachusetts using monoclonal antibodies (MAbs) specific to the S1 glycoprotein of the respective serotype. The assay (designed as a double-antibody sandwich assay) gave the best results when the S1-specific MAb, antigen, and chicken serum were of the same serotype. However, when a group-specific (M glycoprotein-specific) MAb was used for antigen capture, a distinctive pattern of cross-reactivity was observed between the antigens and heterologous chicken sera, suggesting a complex distribution of epitopes on the IBV M glycoproteins. Treatment of antigen with NP40 enhanced the ELISA signal only when the M glycoprotein-specific MAb was used for antigen capture. Although C-ELISA was inconsistent in detecting IBV in chicken tissue homogenates, it was highly effective in detecting the virus in allantoic fluid after the homogenates were given one chicken embryo passage.     

Modification of rapid human immunodeficiency virus (HIV) antibody assay protocols for detecting recent HIV seroconversion

Assay protocols of three rapid human immunodeficiency virus (HIV) assays, OraQuick-1/2, SeroStrip-1/2, and Determine-1/2, were modified to detect recent HIV seroconversion using a higher dilution of serum specimens. Optimal predilution of specimens resulted in negative test results during early periods of seroconversion (about 6 months), when antibody levels were low. A total of 269 seropositive specimens from routine HIV type 1 testing and from commercial sources (low-titer and seroconversion panels) were tested, and results were recorded as negative (score=0) or positive using intensity scores from 0.5 (weak positive) to 4 (strongly positive). The same specimens were previously tested by a less sensitive (LS) enzyme immunoassay (EIA), Abbott 3A 11-LS, and were classified as recent or long-term infections based on the standardized optical density (SOD) cutoff of 0.75. Overall concordance of >94% was observed between 3A 11-LS and modified rapid tests (RT-LSs) for detecting and distinguishing recent HIV seroconversion from long-term HIV infection (kappa statistics=0.894 to 0.901). Moreover, intensity scores on RT-LSs correlated well with median 3A 11-LS SOD values (R(2)>0.98). Our results indicate that rapid HIV tests can be modified to detect recent seroconversion with results comparable to those from less sensitive EIA.     

Seroincidence of recent human immunodeficiency virus type 1 infections in China

A subtype B, E, and D immunoglobulin G capture immunoassay shows promise as a tool for estimating human immunodeficiency virus type 1 seroincidence from cross-sectional surveys, but the test-specific limitations suggest that an adjustment is necessary, and further validation of the assay with populations with divergent subtypes is needed.     

Isolation of the major herpes simplex virus type 1 (HSV-1)-specific glycoprotein by hydroxylapatite chromatography and its use in enzyme-linked immunosorbent assay for titration of human HSV-1-specific antibodies.

A 131,000 molecular weight herpes simplex virus type 1 (HSV-1) glycoprotein designated antigen number 6 (Ag-6) was previously shown to possess almost exclusively HSV-1-specific antigenic sites. Fused rocket and crossed immunoelectrophoresis of fractions obtained from hydroxylapatite chromatography of crude HSV-1 antigen (Triton X-100-solubilized, infected tissue culture cells) showed that a subfraction of Ag-6 could be separated from the other HSV antigens. Enzyme-linked immunosorbent assay with the isolated Ag-6 showed that sera from rabbits infected with HSV-1 and HSV-1 human antisera contained antibodies to Ag-6, whereas sera from HSV-2-infected rabbits and sera from patients with primary HSV-2 infections did not react with Ag-6. Enzyme-linked immunosorbent assay of 852 human sera for antibodies to HSV type-common glycoproteins, Ag-6, and HSV 2-specific antigens showed that 139 sera which reacted negatively with HSV type-common glycoproteins also did not react with Ag-6 with HSV-2 specific antigens. The 713 sera reacting positively to HSV type-common antigens either reacted with Ag-6 (328 sera) or with HSV-2-specific antigens (31 sera) or both (354 sera). This means that Ag-6 might be useful in large-scale human serology for the detection of past infection with HSV-1, irrespective of whether or not past infection with HSV-2 has occurred.     

Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.