An exonuclease-amplification coupled capture technique improves detection of PCR product.
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Base Sequence↗
Biomedical subjects
D D Erdman
Publications and source records attributed to D D Erdman.
An exonuclease-amplification coupled capture technique improves detection of PCR product.
Explore the source record for details and available documents.
Base Sequence↗
Immunoglobulin M antibody response to measles virus following primary and secondary vaccination and natural virus infection.
The use of IgM antibody detection for the classification of the primary and secondary measles antibody response in persons following primary and secondary vaccination and natural measles virus infection was examined. Of 32 nonimmune children receiving primary measles vaccination, 31 (97%) developed IgM antibodies, consistent with a primary antibody response. Of 21 previously vaccinated children with low levels of preexisting IgG antibodies who responded to revaccination, none developed detectable IgM antibodies, whereas 33 of 35 (94%) with no detectable preexisting IgG antibodies developed an IgM response. Of a sample of 57 measles cases with a prior history of vaccination, 55 (96%) had detectable IgM antibodies. Of these, 30 (55%) were classified as having a primary antibody response and 25 (45%) a secondary antibody response based on differences in their ratios of IgM to IgG antibodies. Differences in the severity of clinical symptoms between these 2 groups were consistent with this classification scheme. These findings suggest that 1) an IgM response follows primary measles vaccination in the immunologically naive, 2) an IgM response is absent on revaccination of those previously immunized, and 3) an IgM response may follow clinical measles virus infection independent of prior immunization status.
Adolescent↗
Multiple primer pairs for polymerase chain reaction (PCR) amplification of human parvovirus B19 DNA.
Human parvovirus B19 is the etiologic agent of erythema infectiosum and transient aplastic crisis in patients with hemolytic anemias and has been associated with fetal death, arthritis, and chronic anemia. Acute B19 infection is best diagnosed by detection of IgM antibodies, whereas the diagnosis of chronic infection often requires the sensitivity of PCR to demonstrate presence of virus over time. To improve our ability to detect B19 DNA by polymerase chain reaction (PCR), we evaluated 19 primers combined into 16 different primer pairs for their ability to detect temporally and geographically diverse B19 isolates. All 16 pairs reacted with all isolates tested but with different sensitivity. Sequence analysis showed few nucleotide changes compared with published sequences. These changes did not explain observed differences in sensitivity between primer pairs. The most sensitive primer pairs detected 350 to 3500 DNA copies after 35 cycles. A second amplification cycle with nested primers improved the sensitivity 100-fold. These 16 primer pairs provide the diagnostic virologist with multiple options for B19 PCR assays.
Acute Disease↗
Baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays.
A recombinant baculovirus that expresses the nucleoprotein gene of measles virus (Edmonston vaccine strain) under the transcriptional control of the polyhedrin promoter was generated. The expressed protein (B-MVN) comigrated with the authentic viral nucleoprotein as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was phosphorylated. The B-MVN protein proved to be reactive with monoclonal antibodies in radioimmunoprecipitations, and it was immunogenic, eliciting in mice antisera that recognized the native nucleoprotein. In addition, the B-MVN protein was evaluated as a replacement source of antigen for whole virus in enzyme immunoassays (EIAs) for detection of measles virus-specific immunoglobulin M (IgM) and IgG antibodies. A capture IgM EIA with the B-MVN protein as antigen detected specific IgM antibodies in 18 (72%) acute- and all convalescent-phase specimens from 25 clinical measles cases and exceeded 99% specificity with 120 control specimens. An indirect IgG EIA with the B-MVN protein detected specific IgG antibodies in 129 of 131 (98%) serum specimens with antibodies to measles virus, and results obtained from testing 268 additional serum specimens were better correlated with measles virus-neutralizing antibodies than those obtained with a commercial EIA.
Antibodies, Viral↗
Human parvovirus B19 specific IgG, IgA, and IgM antibodies and DNA in serum specimens from persons with erythema infectiosum.
To determine the diagnostic use of different markers of acute parvovirus B19 infection, serum specimens obtained from 128 persons with erythema infectiosum were tested for specific immunoglobulin G (IgG), IgA, and IgM antibodies by capture enzyme immunoassay (EIA) using Chinese hamster ovary (CHO) cell-expressed B19 antigen, and tested for circulating B19 DNA by polymerase chain reaction (PCR). A significant rise in specific IgG and IgA antibodies was detected in 87% and 77%, respectively, of persons from whom acute- and convalescent-phase serum specimens were available. Specific IgA antibodies were detected in single serum specimens from 90% of cases and were present in 22 (18%) of 120 persons from a control group without a history of recent exposure to B19. Specific IgM antibodies were detected in 97% of cases and one person (1%) from the control group. B19 DNA was detected in 94% of cases and was absent in 20 persons from the control group positive for both IgG and IgA antibodies. Serum specimens obtained between 4 and 6 months after onset of illness from six additional persons were also tested. All had specific IgG antibodies, four (67%) had IgA, five (83%) had IgM, and none had detectable B19 DNA. Our data indicate that 1) specific IgA antibodies are too persistent to be a useful indicator of recent B19 infection; 2) specific IgM antibodies are the most sensitive indicator of acute B19 infection in immunologically normal persons but can persist up to 6 months; and 3) B19 DNA can often be detected up to 2 months after onset of illness even in immunologically normal hosts and might be a useful adjunct test for diagnosis of acute B19 infection.
Adolescent↗
Evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus.
Monoclonal antibodies to the hemagglutinin protein, fusion protein, phosphoprotein, matrix protein, and nucleoprotein of measles virus were evaluated as detector antibodies in capture enzyme immunoassays (EIAs) for the detection of specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to measles virus. A pool of monoclonal antibodies to hemagglutinin protein and nucleoprotein proved optimal and was further evaluated. Specific IgM was detected in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of clinical measles cases and vaccinees, in 26% of healthy persons, and in 36% of infants 8 months postvaccination; consequently, IgA antibodies were not a useful indicator of recent measles infection. A significant increase in IgG antibodies between paired specimens was detected in 92% of clinical cases and all vaccinees. Only 59% of infant specimens had persistent IgG antibodies as detected by capture EIA at 8 months postvaccination, whereas all specimens had antibodies as detected by hemagglutination inhibition and plaque neutralization. An alternative indirect EIA, in which antigen was directly absorbed to the solid phase, was more sensitive than the capture design, detecting IgG antibodies in all infants postvaccination. When standardized with a microneutralization assay for the detection of persistent antibodies, the indirect IgG EIA gave predictive values for positive and negative tests exceeding 90%. Our capture IgM and indirect IgG EIAs provide a practical combination of serologic tests for the determination of acute measles virus infection and past exposure to measles virus or vaccine, respectively.
Antibodies, Monoclonal↗
Use of mixture models in determining laboratory criterion for identification of seropositive individuals: application to parvovirus B19 serology.
Selecting the cut-off value to identify infected individuals requires a trade-off between sensitivity and specificity. The standard technique for selecting a cut-off uses results of a population either known or assumed uninfected. Using mixture models, however, it is not necessary to identify an uninfected population. This technique also provides methods to estimate the probability that an individual will be accurately classified. We illustrate this technique in determining cut-off values for the serological diagnosis of human parvovirus B19 infection.
Antibodies, Viral↗
Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus.
Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.
Adult↗
Serum immunoglobulin A response to Norwalk virus infection.
We describe the serum immunoglobulin A (IgA) antibody response to Norwalk virus infection in human volunteers and compare it with previously described IgM and total antibody responses. Whereas specific IgA and IgM peak within 2 weeks after onset of symptoms, titers of total blocking antibody continue to rise, implying mediation by IgG antibody.
Antibodies, Viral↗
Clinical comparison of ethyl acetate and diethyl ether in the formalin-ether sedimentation technique.
A substitute for the volatile solvent diethyl ether has been actively sought for the Formalin-ether sedimentation technique. Ethyl acetate has recently been shown to be a comparable substitute. In an effort to verify these findings and evaluate ethyl acetate under clinical conditions, comparison studies with 62 fresh human stool specimens were performed. Parallel concentrates with diethyl ether and ethyl acetate were prepared for each specimen, and the quantity and appearance of recovered parasite species were determined. Ethyl acetate was found comparable to diethyl ether in the quantitative recovery of parasite eggs, cysts, and larvae; no distortion or alteration of parasite morphology was observed with either solvent. More care was required, however, to completely remove the interface plugs of ethyl acetate and prevent their remixing the concentrate sediment. In addition, wet mounts prepared from ethyl acetate concentrates were occasionally obscured by liquid bubbles probably composed of remaining insoluble ethyl acetate. Clinical laboratories considering substituting ethyl acetate for diethyl ether in the Formalin-ether sedimentation technique should be aware of these problems and take the appropriate precautions to avoid them.
Acetates↗
Development and evaluation of an IgM capture enzyme immunoassay for diagnosis of recent Norwalk virus infection.
A capture enzyme immunoassay specific for Norwalk IgM class antibody was developed. The assay was moderately sensitive, identifying 33/53 (62%) of patients with naturally acquired Norwalk virus infection and 17/18 (94%) of experimentally infected volunteers. The assay was also specific for IgM class antibody and acute Norwalk virus infection and results were generally reproducible. A specific IgM response correlated with seroconversion by total antibody blocking assay and occurred independently of clinical symptoms. Among 81 symptomatic cases composing seven Norwalk outbreaks, specific IgM was absent from acute phase sera collected less than or equal to three days post onset, and was uncommon in sera collected within one week and after five weeks, with an optimal collection time at about two to three weeks. The Norwalk IgM capture immunoassay may be used to augment paired sera assays in the identification of Norwalk-associated outbreaks of acute gastroenteritis.
Antibodies, Viral↗