Specific Serological Assays Research Articles

New strip immunoblot for the confirmation of HTLV-I/II infection.

In The Netherlands, anti-human T-cell lymphotropic virus type I (HTLV-I) blood donor screening became mandatory in January 1993. Donations reactive in the enzymelinked immunosorbent assay (ELISA) screening test are confirmed with Western blot analysis (WB). Only WB-positive or indeterminate blood donors are notified and retested by polymerase chain reaction (PCR) [1]. In accumulated data of the Dutch Blood Banks, only 2% of donors repeatedly positive in the anti-HTLV-I/II ELISA were WB-positive and all of these were also PCR-positive. However, 75% of the ELISA-positive blood donors have indeterminate WB results. In these cases the ELISA reactivities are nonspecific since all WB-indeterminate donors are negative in PCR [2]. Current WB confirmation thus leads to the notification and often to the deferral of many WB-indeterminate blood donors as well as to high costs for PCR testing, illustrating the need for a more specific serological confirmatory assay. The aim of our study was to evaluate a newly developed anti-HTLV-I/II Recombinant Immunoblot Assay (RIBA) to confirm samples reactive to screening tests with a special emphasis on the ability of this test to resolve WB-indeterminate results in blood donors, without compromising the sensitivity.

Delineation of Immunodominant Epitopes of Human T-Lymphotropic Virus Types I and II and Their Usefulness in Developing Serologic Assays for Detection of Antibodies to HTLV-I and HTLV-II

Several immunodominant B-cell epitopes have been mapped in the structural (gag, env) and regulatory (tax, rex) proteins of human T-lymphotropic virus type I (HTLV-I) and HTLV-II. Identification of these immunogenic epitopes not only has allowed the development of sensitive and specific serologic assays, but also has provided a tool to study correlates of host pathogenesis, viral transmission, and protection. The major immunogenic epitopes for HTLV-I are located at the C terminus of p19gag, the central and carboxyl terminus of gp46env, the central region of transmembrane envelope glycoprotein (p21e), and the carboxyl terminus of p40tax. Similarly, HTLV-II epitopes are located at the amino terminus and central region of gp46env, the central region of p21e, and the carboxyl terminus of p40tax. The transmembrane epitopes of HTLV-I and HTLV-II share extensive homologies and represent a highly cross-reactive region, while the remaining regions represent HTLV type-specific epitopes. The transmembrane epitope has been commonly used for detecting antibodies in both HTLV-I and HTLV-II-infected persons, whereas central envelope epitopes have been exploited for serologic differentiation between HTLV-I and HTLV-II. This detailed analysis at the epitope level has resulted in the development of highly sensitive and specific screening and confirmation assays for detection of antibodies to HTLVs.

Serodiagnosis of Mycobacterium bovis infection in badgers: development of an indirect ELISA using a 25 kDa antigen

A 25 kDa antigen of Mycobacterium bovis has previously been identified as immunodominant during badger infections. This 25 kDa antigen was partially purified from sonicated M bovis bacilli by using water precipitation and ion exchange chromatography, and its purification was monitored with a mouse monoclonal antibody, MBS43, which was specific for the antigen. The partly purified antigen was used to develop an ELISA for the assay of badger sera for the presence of specific antibodies. A presumed negative badger population was used to calculate the assay's threshold of seropositivity and using this value, its sensitivity (37 percent) and specificity (98 percent) were determined in a second population of known culture status. The results indicate that it may be possible to develop a specific and cost effective serological field assay for the diagnosis of M bovis infection in living badgers.

Competitive inhibition enzyme-linked immunosorbent assay for antibody in sheep and other ruminants to a conserved epitope of malignant catarrhal fever virus

Malignant catarrhal fever (MCF) is a severe, usually fatal, acute systemic disease syndrome of certain domestic and wild ruminants caused by members of the family Gammaherpesvirinae. Two distinct but closely related viruses cause clinically indistinguishable syndromes: one that is indigenous to the widebeest and the other that apparently is indigenous to domestic sheep. Neither the pathogenesis nor the epidemiology of sheep-associated MCF (SA-MCF) is understood, primarily because of a lack of adequate detection methods for the etiologic agent or antibody against it. No acceptably documented isolates of SA-MCF virus have been reported, and existing antibody assays suffer from significant cross-reactivity with other viruses. As a basis for a specific serologic assay, an attempt was made to identify an epitope conserved among all isolates of MCF viruses, by using a monoclonal antibody (MAb) produced against a previously reported U.S. isolate of MCF virus. A MAb (15-A) which bound a conserved epitope present on all four isolates of MCF virus examined was found. MAb 15-A did not react with eight common sheep and goat viruses or five common bovine viruses. Immunoprecipitation revealed that the 15-A epitope was located on the viral glycoprotein complex, with molecular masses of 115, 110, 105, 78, and 45 kDa. Sera from experimentally and naturally infected animals which yielded a similar glycoprotein complex immunoprecipitation pattern competed with MAb 15-A for its epitope. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) based on MAb 15-A was therefore developed. The assay detected antibody in inapparently infected sheep and in cattle, deer, and bison with clinical MCF. Of the 149 serum samples from sheep associated with MCF outbreaks, 88 (55%) were seropositive by competitive inhibition ELISA.

Detection of antibodies to herpes simplex virus type 2 with a mammalian cell line expressing glycoprotein gG-2

The gene (US4) coding for herpes simplex virus type 2 (HSV-2) glycoprotein G (gG-2) was cloned and constitutively expressed in Chinese hamster ovary (CHO) cells. The expression vector containing the dihydrofolate reductase (dhfr) gene, and the HSV-2 US4 gene under the control of the Simian virus 40 early promoter (SV40 EP), was transfected into dhfr-deficient CHO cells. The transfected cells were selected and amplified using methotrexate (MTX). To demonstrate that the gG-2 produced in these transformed cells had antigenic determinants in common with the native glycoprotein, CHO cells expressing gG-2 were used in an immunofluorescent assay (IFA) for the detection of HSV-2 type-specific antibodies in human serum samples. Seven of eight serum samples from adults with prior episodes of culture proven HSV-2 infections were found to be positive by the IFA method whereas none of seven serum samples from young children with culture documented HSV-1 infections were positive by IFA. Thus the recombinant CHO : gG-2 cells have diagnostic utility in an HSV-2 specific serologic assay.

Human T-cell lymphotropic virus type I and II DNA amplification in seropositive and seronegative at-risk individuals.

To determine the presence or the absence of human T-lymphotropic virus type I and/or II (HTLV-I/II) DNA in at-risk individuals who were persistently negative for specific serologic assays, polymerase chain reaction with two primer pairs in common and conserved regions of HTLV-I and -II genomes was used. Seronegative individuals at risk for HTLV-I/II infection (15 heterosexual partners of seropositive individuals, 17 breastfed children born to HTLV-I-infected mothers, 47 multiply transfused patients, 22 intravenous drug users) were studied (n = 101); 35 seropositive individuals and 25 seronegative low-risk individuals were used as positive and negative controls, respectively. No positive polymerase chain reaction was observed in the seronegative at-risk individuals or in the negative controls. Positive controls gave positive results with at least one primer pair in all cases except one. A latent HTLV-I/II infection with a persistently negative serologic test for HTLV-I/II seems unlikely.

Analysis and expression of the Borrelia burgdorferi P/Gau fla gene: Identification of heterogeneity with the B31 strain

The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190–234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIs.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.

Analysis and expression of theBorrelia burgdorferiP/Gau fla gene: Identification of heterogeneity with the B31 strain

The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190–234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIs.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.

Complications of Lyme borreliosis.

Lyme borreliosis is the multisystem infectious disease caused by the spirochete Borrelia burgdorferi. Complications of this infection can involve many organ systems, especially the skin, joints, nervous system, and heart. These manifestations may be acute, or evolve slowly over months or years. Diagnosis is not always straightforward, and is currently hampered by lack of a specific serologic assay. This review discusses the syndromes associated with Lyme borreliosis and addresses issues of diagnosis and treatment.

Association of Infection Due to Helicobacter pylori with Specific Upper Gastrointestinal Pathology

The association of infection with Helicobacter pylori and antral (type B) gastritis now is clear, and the development of sensitive and specific serologic assays for IgA and IgG allows for diagnosis of this infection by noninvasive means. With use of these assays, we studied the association of infection with H. pylori and four other upper gastrointestinal inflammatory conditions: Barrett's esophagus, pernicious anemia (which accompanies type A gastritis), and duodenal and gastric ulcers. H. pylori was present in only 39% of 41 patients with Barrett's esophagus whose gastric biopsy specimens were examined histologically. Each serologic assay correctly categorized 39 (95.1%) of the 41 patients. For both assays the frequency of seropositivity noted for 58 patients with Barrett's esophagus was not different from that noted for age- and sex-matched healthy controls. Among 40 patients with pernicious anemia, the results of assays for IgA and IgG were positive for 17.5% and 0%, respectively; these prevalences were significantly less than the 50% (IgA) and 40% (IgG) positivities noted for matched controls (P less than .01 for each; McNemar's test). Among 57 patients with documented duodenal or gastric ulcers, the results of assays for IgG and IgA were positive for 100% and 98.2%, respectively; these prevalences were significantly higher than the rate noted for matched controls (P less than .001 for duodenal ulcers and P = .02 for gastric ulcers for IgA assay). These data suggest that infection with H. pylori is strongly associated with duodenal and gastric ulcers, negatively associated with pernicious anemia, and independent of Barrett's esophagus.

Molecular diagnosis of parasites

New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene cloning and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.

Helicobacter pylori and Zollinger-Ellison syndrome

Helicobacter pylori (previously Campylobacter pylori) is almost invariably associated with chronic duodenal ulcer disease. The relationship between H. pylori infection and duodenal ulcer in Zollinger-Ellison syndrome is unknown. We investigated the frequency of H. pylori infection in Zollinger-Ellison syndrome and also what effect H. pylori infection had on gastric function in patients with Zollinger-Ellison syndrome. H. pylori infection was diagnosed based on a specific serologic (ELISA) assay based on high-molecular-weight cell-associated proteins of H. pylori. We studied 20 patients with Zollinger-Ellison syndrome; 15 men and 5 women ranging in age from 24 to 71 years, median age 51. Six Zollinger-Ellison syndrome patients had H. pylori infection compared to 100 consecutive patients with chronic recurrent duodenal ulcer disease (P less than 0.05). Pretreatment basal acid output in Zollinger-Ellison syndrome patients ranged from 7.9 to 95.0 mmol/hr, median 35.2. Pentagastrin-stimulated maximal acid output ranged from 8.5 to 132 mmol/hr; median 52.7. Acid secretion was lower in the H. pylori-infected patients than the uninfected patients (BAO 24.5 +/- 6.5 vs 45.4 +/- 6.6, and MAO 44.3 +/- 11.8 vs 67.9 +/- 10.7, for H. pylori infected vs uninfected patients, respectively). The difference in BAO was statistically significant (P less than 0.05). The present results indicate that H. pylori is not a major contributing factor in duodenal ulcer associated with Zollinger-Ellison syndrome. The association of a reduced BAO with H. pylori suggests that these findings may be related.

Simultaneous infection with human herpesvirus‐6 and measles virus in infants

Two infants (4 and 5 months of age) with a febrile episode for 3 and 5 days, respectively, developed skin rashes after the fever subsided and were diagnosed as exanthem subitum. The rash continued for 5 days followed by mild-to-moderate pigmentation. Human herpesvirus-6 and measles virus, which were confirmed by a specific immunofluorescence assay and by electron microscopy, were isolated simultaneously from blood in the acute stage of the disease but not from the convalescent stage. The titer of the herpesvirus-6 in blood was greater than that of measles. Specific serologic assays showed marked seroconversion against human herpesvirus-6 but not to measles virus. The results suggest that dual infection with human herpesvirus-6 and measles virus results in atypical exanthem subitum or modified measles with unique immunologic responses.

Seroprevalence of Helicobacter pylori Infections in Thailand

Serologic studies in developed countries indicate that Helicobacter (formerly Campylobacter) pylori infection is uncommon until the third decade of life and achieves a peak prevalence of 50% in the seventh decade. In developing countries the epidemiology of H. pylori has not well been described. A sensitive and specific serologic assay for H. pylori infection was validated in Thai patients also studied by culture and histologic examination of biopsy specimens. The prevalence of H. pylori antibodies in persons from a rural Thai community began early (17.5% of children 5-9 years old), increased to 55% during the third decade of life, and peaked (75%) in the 30- to 49-year age group. At a Bangkok orphanage where enteric infections are hyperendemic, 74% of children 1-4 years old were seropositive. This study shows that the prevalence of H. pylori infection in Thailand is higher than in industrialized countries. The high infection rate at the orphanage suggests that person-to-person transmission of H. pylori may be occurring.

Serologic evaluation of viral hepatitis.

Efforts to prevent transmission of viral hepatitis have been complicated by the identification of two “new” types of hepatitis: non-A, non-B and delta (a serious, sometimes fatal infection). The viral types are rarely distinguishable by clinical means; however, a number of rapid, sensitive, and specific serologic assays permit precise differentiation of etiology, chronicity, and infectivity.

Evaluation of an enzyme-linked immunosorbent assay for the detection of ectromelia (mousepox) antibody.

Ectromelia virus, an orthopoxvirus that can cause extensive morbidity and mortality (mousepox) in colonized mice, has been epizootically responsible for serious disruption of biomedical research since 1930. The lack of a sensitive and specific serological assay for infection with this virus became apparent during outbreaks of mousepox at the National Institutes of Health, Bethesda, Md., and other biomedical research institutions in 1979 and 1980. To fill this need, we evaluated an enzyme-linked immunosorbent assay. Sucrose gradient-purified ectromelia and vaccinia viruses were compared as antigens in tests on approximately 1,000 mouse sera from experimentally infected mice and conventional colonies of uninfected mice. A statistical analysis based on the frequency distribution of the absorbance values for 152 mouse sera (free of ectromelia antibody) gave 0.22 as a value to differentiate ectromelia-positive sera from ectromelia-negative sera. When enzyme-linked immunosorbent assay results were compared with those obtained by an indirect immunofluorescence assay, the former was found to be at least 10-fold more sensitive. With the procedures employed, including the use of purified vaccinia virions as antigen, the enzyme-linked immunosorbent assay proved to be highly sensitive and specific for detecting antibodies to ectromelia and vaccinia viruses. False-positive results were not encountered. False-negative results were observed in 3% of 108 separate tests of a known positive serum. Although data indicated that ectromelia antibody can be differentiated from vaccinia antibody with homologous and heterologous antigen, this procedure probably cannot be generally used because of unavailability of ectromelia antigens.

Autoantibodies and the Spectrum of Sjögren's Syndrome

In studies reported recently, the sera of patients with Sjögren's syndrome were found to contain precipitating antibodies to nuclear antigens that can be identified by immunodiffusion analysis. These precipitating autoantibodies have been termed SS-A and SS-B antibodies. We show that identification of these autoantibodies helped in establishing the diagnosis of Sjögren's syndrome in 12 of 30 patients in whom the diagnosis had not been considered at the time of the physician's initial examination. The reasons for this were related to lack of spontaneous complaints of keratoconjunctivitis sicca and xerostomia and prominence of symptoms associated with arthritis, myalgia, pulmonary fibrosis, and cardiac disease. This study re-emphasizes the importance of multisystem disease in Sjögren's syndrome and shows that specific serologic assays for autoantibodies aided in diagnosis.

Phase-dependent Changes of DNA, RNA and Protein Accumulation during the Ontogenesis of Broad Bean Seeds (Vicia faba L., var. minor)

Broad bean seeds were harvested at different periods after flowering and analyzed for fresh weight dry weight, cell numbers per cotyledon, DNA, RNA, soluble and insoluble nitrogen, total albumins, total globulins, vicilin, and legumin, respectively. Seed development was characterized mainly by the transition from cell division to cell expansion about 21 days after flowering and by desiccation after day 41. Vicilin and legumin, determined by specific serological assays, appeared from day 18 on, although legumin only in trace amounts. Vicilin biosynthesis was almost completely stopped 28 days after flowering, whereas legumin synthesis continued at least until day 41. The amount of legumin never reached the vicilin level, presumably as a result of prevailing dry conditions. The amounts of DNA and RNA per cell, after a rather constant period, were increased between days 21 and 34 about four times without further cell divisions, the ratio DNA: RNA remaining almost constant. The increase of protein content followed after a short delay. The results do not support the gene dosis concept with respect to biosynthesis of RNAs.