bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conservation of the 17-Kilodalton Antigen Gene within the Genus
Bartonella
Authors: Sweger Debra , Resto-Ruiz Sandra , Johnson David P. , Schmiederer Michael , Hawke Noel , Anderson Burt 
Published: July 2002

ABSTRACT

The 17-kDa antigen of
Bartonella henselae
has previously been shown to elicit a strong humoral immune response in patients with cat scratch disease (CSD) and to be useful in screening human serum samples for CSD. In this study, PCR amplification of genes homologous to the 17-kDa antigen gene of
B. henselae
was performed using genomic DNAs from several species of
Bartonella
, including the currently recognized human pathogens. Amplicons of similar size were demonstrated using the following chromosomal DNA templates:
B. henselae
(two strains),
B. quintana
(two strains),
B. elizabethae
,
B. clarridgeiae
,
B. vinsonii
subsp.
vinsonii
, and
B. vinsonii
subsp.
berkhoffii
. No evidence of a
B. bacilliformis
homolog of the 17-kDa antigen gene was obtained using multiple primer pairs. DNA sequencing revealed open reading frames capable of coding for proteins with sizes similar to that of the 17-kDa antigen of
B. henselae
in all of the amplicons; however, extensive sequence divergence across the genus was noted. Cloning of the amplified products into pUC19 resulted in recombinants that directed synthesis of homologs of the 17-kDa protein. Immunoblot analysis using human sera from CSD cases demonstrated very little cross-reactivity among different species for this protein. In contrast, immunoblots using rabbit serum raised to the recombinant
B. henselae
antigen showed extensive cross-reactivity with the proteins of other
Bartonella
species. The data suggest that the use of the 17-kDa antigen as a serologic reagent may allow the development of more specific diagnostic assays. Furthermore, the nucleotide sequences from the various versions of the 17-kDa antigen gene should be useful for rapid identification of
Bartonella
at the species level.
Improved Repetitive-Element PCR Fingerprinting of
Salmonella enterica
with the Use of Extremely Elevated Annealing Temperatures
Authors: Johnson James R. , Clabots Connie 
Published: July 2002

ABSTRACT

Modified thermal cycling conditions were explored in an effort to improve the reproducibility and resolving power of repetitive-element PCR (rep-PCR) fingerprinting. Assay performance was rigorously evaluated under standard and modified cycling conditions, using as a test set 12 strains putatively representing 12 serovars of
Salmonella enterica
. For all three fingerprint types (ERIC2, BOXA1R, and composite fingerprints), the use of extremely elevated annealing temperatures plus an initial “touchdown” cycling routine yielded significant improvements in day-to-day reproducibility and discriminating power despite the somewhat sparser appearance of the fingerprints. Modified cycling conditions markedly reduced the variability of fingerprints between cyclers, allowing fingerprints from different cyclers to be analyzed together without the degradation of assay performance that occurred with between-cycler analyses under standard cycling conditions. With modified cycling, composite fingerprints exhibited the lowest reproducibility but the highest net discriminating power of the three fingerprint types. rep-PCR fingerprints led to the discovery of a serotyping error involving one of the 12 test strains. These data demonstrate that modified cycling regimens that incorporate elevated annealing temperatures (with or without an initial touchdown routine) may markedly improve the performance of rep-PCR fingerprinting as a bacterial typing tool.
Relevance of the Viral RAK Alpha Gene in Diagnosis of Malignant Versus Nonmalignant Tumors of the Ovary and Uterus
Authors: Rakowicz-Szulczynska Eva M. 
Published: July 2002

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1)-like antigens RAK (named after the inventor E. M. Rakowicz) p120, p42, and p25, as well as HIV-1-like segments of cancer DNA (RAK gene alpha), have been found before in breast and prostate cancers. The present study focused on determining the value of markers RAK in the diagnosis and prognosis of gynecological cancer. Expression of RAK antigens in ovarian, uterine, cervical, and vulvar cancer, in benign tumors, in tissues adjacent to cancer, and in normal tissues was tested by Western blot hybridization of the electrophoretically separated proteins with monoclonal antibody RAK BrI. The RAK alpha gene was PCR amplified with HIV-1-derived primers SK68 and SK69. RAK antigens p120, p42, and p25 were found in 95% of ovarian, uterine, and cervical cancer cases and in 75% of vulvar cancer cases. The RAK alpha gene was expressed in 100% of cancer cases, in approximately 25% of benign ovarian tumors, and in 40% of benign tumors of the uterus. DNA sequences amplified in all cancer cases exhibited more than 90% homology to HIV-1 gp41 and were encoded for the functional peptide. DNA sequences found in benign tumors contained frameshift mutations and encoded truncated or nonfunctional peptides. Such sequences have not been amplified in normal tissues. RAK antigens and the RAK alpha gene seem to belong to a lentivirus type that is highly related to HIV-1. Beyond the diagnostic value of RAK markers, future cloning of the full viral genome would lead to a better understanding of the etiology of malignant and nonmalignant tumors of reproductive organs and to the development of novel therapeutic approaches.
Development of an Indirect Tams1 Enzyme-Linked Immunosorbent Assay for Diagnosis ofTheileria annulataInfection in Cattle
Authors: Gubbels Marc-Jan , d'Oliveira Christine , Jongejan Frans 
Published: July 2002

ABSTRACTAn enzyme-linked immunosorbent assay (ELISA) was developed based on a recombinant majorTheileria annulatamerozoite surface antigen, Tams1. Four different recombinant proteins derived from two different Tams1 alleles, both in two different truncated forms, were tested for their performance in the ELISA. Furthermore, antigen concentration, various buffers, washing protocol, and the choice of anti-total-immunoglobulin G (IgG), anti-IgG1, or anti-IgG2 as second antibody were evaluated. The performance of the resulting ELISA was analyzed by measuring the coefficient of variation (CV). A total of 22 sera were analyzed over the measurement range, resulting in a CV of ca. 10%, whereas 30% variation is the maximum acceptable. The cutoff value was determined by the two-graph receiver operating characteristic (TG-ROC), using the indirect fluorescent antibody test (IFAT) as a reference. It was shown that up to 3 months postinfection (p.i.) IFAT is more sensitive and specific, whereas beyond 3 months p.i. ELISA performed as well as IFAT. The cutoff was determined at maximal sensitivity, based on the TG-ROC after 3 months p.i. Nine calves experimentally infected with four differentT. annulatastocks remained positive in the ELISA for at least 1 year p.i. Finally, limited cross-reaction was found only withT. parvaantisera, but not with any otherTheileriaorBabesiaspecies. Since theT. parvaendemic area hardly overlaps withT. annulata, the Tams1 ELISA has the potential to become a useful tool in the epidemiology of tropical theileriosis.
Secretory Leukocyte Protease Inhibitor Inhibits Infection of Monocytes and Lymphocytes with Human Immunodeficiency Virus Type 1 but Does Not Interfere with Transcytosis of Cell-Associated Virus across Tight Epithelial Barriers
Authors: Hocini Hakim , Becquart Pierre , Bouhlal Hicham , Adle-Biassette Homa , Kazatchkine Michel D. , Bélec Laurent 
Published: July 2002

ABSTRACT
In the present study, we demonstrate that recombinant human secretory leukocyte protease inhibitor (rhSLPI) inhibits infection of lymphocyte- and monocyte-derived tumor cell lines and peripheral blood lymphocytes with laboratory-adapted isolates and with the primary isolate, NDK, of free human immunodeficiency virus type 1 (HIV-1). In contrast, rhSLPI did not exhibit inhibitory activity toward transcytosis of cell-associated HIV-1 through a tight monolayer of endometrial epithelial cells. These observations indicate that the inhibitory effect of SLPI is restricted to free HIV-1 in corporal fluids.
Reactivity to p52 and CM2 Recombinant Proteins in Primary Human Cytomegalovirus Infection with a Microparticle Agglutination Assay
Authors: Nulens Eric , Bodéus Monique , Bonelli Fabrizio , Soleti Antonio , Goubau Patrick 
Published: July 2002

ABSTRACT
We evaluated the reactivities of sera against p52 and CM2 recombinant antigens of human cytomegalovirus (HCMV), coated on microparticles, for the differentiation of primary HCMV infection from an established infection. Two different test formats of the CMV Multiplex Copalis assay were evaluated. The 214 serum samples tested were immunoglobulin M (IgM) positive or equivocal by our reference assay. Reactivities against p52 and CM2 antigens were tested for sera from 37 patients with a well-documented seroconversion within the preceding 3 months (119 serum specimens), 31 patients known to have had a seroconversion at least 8 months earlier (31 serum specimens), and 57 patients without a documented seroconversion (64 serum specimens). The assay had a sensitivity for the detection of a primary infection of 70 or 86% by the first test format and a sensitivity of 88 or 94% by the second test format, according to the criteria used to indicate a primary infection by this test. A good correlation of the results of the assay with our in-house avidity index was found. The specificity of the assay warrants further evaluation. With IgM-positive sera, the assay was not sufficiently specific to make a distinction between a primary infection and an established infection.
Need for an External Proficiency Testing Program for Cytokines, Chemokines, and Plasma Markers of Immune Activation
Authors: Fahey John L. , Aziz Najib , Spritzler John , Plaeger Susan , Nishanian Parunag , Lathey Janet L. , Seigel Joan , Landay Alan L. , Kilarui Rakhi , Schmitz John L. , White Carmen , Wara Diane W. , Akridge Robert , Cutili Joie , Douglas Steven D. , Reuben James , Shearer William T. , Nokta Mustafa , Polland Richard , Schooley Robert , Asthana Deshratn , Mizrachi Yaffa , Waxdal Myron 
Published: July 2002

ABSTRACT

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Iβ, soluble TNF receptor II, soluble IL-2 receptor alpha, β
2
-microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.
Development of a Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Quantification of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus
Authors: Witte Steven B. , Chard-Bergstrom Cindy , Loughin Thomas A. , Kapil Sanjay 
Published: July 2002

ABSTRACT
A rapid, inexpensive enzyme-linked immunosorbent assay (ELISA) to quantitate antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) in serum was developed using a recombinant PRRSV nucleoprotein (rN). The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA.
Combined Cell Culture Enzyme-Linked Immunosorbent Assay for Quantification of Poliovirus Neutralization- Relevant Antibodies
Authors: Wahby A. F. 
Published: July 2002

ABSTRACT
A combined cell culture enzyme-linked immunosorbent assay (CCC-ELISA) was developed for measuring the neutralizing antipoliovirus antibodies in human sera. The binding of different concentrations of each of the three poliovirus types to BGM cells in the presence and absence of a constant dilution from each test and reference serum was measured in the CCC-ELISA. The titers of the viruses neutralized by each serum were measured with the titration curves and used for interpretation of neutralizing titers to the three poliovirus types. Analysis of human sera revealed that the sensitivity and specificity of the CCC-ELISA and the microneutralization assay were comparable. The CCC-ELISA is nonsubjective, rapid, and highly reproducible. Furthermore, the CCC-ELISA could potentially be used as a seroepidemiologic tool for assessment of the humoral response to the cell culture infectious viruses.
Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1
Authors: Hashinaka Kazuya , Hashida Seiichi , Nishikata Ichiro , Adachi Akio , Oka Shinichi , Ishikawa Eiji 
Published: July 2002

ABSTRACT

An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of
Escherichia coli
using a rather long DNA fragment (3,012 bp) of the whole HIV-1
pol
gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in
E. coli
as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to β-
d
-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (
Y
) were well correlated to those obtained using rRT as antigen (
X
) (log
Y
= 0.99 log
X
+ 0.23;
r
= 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.
Monoclonal Antibody Binding to a Surface-Exposed Epitope on
Cowdria ruminantium
That Is Conserved among Eight Strains
Authors: Shompole Sankale , Rurangirwa Fred R. , Wambugu Anderson , Sitienei John , Mwangi Duncan M. , Musoke Anthony J. , Mahan Suman , Wells Clive W. , McGuire Travis C. 
Published: July 2002

ABSTRACT

Monoclonal antibodies (MAb) binding to
Cowdria ruminantium
elementary bodies (EB) were identified by enzyme-linked immunosorbent assay, and surface binding of one MAb (446.15) to intact EB was determined by immunofluorescence, immunogold labeling, and transmission electron microscopy. MAb 446.15 bound an antigen of approximately 43 kDa in immunoblots of eight geographically distinct strains. The MAb did not react with
Ehrlichia canis
antigens or uninfected bovine endothelial cell lysate and may be useful in diagnostic assays and vaccine development.
Correlation between Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay with Lytic Antigens for Detection of Antibodies to Human Herpesvirus 8
Authors: Topino Simone , Vincenzi Laura , Mezzaroma Ivano , Nicastri Emanuele , Andreoni Massimo , Sirianni Maria C. 
Published: July 2002

ABSTRACT

The purpose of this study was to evaluate, in Kaposi's sarcoma patients, the correlation between antibody titers to the lytic antigens of human herpesvirus 8, as assessed by immunofluorescence assay, and values obtained by an enzyme immunoassay. The methods showed a stringent correlation,
r
= 0.625 (
P
< 0.001).
Monoclonal Antibodies to Conformational Epitopes of the Surface Glycoprotein of Caprine Arthritis-Encephalitis Virus: Potential Application to Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detecting Antibodies in Goat Sera
Authors: Özyörük Fuat , Cheevers William P. , Hullinger Gordon A. , McGuire Travis C. , Hutton Melinda , Knowles Donald P. 
Published: July 2002

ABSTRACT

Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79–63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-
N
-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with
N
-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection.
Development of an Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay for Detection of Equine and Swine IgM Antibodies to Vesicular Stomatitis Virus
Authors: Zhou En-min , Riva Jose , Clavijo Alfonso 
Published: July 2002

ABSTRACT
An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competitive ELISA (C-ELISA) and the standard microtiter serum neutralization (MTSN) assay by testing serum samples from horses and pigs experimentally infected with VSV-NJ or VSV-IN. The MC-ELISA detected specific homologous IgM antibodies from equine and swine sera as early as 5 and 4 days postinfection (DPI), respectively, and as late as 35 DPI. The MTSN test also detected antibodies as early as 5 DPI and as late as 160 DPI. In a similar fashion, the C-ELISA detected antibodies from 6 to 7 DPI and as late as 160 DPI. These results demonstrated that the MC-ELISA is a useful test for serodiagnosis of primary VSV infection in horses and pigs.
Chromogenic Assay Measuring Opsonophagocytic Killing Capacities of Antipneumococcal Antisera
Authors: Lin J. S. , Park M. K. , Nahm M. H. 
Published: July 2002

ABSTRACT

Assays measuring opsonophagocytic killing capacity of immune sera are good surrogate assays for assessing pneumococcal vaccine responses, but they are tedious to perform primarily because the enumeration of surviving bacteria requires the counting of individual bacterial colonies. To overcome this limitation, we have developed a simple and rapid chromogenic assay for estimating the number of surviving bacteria. In this method, the conventional opsonophagocytic killing assays were performed in microtiter wells with differentiated HL-60 cells as phagocytes. At the end of the assay the reaction mixture was cultured for an additional 4.5 h to increase the number of bacteria. After the short culture, XTT (3,3′-[1{(phenylamino)carbonyl}-3,4-tetrazolium]-bis[4-methoxy-6-nitro] benzene sulfonic acid hydrate) and coenzyme Q were added to the wells and the optical density at 450 nm was measured. Our study shows that changes in the optical density were proportional to the number of CFU of live bacteria in the wells. Also, the number of bacteria at the end of the 4.5-h culture was found to be proportional to the original number of bacteria in the wells. When the performance of the chromogenic assay was evaluated by measuring the opsonizing titers of
Streptococcus pneumoniae
serotypes 6B and 19F, the sensitivity and precision of the new method were similar to those of the conventional opsonization assay employing the colony counting method. Furthermore, the results of this chromogenic assay obtained with 33 human sera correlate well with those obtained with the conventional colony counting method (
R
> 0.90) for the two serotypes (6B and 19F). Thus, this simple chromogenic assay would be useful in rapidly measuring the capacities of antisera to opsonize pneumococci.
Antenatal Screening for Hepatitis B and Antibodies to
Toxoplasma gondii
and Rubella Virus: Evaluation of Two Commercial Immunoassay Systems
Authors: Diepersloot Robert J. A. , Dunnewold-Hoekstra Hiske , Kruit-den Hollander Jorien , Vlaspolder Fer 
Published: July 2002

ABSTRACT

A comparative evaluation of the Abbott AxSYM and DPC Immulite random-access analyzers was performed using 497 prospectively collected serum samples. These samples were sent to the laboratory for routine antenatal screening for hepatitis B surface antigen and immunoglobulin G (IgG) and IgM antibodies to
Toxoplasma gondii
and rubella virus. The overall agreement between the two assay systems ranged from 97.4 to 100%. After discrepancy analysis, the outcome in terms of sensitivity and specificity varied from 98.2 to 100% for all but one of the assays tested. The AxSYM rubella virus IgG assay tended to report protective or indeterminate antibody levels in 1% of the samples. This shortcoming might be overcome by raising the cutoff of the microparticle enzyme immunoassay system.