scholarly journals Improved Serodiagnostic Performance for Lyme Disease by Use of Two Recombinant Proteins in Enzyme-Linked Immunosorbent Assay Compared to Standardized Two-Tier Testing

2017 ◽  
Vol 55 (10) ◽  
pp. 3046-3056 ◽  
Author(s):  
Gary L. Bradshaw ◽  
R. Kelley Thueson ◽  
Todd J. Uriona
Keyword(s):  
Lyme Disease ◽  
Recombinant Proteins ◽  
Immunosorbent Assay ◽  
Negative Control ◽  
Full Length ◽  
Test Method ◽  
Stage I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blots ◽  
Content Type

ABSTRACTThe most reliable test method for the serological confirmation of Lyme disease (LD) is a 2-tier method recommended by the CDC in 1995. The first-tier test is a low-specificity enzyme-linked immunosorbent assay (ELISA), and the second-tier tests are higher-specificity IgG and IgM Western blots. This study describes the selection of twoBorrelia burgdorferirecombinant proteins and evaluation of their performance in a simple 1-tier test for the serological confirmation of LD. These two proteins were generated from (i) the full-lengthdbpAgene combined with the invariable region 6 of thevlsEgene (DbpA/C6) and (b) the full-lengthospCgene (OspC). The expressed DbpA/C6 and OspC proteins were useful in detecting anti-BorreliaIgG and IgM antibodies, respectively. A blind study was conducted on a well-characterized panel of 279 human sera from the CDC, comparing ELISAs using these two recombinant antigens with the 2-tier test method. The two methods (DbpA/C6-OspC versus 2-tier test) were equivalent in identifying sera from negative-control subjects (99% and 100% specificity, respectively) and in detecting stage II and III LD patient sera (100% and 100% sensitivity). However, the DbpA/C6-OspC ELISA was markedly better (80% versus 63%) than the 2-tier test method in detecting anti-Borreliaantibodies in stage I LD patients. The findings suggest that these antigens could be used in a simple 1-tier ELISA that is faster to perform, easier to interpret, and less expensive than the 2-tier test method and which is better at detectingBorrelia-specific antibodies in sera from patients with stage I LD.

scholarly journals Dynamics ofMycobacterium tuberculosisAg85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Joel D. Ernst ◽  
Amber Cornelius ◽  
Miriam Bolz
Keyword(s):  
Protein Secretion ◽  
Immunosorbent Assay ◽  
Bacterial Population ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Protein ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Bacterial Proteins

ABSTRACTSecretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteinsin vivoare poorly understood. We generated new monoclonal antibodies that recognize theMycobacteriumtuberculosissecreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generatedin vitroandin vivo. We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in theM. tuberculosiscomplex: compared with the commonly used H37Rv strain (lineage 4),Mycobacteriumafricanum(lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates ofM. tuberculosisH37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretionin vivois dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCEBacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted byM. tuberculosisand used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

scholarly journals Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

2012 ◽  
Vol 19 (8) ◽  
pp. 1193-1198 ◽  
Author(s):  
Vijai Pal ◽  
Subodh Kumar ◽  
Praveen Malik ◽  
Ganga Prasad Rai
Keyword(s):  
Sensitivity And Specificity ◽  
Recombinant Proteins ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Burkholderia Mallei ◽  
Content Type ◽  
Whole Cells ◽  
Negative Results ◽  
Elisa Format ◽  
Low Sensitivity

ABSTRACTGlanders is a contagious disease caused by the Gram-negative bacillusBurkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins ofB. malleiwere used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

A Comparison of an Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting for Detection of Peanut Mottle Virus and Peanut Stripe Virus1

1986 ◽  
Vol 13 (2) ◽  
pp. 64-67 ◽  
Author(s):  
J. L. Sherwood ◽  
H. A. Melouk
Keyword(s):  
Western Blotting ◽  
Immunosorbent Assay ◽  
Protein A ◽  
Mottle Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coat Proteins ◽  
Western Blots ◽  
Peanut Stripe Virus ◽  
Dry Milk ◽  
The Difference

Abstract Western blotting was used to detect infections of peanut cv. Tamnut 74 with peanut mottle virus (PMV) and/or peanut stripe virus (PStV). Leaf samples were ground in electrophoresis sample buffer and heated for 5 min at 95 C prior to electrophoresis in 12% polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose sheets at 100V for 45 min. Western blots were performed by first blocking unbound sites on the nitrocellulose with 5% non-fat dry milk in Tris-buffered saline (TBS), pH 7.4 for 30 min, followed by incubation in a 1/200 dilution of PMV and/or PStV antiserum in TBS (the latter antiserum provided by J. W. Demski, U. of GA) for 45 min. This was followed by incubation in protein-A-peroxidase (2 μg/mL in TBS) for 45 min, followed by 4-chloro-1-napthol plus hydrogen peroxide in TBS. As little as 25 ng of either purified PMV or PStV was detected. This was similar to the limits of detection fo the double sandwich enzyme linked immunosorbent assay (ELISA). Because of the difference in migration of the coat proteins of PMV and PStV, both viruses may be detected in plants infected with PMV and PStV. This assay can be performed in approximately 6 h when mini-gels are used for the initial electrophoretic seperation and does not require the antiserum to be fractionated or bound to an enzyme as is the case with ELISA.

scholarly journals Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

2013 ◽  
Vol 21 (2) ◽  
pp. 196-202 ◽  
Author(s):  
Nadeeka K. Wawegama ◽  
Glenn F. Browning ◽  
Anna Kanci ◽  
Marc S. Marenda ◽  
Philip F. Markham
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Mycoplasma Bovis ◽  
Western Blots ◽  
Content Type ◽  
Amino Terminal ◽  
Igm Elisa ◽  
Specific Igg ◽  
Lipase A ◽  
Strong Immunoreactivity ◽  
Gst Fusion Proteins

ABSTRACTMycoplasma boviscauses a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected withM. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed inEscherichia colias glutathioneS-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity withM. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detectedM. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detectedM. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

scholarly journals Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

2016 ◽  
Vol 54 (6) ◽  
pp. 1557-1565 ◽  
Author(s):  
Martin Heller ◽  
Nimmo Gicheru ◽  
Georgina Tjipura-Zaire ◽  
Cecilia Muriuki ◽  
Mingyan Yu ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Rapid Test ◽  
Lateral Flow ◽  
Sub Saharan Africa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Contagious Bovine Pleuropneumonia ◽  
Serum Samples ◽  
Content Type ◽  
Mycoplasma Mycoides ◽  
Sub Saharan

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused byMycoplasma mycoidessubsp.mycoides, a bacterium belonging to theMycoplasma mycoidescluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenicMycoplasma mycoidessubsp.mycoidesproteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinantMycoplasmaimmunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

scholarly journals 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

2001 ◽  
Vol 8 (1) ◽  
pp. 44-51 ◽  
Author(s):  
Fuat Özyörük ◽  
William P. Cheevers ◽  
Gordon A. Hullinger ◽  
Travis C. McGuire ◽  
Melinda Hutton ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Encephalitis Virus ◽  
Surface Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blots ◽  
Caprine Arthritis Encephalitis Virus ◽  
Potential Applicability ◽  
Surface Envelope

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 withN-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.

scholarly journals Comparison of Western Immunobloting to an Enzyme-Linked Immunosorbent Assay for the Determination of Anti-Bordetella pertussis Antibodies

2011 ◽  
Vol 18 (4) ◽  
pp. 615-620 ◽  
Author(s):  
Stephen D. Merrigan ◽  
Ryan J. Welch ◽  
Christine M. Litwin
Keyword(s):  
Immunosorbent Assay ◽  
Bordetella Pertussis ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recent Infection ◽  
Content Type ◽  
Igm Elisa ◽  
Iga Antibodies ◽  
Health Organization

ABSTRACTDuringBordetella pertussisinfection, it has been established that an increase of anti-pertussis toxin (PT) and anti-filamentous hemagglutinin (FHA) antibodies occurs. Immunoblots from two manufacturers using FHA and PT antigens were compared with an enzyme-linked immunosorbent assay (ELISA) that used both FHA and PT. One manufacturer used two concentrations of PT bands for the IgG immunoblot, calibrated to the World Health Organization standard for PT in international units (IU/ml), 100 IU/ml (PT-100) and 8 IU/ml (PT). The second immunoblot kit measured antibodies to a single calibrated PT band. Both kits measured IgA antibodies, and one additionally measured IgM antibodies. Two of 41 (5%) ELISA IgM positives were confirmed positive by IgM immunoblotting, suggesting poor specificity of the IgM ELISA. The agreements of the IgG and IgA immunoblots with the ELISA ranged from 72.5% to 85.3%, with only 38 to 51% of IgA positives confirmed by immunoblotting and only 61 to 68% of IgG positives confirmed by immunoblotting. The two immunoblots correlated well with each other, with 91.7% and 94.3% agreement for IgG and IgA, respectively. When the FHA band was used with the PT band as the criterion for positivity, significant differences existed in specificity compared to the ELISA (IgG, 84.1% versus 33.3%; IgA, 82.4% versus 71.0%). When the positive IgA immunoblots (evidence of natural recent infection) were compared to the positive PT-100 IgG immunoblots (evidence of recent infection or vaccination), the PT-100 blot showed a 71% sensitivity in detecting natural recent infection.B. pertussisimmunoblots, alone or in combination with ELISAs, can aid in the diagnosis ofB. pertussisinfection.

scholarly journals Detection of Antibodies to the Biofilm Exopolysaccharide of Histophilus somni following Infection in Cattle by Enzyme-Linked Immunosorbent Assay

2014 ◽  
Vol 21 (10) ◽  
pp. 1463-1467 ◽  
Author(s):  
Yu Pan ◽  
Taylor Fisher ◽  
Christina Olk ◽  
Thomas J. Inzana
Keyword(s):  
Immunosorbent Assay ◽  
Biofilm Formation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Histophilus Somni ◽  
Index Value

ABSTRACTAn enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibodies toHistophilus somniexopolysaccharide (EPS), which is created during biofilm formation. When an index value of 0.268 was used, the sensitivity of the assay for infected calves was 90.5% at 3 weeks postinfection, but the number of positive animals increased by week 4. The specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves withH. somnidiseases.

scholarly journals Validation of an Enzyme-Linked Immunosorbent Assay That Detects Histoplasma capsulatum Antigenuria in Colombian Patients with AIDS for Diagnosis and Follow-Up during Therapy

2014 ◽  
Vol 21 (9) ◽  
pp. 1364-1368 ◽  
Author(s):  
Diego H. Caceres ◽  
Christina M. Scheel ◽  
Ángela M. Tobón ◽  
Angela Ahlquist Cleveland ◽  
Ángela Restrepo ◽  
...  
Keyword(s):  
Clinical Improvement ◽  
Immunosorbent Assay ◽  
Histoplasma Capsulatum ◽  
Response To Therapy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Test ◽  
Content Type ◽  
Antigen Capture

ABSTRACTWe validated an antigen capture enzyme-linked immunosorbent assay (ELISA) in Colombian persons with AIDS and proven histoplasmosis and evaluated the correlation between antigenuria and clinical improvement during follow-up. The sensitivity of theHistoplasma capsulatumELISA was 86%, and the overall specificity was 94%. The antigen test successfully monitored the response to therapy.

Epitope Mapping of a Monoclonal Antibody against VWF Which Diminishes the Proteolytic Cleavage of VWF by ADAMTS13 Under Flow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2133-2133
Author(s):  
Jingyu Zhang ◽  
Zhenni Ma ◽  
Ningzheng Dong ◽  
Jian Su ◽  
Anyou Wang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Proteolytic Activity ◽  
Recombinant Proteins ◽  
Expression System ◽  
Guanidine Hydrochloride ◽  
Proteolytic Cleavage ◽  
Terminal Region ◽  
Full Length ◽  
Western Blots ◽  
A2 Domain

Abstract Abstract 2133 Poster Board II-110 Introduction: In our former study, we have found that SZ-34, a monoclonal antibody to Von Willebrand factor (VWF), can inhibit the proteolysis of VWF by ADAMTS13 under shear stress. But the precise epitope of this antibody (SZ-34) on VWF is not clear for it is generated by immunizing mouse with native full-length VWF purified from pooled human normal plasmas. Thus, the objective of this study is to map the epitope of SZ-34 and to explore the effect of VWF structrue on the proteolytic activity by ADAMTS13. Materials and Methods: Firstly we constructed and expressed a series of recombinant proteins of different domains or polypeptide fragments of human VWF in prokaryotic cell expression system, including A1A2A3, D′D3, A1, A2, A3, A1A2, A2A3 and five sub-fragments of A2 domain. Then native VWF and these recombinant proteins or polypeptide fragments were subjected to polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blots with SZ-34. Results: Different recombinant proteins of VWF were successfully expressed and purified. Results of Western blot showed that SZ-34 could bind specifically some recombinant proteins, such as full-length VWF, A1A2A3, A2 and GST-D1459D1596 in which the last was a fusion protein of a sub-fragment of A2 domain with GST. But SZ-34 couldn't bind to others, including A1, A3, D′D3, GST-D1459E1554, GST-E1554D1596, GST-D1596R1668 (VWF73) and GST- E1554R1668. In addition, the reacting activity of SZ-34 with native VWF was significantly stronger than with unfolded VWF, such as heat-treated or 1.5M guanidine hydrochloride-treated VWF. Conclusions: The epitope of SZ-34 is located within N-terminal region fore-VWF73 inside VWF-A2 domain. Besides, SZ-34 maybe is a conformation-specific monoclonal antibody. Combining with our former findings that SZ-34 inhibits the proteolytic cleavage of VWF by ADAMTS13, we can conclude that N-terminal region fore-VWF73 inside VWF-A2 domain also regulates the proteolytic activity of VWF by ADAMTS13, although VWF73 is considered as the minimal substrate for ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document