Direct hapten-linked competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of O-pinacolyl methylphosphonic acid

The Analyst ◽  
2012 ◽  
Vol 137 (2) ◽  
pp. 406-413 ◽  
Author(s):  
Manisha Sathe ◽  
R. Ghorpade ◽  
S. Merwyn ◽  
G. S. Agarwal ◽  
M. P. Kaushik
Keyword(s):  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Methylphosphonic Acid ◽  
Enzyme Linked Immunosorbent Assay

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.

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

2016 ◽  
Vol 66 (5) ◽  
pp. 471
Author(s):  
Manisha Sathe ◽  
Shruti Srivastava ◽  
Sumit Agrawal ◽  
Ramrao Ghorpade
Keyword(s):  
Immunosorbent Assay ◽  
Inhibitory Concentration ◽  
Methylphosphonic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunoassay ◽  
Ic50 Value ◽  
Lower Detection ◽  
Enzyme Conjugate ◽  
Enzyme Label ◽  
Free Antigen

The effect of spacers and the enzyme-linked immunosorbent assay (ELISA) formats on the functional parameters of assays such as lower detection limit, inhibitory concentration at 50 per cent (IC50), and specificity were studied. Enzyme conjugates having hydrophobic and hydrophilic spacers were prepared using O-isopropyl methylphosphonic acid (IMPA) and horseradish peroxidase (HRP) as an enzyme label. Comparison was made with reference to enzyme conjugate without any spacer. The present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate significantly improves the sensitivity of assays. An enhanced IC50 value achieved was 0.01 μg mL−1 for free antigen detection by direct immunoassay using hydrophilic spacers and precoating of ELISA plates by secondary antibody. The use of a hydrophilic spacer might have helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay.

scholarly journals Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

2003 ◽  
Vol 10 (5) ◽  
pp. 862-865 ◽  
Author(s):  
Lynn M. Herrmann ◽  
William P. Cheevers ◽  
Katherine L. Marshall ◽  
Travis C. McGuire ◽  
Melinda M. Hutton ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
False Negative ◽  
High Sensitivity ◽  
The United States ◽  
Encephalitis Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Caprine Arthritis Encephalitis Virus ◽  
Ovine Progressive Pneumonia

ABSTRACT A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.

scholarly journals Quantitation of Indian Krait (Bungarus caeruleus) Venom in Human Specimens of Forensic Origin by Indirect Competitive Inhibition Enzyme-Linked Immunosorbent Assay

2006 ◽  
Vol 89 (5) ◽  
pp. 1360-1366 ◽  
Author(s):  
Ganneru Brunda ◽  
Beedu Sashidhar Rao ◽  
Rajendra Kumar Sarin
Keyword(s):  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Limit Of Detection ◽  
Linear Regression Analysis ◽  
Cross Reactivity ◽  
Assay System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Calibration Plot ◽  
Rat Tissues ◽  
Capture Assay

Abstract An indirect competitive inhibition enzyme-linked immunosorbent assay was reported to detect krait venom in human specimens of forensic origin. Polyclonal anti-krait venom antibodies were characterized by indirect antibody capture assay. The calibration plot was constructed based on linear regression analysis (y = 72.85 12.29x, r2 = 0.98) with concentration ranges from 0.013 to 1000 ng/well of krait venom with a limit of detection of 0.2 ng/mL in the assay system. The IC50 (inhibitory concentration at 50% displacement) value of krait venom was observed to be 70 ng. Spiking studies indicated recoveries of 95100% and 94100% when various concentrations of krait venom were spiked to rat tissues (skin, liver, and kidneys) and pooled human serum, respectively. Polyclonal anti-krait venom antibodies showed no cross-reactivity with cobra and viper venom when tested in the assay system. The coefficient of variation of various concentrations of working range in intra-assay (n = 6) was &lt;5%, whereas in interassay (n = 6) it was observed to be 7%. Further, the method was used to quantitate krait venom in human autopsy and biopsy specimens of forensic origin. Concentration of krait venom was found to be in the range of 4172 ng/100 mg skin or skin scrapings and 64378 ng/mL blood or serum. The methodology may find application in forensic laboratories to assess the cause of death in the cases of krait-bite victims.

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