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.

Immunoglobulin G- and M-specific enzyme-linked immunosorbent assay for detection of dengue antibodies

1979 ◽  
Vol 9 (4) ◽  
pp. 498-502
Author(s):  
D Dittmar ◽  
T J Cleary ◽  
A Castro
Keyword(s):  
Dengue Virus ◽  
Immunosorbent Assay ◽  
Immunoglobulin G ◽  
Hemagglutination Inhibition ◽  
Dengue Infection ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Enzyme ◽  
Enzyme Conjugate ◽  
Inhibition Technique

An enzyme-linked immunosorbent assay for the detection of antibodies to dengue virus is described. This method correlates well with a hemagglutination inhibition technique. The enzyme-linked immunosorbent assay can also be specific for human immunoglobulin M antibodies when a mu-chain-specific antiglobulin-enzyme conjugate and fractionated serum are employed. By using this technique, dengue immunoglobulin M antibodies were demonstrated in an infant suspected of having a recent dengue infection.

Centrifugal analyzer used for enzyme immunoassay of progesterone and choriomammotropin.

1983 ◽  
Vol 29 (2) ◽  
pp. 302-304 ◽  
Author(s):  
B Terouanne ◽  
J Marchand ◽  
C Calzolari ◽  
J Monnier ◽  
J C Nicolas ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Enzyme Immunoassay ◽  
Gel Filtration ◽  
Placental Lactogen ◽  
Human Placental Lactogen ◽  
Rapid Separation ◽  
Enzyme Conjugate ◽  
Enzyme Label ◽  
Free Antigen

Abstract Recently we developed an enzyme immunoassay involving the use of steroid delta-isomerase (EC 5.3.3.1) as enzyme label and exclusion-affinity chromatography for rapid separation of free antigen-enzyme conjugate that bound to antibodies (J. Immunol. Methods 35: 267-284, 1980). Here we describe an automated version of this procedure, for immunoassay of progesterone and choriomammotropin (human placental lactogen) in serum with the use of a centrifugal analyzer. After incubation, suitable dilutions of sera or extract plus antiserum, conjugate, and double antibody were filtered on an estradiol affinity gel-filtration column; the enzyme activity of the filtrates was determined with the centrifugal analyzer. Results correlate well with those obtained by radioimmunoassay: r (progesterone) = 0.980, r (choriomammotropin) = 0.940. The within-run and between-run precision, specificity, sensitivity, accuracy, and speed of this system make it a useful tool for immunoassay.

Indirect Enzyme-Linked Immunosorbent Assay for Saxitoxin in Shellfish

1985 ◽  
Vol 68 (1) ◽  
pp. 13-16 ◽  
Author(s):  
Fun S Chu ◽  
Titan S L Fan
Keyword(s):  
Detection Limit ◽  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Solid Phase ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromogenic Substrate ◽  
Rabbit Igg ◽  
Lower Detection ◽  
Peroxidase Conjugate

Abstract An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of saxitoxin (STX). Antibodies against STX were demonstrated in rabbits 5 weeks after immunizing with STX-bovine serum albumin (STX-HCHO-BSA). In the ELISA, STX-HCHO-BSA or polylysine-STX was coated onto the microtiter plate, followed by incubation with standard toxin and anti-STX antibody. The amount of antibody bound to the solid phase was determined by incubation with goat anti-rabbit IgG peroxidase conjugate and a reaction with chromogenic substrate. Competitive indirect ELISA revealed that the antiserum did not cross-react with either carbamoyl-neo-STX-suIfate or tetrodotoxin. The antibodies for STX cross-reacted with decarbamoyl- STX and neo-STX about 56% and 16% as much as they did with STX, respectively. The lower detection limits for STX, decarbamoyl-STX, and neo-STX in this sytem were about 25, 45, and 156 pg per assay, respectively. When STX added to clams or mussels was assayed, the detection limit for STX was about 50-100 ppb, and recoveries were in the range of 86.8-107%.

Enzyme-linked immunosorbent assay for 17 alpha-hydroxyprogesterone in dried blood spotted on filter paper.

1987 ◽  
Vol 33 (6) ◽  
pp. 761-764 ◽  
Author(s):  
M Maeda ◽  
H Arakawa ◽  
A Tsuji ◽  
Y Yamagami ◽  
A Isozaki ◽  
...  
Keyword(s):  
Detection Limit ◽  
Immunosorbent Assay ◽  
Filter Paper ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adrenal Hyperplasia ◽  
Bovine Serum Albumin Conjugate ◽  
Enzyme Conjugate ◽  
Sensitivity Specificity ◽  
Microwell Plate

Abstract In this rapid, cost-effective, enzyme-linked immunosorbent assay for 17 alpha-hydroxyprogesterone (17-OHP) eluted from dried blood spotted on filter paper, second antibody is coated onto the microwell plate and horseradish peroxidase (EC 1.11.1.7) is the label enzyme. Antiserum to 17-OHP was prepared by using 4-(2-carboxymethylthio)-17-OHP-bovine serum albumin conjugate as immunogen. Enzyme conjugate was prepared from 4-(2-carboxymethylthio)-17-OHP and peroxidase. The blood spots are assayed in the microwells without extraction or centrifugation steps. The detection limit of the assay is 1 microgram/L, equivalent to 3.5 pg (10.6 fmol) per disc. Intra- and interassay CVs at two steroid concentrations (7.38 and 22.79 micrograms/L) ranged from 3.74 to 11.90% (n = 5), and 9.49 and 9.83% (n = 5), respectively. Results correlated well (r = 0.91) with those of a fluorescence enzyme immunoassay. The sensitivity, specificity, and precision of this method make it potentially useful in the mass screening of neonates for congenital adrenal hyperplasia.

scholarly journals Determination and comparative analysis of the catalytic subunit of adenosine 3′,5′-cyclic phosphate-dependent protein kinase by an enzyme-linked immunosorbent assay

1982 ◽  
Vol 208 (1) ◽  
pp. 109-117 ◽  
Author(s):  
G Schwoch ◽  
A Hamann
Keyword(s):  
Immunosorbent Assay ◽  
Animal Species ◽  
Catalytic Subunit ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Protein Kinase ◽  
Bovine Heart ◽  
Lower Detection ◽  
Dependent Protein ◽  
Cyclic Phosphate

A specific antiserum against bovine heart catalytic subunit was used for the determination of the catalytic subunit in an enzyme-linked immunosorbent assay. Under the conditions elaborated the assay has a lower detection limit for catalytic subunit of 0.25 pmol/ml. In crude bovine heart extracts the concentration of catalytic subunit was determined by this method to be 0.18 +/- 0.02 mumol/kg wet wt. The immunochemical comparison of various animal species and cells, including organisms like amoebae and yeast, shows the broad applicability of the assay and provides evidence that the catalytic subunit is a highly conserved molecule.

Gold Nanoparticles-Amplified Indirect Competitive Enzyme-Linked Immunosorbent Assay of BDE-121 Using a Monoclonal Antibody

2021 ◽  
Vol 21 (10) ◽  
pp. 5036-5043
Author(s):  
Yan Qiao ◽  
Qing-Yun Cai
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Gold Nanoparticles ◽  
Immunosorbent Assay ◽  
Inhibitory Concentration ◽  
High Sensitivity ◽  
High Specificity ◽  
Gas Chromatography Mass Spectrometry ◽  
Enzyme Linked Immunosorbent Assay ◽  
Good Recovery

In this study, we developed a monoclonal antibody against 2,3’,4,5’,6-pentabromodiphenylether (BDE-121) using a synthesized hapten, and established an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA), using gold nanoparticles, to amplify the signal. The monoclonal antibody showed high specificity, with a half inhibitory concentration (IC50) value of 2.78 ng/mL, towards BDE-121. The developed IC-ELISA exhibited high sensitivity and stability as well as good recovery. The intra-assay deviation is below 6.8% and the inter-assay deviations range from 6.5% to 8.7%. The assay of the actual samples was found to be consistent with those of gas chromatography/mass spectrometry (GC/MS).

scholarly journals Quantification of bee-derived peptide defensin-1 in honey by competitive enzyme-linked immunosorbent assay, a new approach in honey quality control

2016 ◽  
Vol 34 (No. 3) ◽  
pp. 233-243 ◽  
Author(s):  
Valachová Ivana ◽  
Bučeková Marcela ◽  
Majtán Juraj
Keyword(s):  
Antibacterial Activity ◽  
Immunosorbent Assay ◽  
Inhibitory Concentration ◽  
Rapid Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
New Approach ◽  
Relative Standard ◽  
Dependent Correlation ◽  
Sensitive Method ◽  
Significant Concentration

We established and evaluated a polyclonal antibody based competitive enzyme-linked immunosorbent assay for the quantification of defensin-1 in honey. The assay showed an inhibitory concentration (IC<sub>50</sub>) value of 111.5 ± 15.41 ng/ml with a detection limit of 7.8125 ng/ml. The regaining of defensin-1 in spiked ‘artificial honey’ was between 87.05 and 112.96% with relative standard deviation less than 9.2%. Sensitivity and specificity of the test were experimentally validated on a sample of 20 different honeys. The antibacterial activity of these honey samples showed a significant concentration-dependent correlation with the production of defensin-1 (n = 20; r = −0.6598; P = 0.0016). The assay provides a specific and sensitive method for the screening of defensin-1 in honey. The method to detect honeybee-derived proteins in honey is a promising approach to verifying the authenticity of honey. The defensin-1 ELISA could also be used for the rapid screening of honeys suitable for medicinal purposes.

scholarly journals Simple Histidine-Rich Protein 2 Double-Site Sandwich Enzyme-Linked Immunosorbent Assay for Use in Malaria Drug Sensitivity Testing

2005 ◽  
Vol 49 (8) ◽  
pp. 3575-3577 ◽  
Author(s):  
Harald Noedl ◽  
Jan Bronnert ◽  
Kritsanai Yingyuen ◽  
Bernhard Attlmayr ◽  
Herwig Kollaritsch ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Drug Sensitivity ◽  
Inhibitory Concentration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sensitivity Testing ◽  
Drug Sensitivity Test ◽  
Sensitivity Tests

ABSTRACT A simple double-site sandwich enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum in vitro drug sensitivity tests based on measuring histidine-rich protein 2 (HRP2) is presented. The ELISA uses two commercial monoclonal antibodies and provides a drastically cheaper alternative to the test kits previously used in the HRP2 drug sensitivity test. The assay is simple to establish and perform. The sensitivity is comparable and the drug sensitivity results very closely match those obtained with the commercial ELISA kits (R 2 = 0.979; P < 0.001; mean log difference at the 50% inhibitory concentration = 0.07).

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