HRP-conjugated Anti-EPO antibodies for direct rhEPO detection – proof of concept
Anti-doping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 hours to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish-peroxidase (HRP)-conjugated anti-EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate nonspecific cross-reactivity with secondary antibodies. An in-house, periodate coupled (R&D systems, clone AE7A5), and three commercially available anti-human EPO-HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study.
The in-house anti-EPO-HRP conjugate performed as well as the current two step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 hours with the routine method down to 7 hours with the in-house HRP conjugate. Of the 3 commercially available conjugates tested, only the Santa Cruz anti-EPO-HRP conjugate showed comparable specificity but had lower sensitivity to both the in-house and the antibody combination currently applied routinely.
The other 2 commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP-conjugated anti-EPO antibody to reduce analysis time for EPO in doping control.
Antibody-biotin-streptavidin-horseradish peroxidase (HRP) sensor for rapid and ultra-sensitive detection of fumonisins
Fumonisins (FBs) exist widely in crops, foods and feeds. Consumption of FBs contaminated corn can cause oesophageal cancer. So it is necessary to develop sensitivity methods for its detection. Here, we report an enhanced assay for rapid and ultra-sensitive detection of FBs based on nanomagnetic beads (NMBs) and antibody-biotin-streptavidin-HRP. Because antibody-BNHS can bind with several number of streptavidin-HRP, the signal amplification of the catalytical oxidation of TMB was enhanced.
The detection limit of sensor was down to 0.21 ng mL-1 with a linear range from 0.31 to 162.42 ng mL-1. Since NMBs provide a nearly “in solution” reaction, they lead to a shortened reaction time (22 min) than that of flat solid-phase based traditional assay. Furthermore, the recoveries obtained by standard FBs spiked to maize samples were from 100.6 to 107.3%. This enhanced assay supplied a rapid, sensitive and reliable method for detection of FBs in maize.
Construction of an HRP-streptavidin bound antigen and its application in an ELISA for porcine circovirus 2 antibodies
A fusion protein SBP-Cap∆41, consisting of Cap∆41 (without 41 amino acids at the N-terminus) protein of porcine circovirus 2 (PCV2) and a streptavidin binding peptide (SBP), was constructed. This fusion protein binds to HRP-labeled streptavidin (HRP-SA) through high affinity between SBP and SA, forming an HRP-streptavidin bound antigen (Hsb-Ag) with both immunoreactivity and enzymatic activity, which can be used in a double-antigen sandwich ELISA for detection of PCV2 antibodies.
Comparison of the characteristics of the HSb-Cap∆41 and chemical conjugates of the recombinant Cap∆41 protein showed that the HSb-Cap∆41 based double-antigen sandwich ELISA (HBDS-ELISA) had higher specificity and sensitivity. Use of the HBDS-ELISA detected PCV2-IgG in 9 injected pigs as early as 10 days p.i., 3 days earlier than both a double-antigen sandwich ELISA (DS-ELISA) based on a chemically conjugated antigen, and a commercial indirect ELISA kit.
A Fe3O4 Au-basedpseudo-homogeneous electrochemical immunosensor for AFP measurement using AFP antibody-GNPs-HRP as detection probe
In this study, a Fe3O4@Au-based pseudo-homogeneous electrochemical immunosensor was prepared for detection of alpha fetoprotein (AFP), a well-known hepatocellular carcinoma biomarker. The primary antibody (Ab1) was immobilized on Fe3O4@Au NPs as the capture probe. Horseradish peroxidase (HRP) and secondary antibody (Ab2) were conjugated on gold nanoparticles (GNPs) through electrostatic adsorption to form signal-amplifying labels. In the presence of AFP, a sandwich immunocomplex was formed via specific recognition of antigen-antibody in a Fe3O4@Au-basedpseudo-homogeneousreaction system.
After the immunocomplex was captured to the surface of magnetic glassy carbon electrode (MGCE), the labeling HRP catalyzed the decomposition of H2O2, resulting in a substantial current for the quantitative detection of AFP. The amperometric (i-t) method was employed to record the response signal of the immunosensor based on the catalysis of the immobilized HRP toward the reduction of H2O2 with hydroquinone (HQ) as the redox mediator.
Under the optimal conditions, the amperometric current response presented a linear relationship with AFP concentration over the range of 20 ng/mL-100 ng/mLwith a correlation coefficient of 0.9940, and the detection limit was 0.64 ng/mL at signal/noise [S/N] = 3. Moreover, the electrochemical immunosensor exhibited higher anti-interference ability, acceptable reproducibility and long-term stability for AFP detection.
High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues Using Secondary Antibody Conjugated to Polymer-HRP.
A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody.
Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.
Electrochemical immunosensor for interferon-γ based on disposable ITO detector and HRP–antibody-conjugated nano gold as signal tag
Tuberculosis is the most frequent cause of infection-related death worldwide. A new disposable electrochemical immunosensor with low cost and simple fabrication was proposed to detect interferon-γ (IFN-γ). Diallyldimethylammonium chloride (PDDA) and Au nanoparticle (AuNP) composite were used to provide an efficient biointerface, horseradish peroxidase (HRP)-labeled antibody-conjugated AuNP (HRP-Ab2-AuNP) bioconjugates were used as a novel signal tag.
The large amounts of HRP on the signal tag can catalyze the oxidation of Hydroquinone (HQ) by H2O2, which can induce an amplified reductive current.
HRP 2 (HRP 2) Antibody |
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| abx234012-100ug | Abbexa | 100 ug | 577.2 EUR |
HRP Antibody |
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| 20-abx134340 | Abbexa |
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HRP Antibody |
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| abx015753-100ul | Abbexa | 100 ul | 493.2 EUR |
HRP antibody |
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| 10-3154 | Fitzgerald | 1 mg | 246 EUR |
HRP antibody |
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| 10R-P156a | Fitzgerald | 1 mg | 323 EUR |
HRP antibody |
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| 10R-1969 | Fitzgerald | 100 ul | 435 EUR |
HRP antibody |
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| 20R-3010 | Fitzgerald | 100 µg | 697 EUR |
HRP antibody |
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| 20C-CR7015GP | Fitzgerald | 500 ul | 52 EUR |
HRP Antibody |
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| E10-20162 | EnoGene | 100μg/100μl | 225 EUR |
HRP Antibody |
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| abx015753-1mg | Abbexa | 1 mg | 362.5 EUR |
HRP antibody |
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| MBS831220-1mg | MyBiosource | 1mg | 345 EUR |
HRP antibody |
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| MBS831220-5x1mg | MyBiosource | 5x1mg | 1410 EUR |
HRP antibody |
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| MBS831423-01mL | MyBiosource | 0.1mL | 745 EUR |
HRP antibody |
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| MBS831423-5x01mL | MyBiosource | 5x0.1mL | 3205 EUR |
HRP Antibody |
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| MBS850418-01mg | MyBiosource | 0.1mg | 305 EUR |
HRP Antibody |
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| MBS850418-01mLAF405L | MyBiosource | 0.1mL(AF405L) | 565 EUR |
HRP Antibody |
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| MBS850418-01mLAF405S | MyBiosource | 0.1mL(AF405S) | 565 EUR |
HRP Antibody |
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| MBS850418-01mLAF610 | MyBiosource | 0.1mL(AF610) | 565 EUR |
HRP Antibody |
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| MBS850418-01mLAF635 | MyBiosource | 0.1mL(AF635) | 565 EUR |
HRP antibody |
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| MBS531597-1mg | MyBiosource | 1mg | 590 EUR |
HRP antibody |
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| MBS531597-5x1mg | MyBiosource | 5x1mg | 2510 EUR |
HRP antibody |
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| MBS5310685-01mg | MyBiosource | 0.1mg | 1160 EUR |
HRP antibody |
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| MBS5310685-5x01mg | MyBiosource | 5x0.1mg | 5065 EUR |
HRP antibody |
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| MBS536265-05mL | MyBiosource | 0.5mL | 175 EUR |
HRP antibody |
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| MBS536265-5x05mL | MyBiosource | 5x0.5mL | 635 EUR |
HRP-3 Antibody |
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| C30644-100ul | Assay Biotech | 100μl | 217 EUR |
HRP-3 Antibody |
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| C30644-50ul | Assay Biotech | 50μl | 143.5 EUR |
The catalytic reduction current was related to the amount of HRP immobilized on the surface, which itself was related to the concentration of IFN-γ. Under optimized conditions, the proposed immunosensor showed a high sensitivity and a linear range of 0.1-10,000pg/mL with a detection limit of 0.048pg/mL. The assay results of clinical serum samples obtained by the immunosensor were in acceptable agreement with the reference values. Therefore, the immunosensor possessed excellent clinical value in early diagnosis and control of tuberculosis.