Bioconjugation of Antibodies to Horseradish Peroxidase (HRP)
The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross-linkers to covalently link antibodies to HRP provides a simple and convenient means to maintain antibody affinity while imparting a functional reporter used for antigen detection. In this chapter, we describe a process by which Sulfo-SMCC is used to generate a stable maleimide-activated HRP that is reactive with sulfhydryl groups generated in antibodies by SATA-mediated thiolation.
Nano-magnetic immunosensor based on staphylococcus protein a and the amplification effect of HRP-conjugated phage antibody
In this research, super-paramagnetic Fe3O4 nanoparticles (magnetic particles) were coated with Staphylococcus protein A (SPA) and coupled with polyclonal antibody (pcAb) to construct magnetic capturing probes, and HRP-conjugated phage antibody was then used as specific detecting probe to design a labeled immunosensor for trace detection of Staphylococcus aureus enterotoxin B (SEB). The linear detection range of the sensor was 0.008~125 µg/L, the regression equation was Y = 0.487X + 1.2 (R = 0.996, N = 15, p < 0.0001), the limit of detection (LOD) was 0.008 µg/L, and the limit of quantification (LOQ) was 0.008 µg/L. HRP-conjugated phage antibody, SPA, and magnetic particles can enhance the sensitivity 4-fold, 3-fold and 2.6-fold higher, respectively.
Compared with conventional double-antibody sandwich ELISA, the detection sensitivity of the sensor was 31-fold higher resulting from the integrated amplifying effect. The immunosensor integrates the unique advantages of SPA-oriented antibody as magnetic capturing probe, HRP-conjugated phage antibody as detecting probe, magnetic separation immunoassay technique, and several other advanced techniques, so it achieves high sensitivity, specificity and interference-resistance. It is proven to be well suited for analysis of trace SEB in various environmental samples with high recovery rate and reproducibility.
Improved performance of antigen-HRP conjugate-based immunoassays after the addition of anti-HRP antibody and application of a liposomal chemiluminescence marker
To overcome the sensitivity limit for small molecules (haptens) in immunoassays based on antigen-horseradish peroxidase (HRP) conjugates as labels, a novel approach was established that afforded very low detection limits. Biotinylated anti-HRP antibody was utilized in order to attach, via a streptavidin bridge, liposomaly entrapped HRP. Fentanyl, used as a model antigen, could be determined via the generation of a high-intensity and relatively stable chemiluminescence (CL) signal of a HRP-catalyzed luminol/H(2)O(2)/enhancer system, immediately after the addition of a substrate solution. 4-(1-Imidazolyl)phenol (4-IMP) was used as an enhancer, and the outcome of this combination was a very low detection limit (0.895 pg mL(-1)) in plasma samples. The respective detection limit with the use of just the classical HRP-antigen conjugate was>> 5-times higher. Intra- and inter-assay RSDs of the novel assay were 6.8 – 9.9 and 11 – 17%, respectively. The proposed method could be utilized for a wide range of molecules without replacing existing antigen-HRP based kits.
Signal-amplified platform for electrochemical immunosensor based on TiO2 nanotube arrays using a HRP tagged antibody-Au nanoparticles as probe
In this study, a novel signal-amplified electrochemical immunosensor was proposed by using TiO(2) nanotube (TiNT) arrays as the platform. Due to the distinct tubular features-large surface area, high pore volume and good electrochemical conductivity, the TiNT based electrodes exhibited excellent signal-amplified effects. gold nanoparticle (AuNP) was further utilized to bind horseradish peroxidase (HRP) tagged antibodies as recognition elements. Compared to the immunosensor based on either flat electrode, the immunosensors using TiNT layer as electrode showed higher amplified electrochemical signals from the catalytic reaction of HRP relative to hydrogen peroxide (H(2)O(2)).
Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to antigen in a concentration range from 0.1 ng mL(-1) to 10(5) ng mL(-1) with a detection limit of 0.01 ng mL(-1). The results showed that the TiNT-based electrochemical immunosensing platform could provide a great potential in clinical application for detection of low-abundant proteins.
High-expression of monoclonal nanobodies used in the preparation of HRP-conjugated second antibody
Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy-chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy-chain antibody (VHH) or nanobody. The very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent, so the anti-nanobody HRP conjugate is one of the key components in production, characterization, and application of these molecules in detection and therapy. These antigen-specific fragments are well expressed in Escherichia coli. Here we report high expression and purification of some nanobodies against tumor markers.
The nanobody genes were subcloned into a pET22b(+) vector to overexpress the protein coupled with fusion tag in E. coli. The expressed nanobodies were purified by immobilized metal affinity chromatography and injected into rabbits as an immunogen. Described here are the preparation, purification, and characterization of anti-nanobody HRP conjugate for use in the various nanobody immunoassay systems. Several biochemical modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product. We concluded that the present reagent can detect nanobodies in various detection procedures with great sensitivity and accuracy.
Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system
There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates.
Asprosin Polyclonal Antibody (Human), HRP |
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| 4-PAA332Hu01-HRP | Cloud-Clone |
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Vinculin (VCL) Polyclonal Antibody (Human), HRP |
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| 4-CAB839Hu01-HRP | Cloud-Clone |
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Titin (TTN) Polyclonal Antibody (Human), HRP |
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| 4-PAB667Hu01-HRP | Cloud-Clone |
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Melanotransferrin (MFI2) Polyclonal Antibody (Human), HRP |
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| 4-PAB677Hu01-HRP | Cloud-Clone |
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Cholesterol (CH) Polyclonal Antibody (General), HRP |
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| 4-PAB701Ge01-HRP | Cloud-Clone |
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Glycocalicin (GC) Polyclonal Antibody (Human), HRP |
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| 4-PAB710Hu01-HRP | Cloud-Clone |
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Fascin (FSCN) Polyclonal Antibody (Human), HRP |
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| 4-PAB757Hu01-HRP | Cloud-Clone |
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Fascin (FSCN) Polyclonal Antibody (Mouse), HRP |
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| 4-PAB757Mu01-HRP | Cloud-Clone |
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Neprilysin (NEP) Polyclonal Antibody (Mouse), HRP |
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| 4-PAB785Mu01-HRP | Cloud-Clone |
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Neprilysin (NEP) Polyclonal Antibody (Rat), HRP |
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| 4-PAB785Ra01-HRP | Cloud-Clone |
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Neprilysin (NEP) Polyclonal Antibody (Rat), HRP |
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| 4-PAB785Ra02-HRP | Cloud-Clone |
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Arginine (Arg) Polyclonal Antibody (General), HRP |
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| 4-PAB938Ge01-HRP | Cloud-Clone |
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Epiregulin (EREG) Polyclonal Antibody (Rat), HRP |
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| 4-PAB945Ra01-HRP | Cloud-Clone |
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Acrosin (ACR) Polyclonal Antibody (Rat), HRP |
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| 4-PAB947Ra01-HRP | Cloud-Clone |
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Hepcidin (Hepc) Polyclonal Antibody (Bovine), HRP |
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| 4-PAB979Bo01-HRP | Cloud-Clone |
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Hepcidin (Hepc) Polyclonal Antibody (Human), HRP |
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| 4-PAB979Hu01-HRP | Cloud-Clone |
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Hepcidin (Hepc) Polyclonal Antibody (Mouse), HRP |
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| 4-PAB979Mu01-HRP | Cloud-Clone |
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Hepcidin (Hepc) Polyclonal Antibody (Pig), HRP |
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| 4-PAB979Po01-HRP | Cloud-Clone |
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Hepcidin (Hepc) Polyclonal Antibody (Rat), HRP |
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| 4-PAB979Ra01-HRP | Cloud-Clone |
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Hemopexin (HPX) Polyclonal Antibody (Human), HRP |
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| 4-PAB986Hu01-HRP | Cloud-Clone |
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Hemopexin (HPX) Polyclonal Antibody (Human), HRP |
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| 4-PAB986Hu02-HRP | Cloud-Clone |
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Hemopexin (HPX) Polyclonal Antibody (Rat), HRP |
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| 4-PAB986Ra01-HRP | Cloud-Clone |
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Hemopexin (HPX) Polyclonal Antibody (Rat), HRP |
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| 4-PAB986Ra02-HRP | Cloud-Clone |
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Hemojuvelin (HJV) Polyclonal Antibody (Human), HRP |
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| 4-PAB995Hu01-HRP | Cloud-Clone |
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Lipopolysaccharide (LPS) Polyclonal Antibody (General), HRP |
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| 4-PAB526Ge01-HRP | Cloud-Clone |
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Palladin (PALLD) Polyclonal Antibody (Human), HRP |
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| 4-PAB590Hu01-HRP | Cloud-Clone |
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Neuraminidase (NEU) Polyclonal Antibody (Human), HRP |
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| 4-PAB611Hu01-HRP | Cloud-Clone |
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Myostatin (MSTN) Polyclonal Antibody (Bovine), HRP |
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| 4-PAB653Bo01-HRP | Cloud-Clone |
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Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2′-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.