Horseradish peroxidase: an overview (2023)

Purpose of HRP.6

Research with humans may involve new types of surgical procedures, the use of new drugs, or off-label/label use (without brand) of medications from the current formularies (of currently formulated medications), ouse of adult treatment modalities in pediatric populations and many other research topics and methodologies. Of fundamental importance is the inclusion of research activities in the routine process of the hospital;For example,the process of ordering, dispensing, and administering study drugs. Routine processes also include the reporting of adverse events through quality monitoring and patient safety processes. Therefore, reporting of an adverse event related to a hospital patient in a research protocol should be made to the hospital's quality control mechanism, as well as to the research sponsor or contract research organization. (see alsoQPS.7 y QPS.7.1)

Reporting of events related to research protocols can provide vital information for understanding the overall quality and safety of hospital patient care.For example,a significant adverse event when a drug is used for an off-label/label purpose (without brand) is important patient safety information that should be part of the hospital's ongoing medication management process. Equally important is the management and disposal of certain experimental investigational drugs, which should be a component of hazardous materials management. In addition, the medical equipment used in the experimental procedures must be monitored and maintained.

Therefore, all aspects of the human research program should be evaluated against the hospital quality and safety programs to which they apply, and ongoing reporting and monitoring processes within the hospital should then be incorporated into the research program. . This should also be the case when some research activities are provided by a contract research organization. (see alsoGLD.6)

5.5 Brown or yellow bands on the membrane

HRP turns brown when oxidized and inactive. Within a given amount of conjugated enzyme, there is always a portion that is oxidized. In an optimized system, the amount of HRP oxidized is minuscule and cannot be seen on the blot. The appearance of yellow or brown bands indicates the presence of a large amount of HRP and therefore the oxidized and inactive portion is visible. A blotting system that results in yellow bands requires optimization using much less enzyme conjugate. Also, too much HRP in a localized area produces a large number of free radicals during enzyme activity. Free radicals can inactivate HRP and damage the antibodies, the target and the membrane, preventing effective rejection.

Purpose of HRP.7 and HRP.7.1

A hospital that conducts clinical investigations, clinical investigations, or clinical trials with patients knows that its first responsibility is the health and well-being of patients. The hospital provides information to patients and their families on how to access research relevant to patients' treatment needs.

Safeguards are implemented through the hospital's research review function to protect vulnerable patients who may be at risk of coercion or undue influence to participate in research projects. Vulnerable patients include children, prisoners, pregnant women, people with mental disabilities, people who are economically or educationally disadvantaged, and others with little or no ability to make informed or voluntary decisions to participate in research. Another group that can be considered a vulnerable population is hospital staff. Employees may feel pressured to participate;For example,when the principal investigator is your supervisor.

horseradish peroxidase

horseradish peroxidase(HRP) is a glycoprotein absent from most mammalian cells. It can be used both as a receptor-mediated and fluid phase probe. HRP has been shown to be a ligand for MR (Stahland another., 1978). It is a convenient marker for immunocytochemistry;no premises, HRP can be detected by reaction with diamino-benzidine. Its concentration can be measured by colorimetric assay usingO-phenylenediamine (OPD) and hydrogen peroxide (Strauss, 1964). Polyclonal sera directed against HRP are commercially available and are useful for fluorescence microscopy imaging. HRP has been a standard marker for the endocytic pathway for nearly four decades.Steinmanand another., 1974). It is relatively nontoxic and has only been shown to disrupt the endocytic pathway in thioglycolate-stimulated macrophages.Swansonand another., 1985).

rapid diagnostic tests

While evaluation of Giemsa-stained thick and thin smears remains the accepted standard for diagnosing malaria, rapid diagnostic tests (RDTs) have become increasingly useful.^484–490In situations where specialized microscopic examination is time consuming or difficult to obtain, medical decisions and malaria case management can greatly benefit from the appropriate use of RDTs. In US health care settings, the CDC recommends that all RDT positive and negative results be confirmed with microscopy, which can provide additional information about species, life cycle stages, and level of infection. parasitemia that can be useful in clinical management. Two types of RDTs based on different detection schemes are currently available and are being used more and more.

The first type is based on the detection ofPlasmodiohistidine-rich protein-2 (HRP-2).⁴⁹¹In 556 travelers who returned to France with suspected malaria, a US Food and Drug Administration (FDA)-approved commercial test based on HRP-2 had a sensitivity of 96% and a specificity of 99% forPlasmodioinfection compared to microscopy.⁴⁹²In 32 US Marines who returned from Liberia with febrile illness, this test had a sensitivity of 100% and a specificity of 100% forP sicklesinfection compared to microscopy.⁴⁹³Although the HRP-2 tests are highly sensitive and specific for diagnosing malaria, they have certain limitations. First, HRP-2-based detection is limited toP sicklesmay present negative results in cases where theP sicklesstrain does not express HRP-2 antigen^494–497or produce a prozone effect that interferes with the test.^498.499Other antigen detection schemes are needed toP. vivax, P. ovale,miP. malaria.These are generally less sensitive than HRP-2 detection forP sickles,^492.500making them less useful for diagnosing malaria in returning travelers, who are usually infected withP. vivaxat least as often as withP sicklesSecond, HRP-2 tests have limited use in monitoring therapeutic responses because tests are persistently positive up to 28 days after treatment. Third, the sensitivity of many rapid screening tests forP sicklesmiP. vivaxinfections decrease at parasite densities of less than 100 to 1000/µL,⁵⁰⁰making them less useful for diagnosing malaria in returning non-immune travelers, who may experience malaria symptoms with low parasite densities. A recently improved HRP-2 based RDT format may improve the detection ofP sicklesinfection up to 10 times.^501.502WHO provides a list of prequalified malaria RDT products and manufacturers (https://www.who.int/diagnostics_laboratory/evalues/190527_prequalified_product_list.pdf?ua/=1). In the United States, the BinaxNOW Malaria Test (Abbott, Abbott Park, IL) is FDA-approved for use in hospitals and commercial laboratories, but not in physicians or patients (https://www.cdc.gov/malaria/diagnosis_treatment/diagnostic_tools.html).

4.9 Preparation of the bioCNT-HRP complex

7 Post-functionalization of GCE/Ct-RGO–PAMAM by the enzyme horseradish peroxidase

17.2.3.1 Direct Electronic Transfer of HRP

horseradish peroxidase(HRP) is a member of the large class of peroxidases, which are enzymes defined as oxidoreductases that use hydroperoxide as an electron acceptor. Due to its commercial availability in high purity, HRP has been a representative system to investigate the structure, dynamics and thermodynamic properties of peroxidases, especially to understand their biological behaviors of catalyzing the oxidation of substrates by H₂O₂[125]. HRP can react with H₂O₂to form a powerful enzymatic oxidizing agent known as Compound I, which is a two equivalent oxidized form containing a heme oxyferryl (Fe⁴⁺=O) and a cationic porphyrin radical φ. Compound I is catalytically active and can extract an electron from the substrate to form a second intermediate, called Compound II, which is subsequently reduced to the resting state of the native enzyme, HRP-Fe(III), by accepting an additional electron from the substrate. HRP-Fe(III) can also be further reduced to HRP-Fe(II). Efficient electron transfer between HRP and electrodes has been reported for many years.126]. However, in most cases, direct electrochemistry in the presence of H has been tested.₂O₂or other peroxides by amperometry and attributed to the electrochemical reduction of Compound I or Compound II. Only a few examples of HRP-independent quasi-reversible CVs in the absence of peroxides have been reported, probably due to the large molecular weight and extended structure of HRP, and the inaccessibility of its redox centers.127–130]. ferri [127–128] reported a pair of nearly reversible CV peaks for the HRP Fe(III)/Fe(II) redox couple trapping HRP on a tributylmethylphosphonium chloride (TBMPC) polymer film attached to an anion exchange resin on electrodes. PG. Chen [129–130] then explored the direct electrochemistry of the HRP Fe(III)/Fe(II) pair using CV on DDAB films and DNA on PG electrodes. These films provided a suitable microenvironment for the HRP, which greatly facilitated the exchange of electrons between the HRP and the electrodes. Electrochemical catalytic reduction of H₂O₂by HRP in these films was also described. Recently, a new functional Nafion-cysteine ​​membrane was constructed [131]. Fast and direct HRP electron transfer was performed on the functional membrane modified gold electrode with good stability and repeatability.Poly(sulfonic acid ester), trade name Eastman AQ, is a type of anionic ionomer. Unlike another more popular ionomer, Nafion, this thermoplastic amorphous polymer provides low-viscosity, translucent dispersions in water without the addition of an organic solvent. AQ films placed on the electrodes do not dissolve in water. Like Nafion, AQ films preferentially bind hydrophobic cations and exclude negatively charged species [132]. Enzymes can be added directly to aqueous dispersions of AQ ionomers and deposited on solid surfaces to form films without denaturation or significant loss of activity.133]. Direct electrochemistry of small redox proteins such as cytC[134], MB [135], e Hb [136] in AQ films on PG electrodes was previously studied. Thus, Huang Rong and Hu Naifei [137] produced stable films of poly(sulfonic acid ester) ionomer or Eastman AQ29 on PG electrodes and achieved direct electrochemistry for the incorporated HRP enzyme. Cyclic voltammetry of HRP-AQ films showed a well-defined and nearly reversible pair of peaks at approximately -0.33 V (versus SCE) at pH 7.0 in blank buffers, characteristic of the HRP heme Fe redox couple. (III) /Fe(II) . Electron transfer between the HRP and PG electrodes was greatly facilitated in the AQ films. Infrared absorption, reflectance absorption (RAIR) and ultraviolet visible (UV-Vis) spectra demonstrated that HRP maintained a nearly native conformation in AQ films. HRP embedded in AQ films retained electrocatalytic activity for oxygen, nitrite, and H₂O₂.

Omi⁰' was linearly dependent on the pH of the solution (Bohr redox effect), indicating that the electron transfer was proton-coupled. UV-Vis absorption spectra and RAIR spectra suggested that the conformation of HRP in the agarose film was slightly different from that of the protein alone, and the conformation changed reversibly in the range of pH 3.0–10.0. Atomic force microscopy images of the agarose film indicated a stable crystalline structure formed possibly due to synergistic hydrogen bonding interaction between DMF, agarose hydrogel, and HRP. This suggested a strong interaction between the heme protein and the agarose hydrogel. DMF played an important role in immobilizing proteins and increasing the rate of electron transfer between the protein and the electrode.

It is known that the TiO₂It is widely used in cosmetics, solar cells, batteries, toothpaste additives, and white paint, among others. Recently, there has been considerable interest in the use of TiO₂nanoparticles as a film-forming material, since they have a highsurface, optical transparency, good biocompatibility and relatively good conductivity. Various TiO₂Films have also been used to immobilize proteins or enzymes on the electrode surface for the mechanical study of proteins or the fabrication of electrochemical biosensors. For example, Durrant and his colleagues immobilized a variety of proteins on nanoporous TiO2.₂film-modified electrodes and have successfully used this strategy to develop electrochemical and optical biosensors.141–142]. Luo and his coworkers used nanocrystalline TiO2₂films on electrodes to trap heme proteins such as cytC, Mb, and Hb, and looked at the direct electrochemistry of these proteins [143]. Accumulation and electroactivity of cit.Cin TiO layer-by-layer mesoporous films₂and phytate on ITO electrodes was also studied [144]. Titanium sol-gel matrix films were used to immobilize HRP and, with the help of mediators, HRP-TiO₂Film electrodes were used to detect H₂O₂by amperometry [66,145]. Hu group [146] also incorporated HRP into TiO₂Modified nanoparticle films on electrodes to obtain direct electron transfer from HRP. HRP-TiO₂Film electrodes were fabricated by melting a mixture of HRP solution and an aqueous dispersion of titania nanoparticles onto PG electrodes and allowing the solvent to evaporate. HRP embedded in TiO₂The films exhibited a pair of well-defined and nearly reversible CV peaks at approximately -0.35 V in pH 7.0 buffer, reflecting that the rate of electron exchange between the enzyme and the PG electrodes was greatly improved in the uncle₂nanoparticle film microenvironment. HRP-TiO₂film electrodes were quite stable and suitable for long-term voltammetric experiments. UV-Vis spectroscopy showed that the position and shape of the Soret absorption band of HRP in TiO₂the films were almost unchanged and were different from those for hemin or hemin-TiO₂films, suggesting that HRP retains its native tertiary structure in TiO₂Movie (s. The electrocatalytic activity of HRP embedded in TiO₂movies towards O₂wed H₂O₂was retained

4.1.5 Horseradish peroxidase (HRP)

HRP was introduced for structural analysis based on electron microscopy (Strauss, 1959) because it is possible to make the reaction product an electron-dense material (Brightman y Reese, 1969), allowing microscopic examination of the BBB structure, as the HRP antibody will bind to endothelial cell walls and tight junctions (Brightman y Reese, 1969). In an in vivo study, HRP is typically injected, and in an in vitro system, it is typically introduced into the donor chamber. Once introduced into the in vitro system, the cells are dissected and can be visualized using electron microscopy. The evaluation, in this case, is purely visual or based on image analysis software. The main advantage of HRP is that it requires a very small amount of antibodies and is highly specific for a particular cell type.

1.1 Horseradish peroxidase (HRP)

The use of antibody-HRP or streptavidin-HRP conjugates in peroxidase-catalyzed enhanced chemiluminescent assays may result in one of the most sensitive detection methods for analyzing specific analytes in ELISA and western blot applications. The cascade of reactions that occurs during HRP catalysis can be drastically enhanced by the addition of various enhancer molecules, which create oxidized intermediates that lead to the oxidation and light emission of a chemiluminescent substrate such as luminol.Vdovenkoand another(2012)analyzed this reaction using a multifactorial design of experiments (DOE) to identify the best combination and concentration of H₂O₂, luminol and two different enhancer compounds (3-(10'-phenothiazinyl)propane-1-sulfonate and 4-morpholinopyridine). This combination at an optimized concentration resulted in the best signal-to-noise ratio and the longest chemiluminescent emission.

HRP is a hemoprotein that contains photohemin IX as its prosthetic group. The presence of the heme structure gives the enzyme its characteristic color and its maximum absorption capacity at 403 nm. The ratio between its absorption in solution at 403 nm and its absorption at 275 nm, called the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However, there are at least seven isozymes for HRP (Shannonand another, 1966;kayand another, 1967;Stricklandand another, 1968), and their RZ values ​​range from 2.50 to 4.19. Therefore, unless the RZ ratio for the particular HRP isoenzyme used in the preparation of an antibody-enzyme conjugate is known or accurately determined, subsequent measurement after crosslinking would yield questionable results in determining the amount of HRP present. in the conjugate.

HRP is a glycoprotein that contains significant amounts of carbohydrates. Its polysaccharide chains are often used in cross-linking reactions to couple the enzyme to target molecules. Mild oxidation of its associated glycan sugar residues with sodium periodate generates reactive aldehyde groups that can be used for conjugation with amine-containing molecules. The reductive amination of oxidized HRP to antibody molecules in the presence of sodium cyanoborohydride is perhaps the simplest method for preparing highly active conjugates with this enzyme.Chapter 4, Section 1.4, miChapter 20, Section 1.3).

Other HRP conjugation methods include the use of the homobifunctional glutaraldehyde reagent (Chapter 5, Section 6.2, miChapter 15, Section 2.1) and the heterobifunctional crosslinker, SMCC (succinimidyl-4-(norte-maleimidometil)ciclohexano-1-carboxilato) (Chapter 6, Section 1.3). Using glutaraldehyde, a two-step protocol is generally employed to attempt to limit the extent of oligomer formation. However, even using the most controlled reactions, this method often produces unacceptable amounts of precipitated conjugate. Despite this drawback, glutaraldehyde conjugation is still used routinely, especially in the preparation of some antibody-enzyme reagents that enter into established diagnostic assays. the use ofnorteThe β-hydroxysuccinimide-maleimide (NHS) ester crosslinker, SMCC, provides much better control over the conjugation process. Generally, SMCC is first reacted with HRP to create a derivative containing sulfhydryl-reactive maleimide groups. Activation of HRP by the native enzyme should result in the modification of at most two amino groups in the protein, because HRP contains only two lysines. An increase in the level of activation can be achieved if the enzyme is first modified with ethylenediamine (EDA) using the carbodiimide EDC according to the methods described inchapter 19for the production of cationized bovine serum albumin (cBSA). EDA-modified HRP is also more stable than the unmodified version, so cationization may have benefits in retaining enzyme activity. The maleimide-activated enzyme can be purified and lyophilized, providing a ready source of HRP modified to react with a sulfhydryl-containing antibody. Various preactivated forms of this enzyme are available from Thermo Fisher.

The size of the HRP is an advantage in the preparation of antibody-enzyme conjugates, since the total size of the complex can also be designed to be small. Relatively low molecular weight conjugates can penetrate cell structures better than large polymeric complexes. This is the reason why HRP conjugates are often the best choice for immunohistochemical (IHC) and immunocytochemical staining techniques. The small size of the conjugate means greater accessibility to antigenic structures in tissue sections.

Another distinctive advantage of HRP is its robustness and stability, especially under the conditions used for crosslinking. HRP is stable for years in the lyophilized state, and the purified enzyme can be stored in solution at 4°C for many months without significant loss of activity. The enzyme also retains excellent activity after being modified with a conjugating reagent or after being oxidized with periodate tothey form aldehyde groups on their polysaccharide chains. Depending on the methods used for cross-linking, HRP conjugates can be constructed to have a high enzyme/antibody ratio or a low ratio, both of which retain high specific activity.

The disadvantages associated with HRP are several. The enzyme contains only two available primary ε-amine groups, remarkably low for most proteins, limiting its ability to be activated by amine-reactive heterobifunctionals. HRP is sensitive to the presence of many antibacterial agents, especially azide. It is also reversibly inhibited by cyanide and sulfide (Theorell, 1951). Finally, although the enzymatic activity of HRP is extremely high, its shelf life or practical substrate development time is somewhat limited. After approximately one hour of substrate renewal, in some situations its activity may be severely diminished.

However, HRP is by far the most popular enzyme used in antibody-enzyme conjugates. Research on the use of enzymes indicated that HRP is incorporated into approximately 80% of all antibody conjugates, most of which are used in diagnostic assay systems.