Horseradish peroxidase: an overview (2023)

Purpose of HRP.6

Research in humans may involve new types of surgical procedures, the use of new medications, or off-label/off-label uses (Without brand) of drugs in current formulations (of current drug formulations), ÖUse of adult treatment modalities in pediatric populations and many other research topics and methods. The integration of research activities into daily clinical practice is of fundamental importance;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 associated with inpatients in a research protocol should be made to the hospital's quality control mechanism, as well as to the research sponsor or CRO. (See alsoQPS.7 y QPS.7.1)

Reporting of events related to research protocols can provide important 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 purpose (Without brand) is important patient safety information that should be part of the hospital's ongoing medication management process. Equally important is the handling and disposal of certain experimental investigational drugs, which should be part of hazardous materials management. Additionally, medical devices used in experimental procedures require supervision and maintenance.

Therefore, all aspects of the human research program should be evaluated against the quality and safety programs of the hospital 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 investigative activities are carried out by a CRO. (See alsoGLD.6)

5.5 Brown or yellow streaks on the membrane

HRP turns brown when oxidized and inactive. Within a given amount of conjugated enzyme, there is always a part that is oxidized. In an optimized system, the amount of HRP oxidized is minimal and cannot be visualized 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 part 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, target, and membrane, preventing effective reprobing.

Intent of HRP.7 and HRP.7.1

A hospital conducting clinical investigations, clinical investigations, or clinical trials with patients recognizes that its primary responsibility is the health and well-being of patients. The hospital educates patients and their families on how to access research relevant to patients' treatment needs.

Safeguards are put in place through the hospital's research review function to protect vulnerable patients who are at risk of being coerced or improperly influenced to participate in research projects. Patients at risk include children, inmates, pregnant women, people with intellectual disabilities, those who are economically or educationally disadvantaged, and others who are limited or unable to make informed or voluntary decisions about their participation in research. Another group that can be considered a vulnerable population is hospital staff. Staff may feel pressured to participate;For example,when the principal investigator is your supervisor.

Meerrettich-Peroxidase

Meerrettich-Peroxidase(HRP) is a glycoprotein that is absent from most mammalian cells. It can be used in both liquid phase and receptor-mediated probe. HRP has been shown to be a ligand for MR (Stahlet al., 1978). It is a convenient tracer for immunocytochemistry;in the place, HRP can be detected by reaction with diaminobenzidine. Its concentration can be measured by a colorimetric assay.o-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 (stone manet al., 1974). It is relatively nontoxic and has only been shown to disrupt the endocytic pathway in thioglycolate-stimulated macrophages.Schwansonet al., 1985).

rapid diagnostic tests

Although the evaluation of Giemsa-stained thick and thin swabs remains the accepted standard for malaria diagnosis, rapid diagnostic tests (RDTs) are becoming more useful.^484–490In situations where expert microscopic examination is delayed or difficult, medical decision-making and malaria case management can greatly benefit from the proper use of RDTs. In US healthcare, the CDC recommends that all positive and negative RDT results be confirmed with microscopy, which can provide additional information about species, life-cycle stages, and range of parasitemia that may be useful for clinical management. Two types of RDTs, based on different detection schemes, are currently available and are becoming more common.

The first type is based on the detection ofPlasmodiohistidinreiches Protein-2 (HRP-2).⁴⁹¹In 556 travelers returning 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 versus microscopy.⁴⁹²In 32 US Marines returning from Liberia with febrile illness, this test had a sensitivity of 100% and a specificity of 100% forP sicklesInfection versus 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 sicklesand can in cases where theP sicklesEither strain does not express the HRP-2 antigen^494–497or creates a prozone effect that interferes with the test.^498.499Other antigen detection schemes are requiredP. vivax, P. ovale,YP. malariae.These are generally less sensitive than HRP-2 detection.P sickles,^492.500This makes them less useful for diagnosing malaria in returning travelers who are normally infected with malaria.S. vivaxat least as often as withP sicklesSecond, HRP-2 tests have limited use in monitoring therapeutic response because tests are persistently positive up to 28 days after treatment. Third, the sensitivity of many rapid screening tests, e.g.P sicklesYS. vivaxInfections decrease with parasite densities of less than 100 to 1000/µL,⁵⁰⁰This makes them less useful for diagnosing malaria in non-immune returnees, who may show malaria symptoms when parasite density is low. 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 for 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 transfer of electrons from HRP

Meerrettich-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 long been a representative system to study the structure, dynamic and thermodynamic properties of peroxidases, particularly to understand their biological behavior in catalyzing the oxidation of substrates by H₂o₂[125]. HRP can react with H₂o₂to form a strong enzymatic oxidant known as compound I, a two equivalent oxidized form containing a heme oxyferryl (Fe⁴⁺=O) and a cationic radical φ of porphyrin. Compound I is catalytically active and can withdraw an electron from the substrate to form a second intermediate called compound II, which is subsequently reduced to the resting state of the native HRP-Fe(III) enzyme 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]. In most cases, however, it has been shown that direct electrochemistry in the presence of H₂o₂or other peroxides determined by amperometry and attributed to the electrochemical reduction of compound I or compound II. Few examples of HRP-independent quasi-reversible CVs in the absence of peroxides have been reported, probably due to the large molecular mass 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 when trapping HRP on tributylmethylphosphonium chloride (TBMPC) polymer film attached to PG electrodes on anion exchange resin. Chen [129–130] then studied the direct electrochemistry of the HRP Fe(III)/Fe(II) pair by CV on DDAB and DNA films 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 has also been described. Recently, a new functional cysteine ​​membrane was constructed from Nafion [131]. Fast and direct electron transfer from HRP was performed on the membrane-functional modified gold electrode with good stability and repeatability.Poly(ester sulfonic acid) under the tradename Eastman AQ is a type of anionic ionomer. Unlike another more popular ionomer, Nafion, this thermoplastic amorphous polymer produces low-viscosity, translucent dispersions in water without the addition of organic solvents. AQ films cast on 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] y Hb [136] in AQ films on PG electrodes has been previously studied. So Huang Rong and Hu Naifei [137] prepared stable ionomeric poly(sulfonic acid ester) or Eastman AQ29 films on PG electrodes and achieved direct electrochemistry for the incorporated HRP enzyme. Cyclic voltammetry of HRP-AQ films revealed a pair of well-defined and nearly reversible peaks around -0.33 V (versus SCE) at pH 7.0 in blank buffers, characteristic of HRP-heme redox. Fe(III)/Fe(II). couple. Electron transfer between the HRP and PG electrodes was greatly facilitated in the AQ films. Reflectance-infrared absorption (RAIR) and UV-visible (UV-Vis) absorption spectra showed that HRP retained a nearly native conformation in AQ films. HRP embedded in AQ films retained electrocatalytic activity for oxygen, nitrite, and H₂o₂.

Hemi⁰' was linearly dependent on the pH of the solution (redox-Bohr effect), indicating that the electron transfer was proton-coupled. UV-Vis absorption spectra and RAIR spectra indicated that the conformation of HRP in the agarose film differed little from that protein alone and that the conformation changed reversibly in the pH range 3.0-10.0. Atomic force microscopy images of the agarose film revealed a stable, crystal-like structure, possibly formed due to the synergistic interaction of hydrogen bonds 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, additives in toothpaste and white paint and others. Recently, there has been considerable interest in the use of TiO₂Nanoparticles as a film-forming material due to its highsurface, optical transparency, good biocompatibility and relatively good conductivity. Various TiO₂The films have also been used to immobilize proteins or enzymes on the electrode surface for mechanistic studies of proteins or to fabricate electrochemical biosensors. For example, Durrant and his colleagues immobilized a variety of proteins on nanoporous TiO2.₂developed film-modified electrodes and successfully used this strategy to develop electrochemical and optical biosensors.141–142]. Luo and his collaborators 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]. Cyt accumulation and electroactivityCin TiO layer-by-layer mesoporous films₂and phytate in ITO electrodes has also been studied [144]. Titanium oxide 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]. House group [146] also incorporated HRP into TiO₂Modified nanoparticle films on electrodes to achieve direct electron transfer from HRP. HRPTiO₂Film electrodes were prepared by pouring the mixture of HRP solution and aqueous dispersion of titanium oxide nanoparticles onto PG electrodes and evaporating the solvent. The HRP embedded in TiO₂The movies showed a pair of well-defined and nearly reversible CV peaks at around -0.35 V in pH 7.0 buffer, reflecting that the rate of electron exchange between the enzyme and the PG electrodes was increased. considerably in TiO₂Nanoparticle film microenvironment. TiO HRP₂The film electrodes were quite stable and suitable for long-term voltammetry experiments. UV-Vis spectroscopy showed that the position and shape of the Soret absorption band of HRP in TiO₂The films were almost unchanged and differed from those of hemin or hemin-TiO₂films, suggesting that HRP retains its native-like tertiary structure in TiO₂films. The electrocatalytic activity of HRP embedded in TiO₂Movies about O₂yh₂o₂It has remained.

4.1.5 Meerrettichperoxidasa (HRP)

HRP was introduced for the analysis of electron microscopic structures (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 binds 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 used for visualization using electron microscopy. The evaluation is purely visual or based on image analysis software. The main advantage of HRP is that it requires an extremely small amount of antibodies and is highly specific for a given cell type.

1.1 Meerrettichperoxidasa (HRP)

The use of antibody-HRP or streptavidin-HRP conjugates in peroxidase-catalyzed enhanced chemiluminescence assays may result in one of the most sensitive detection methods for target analyte determination 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 that generate oxidized intermediates that lead to the oxidation and light emission of a chemiluminescent substrate such as luminol.Vdovenkoet al.(2012)analyzed this response using a multifactorial design of experiments (DOE) approach to find the best combination and concentration of H₂o₂, luminol, and two different enhancer compounds (3-(10′-phenothiazinyl)propane-1-sulfonate and 4-morpholinopyridine. This combination resulted in the best signal-to-noise ratio and longest chemiluminescence emission at optimized concentration.

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 absorbance maximum at 403 nm. The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called the RZ or purity number ratio, can be used to determine the purity of the to approach the enzyme. However, there are at least seven isoenzymes for HRP (Shannonet al., 1966;kayet al., 1967;Stricklandet al., 1968) and their RZ values ​​vary from 2.50 to 4.19. Therefore, unless the RZ ratio is precisely known, or precisely known for the particular HRP isoenzyme used in the preparation of an antibody-enzyme conjugate, subsequent measurement after crosslinking would give questionable results for determining the levels present in the existing HRP delivering conjugate. amount.

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 glycan sugar residues associated with sodium periodate generates reactive aldehyde groups that can be used for conjugation with amine-containing molecules. The reductive amination of oxidized HRP on 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, YChapter 20, Section 1.3).

Other methods of HRP conjugation include the use of the homobifunctional reagent glutaraldehyde (Chapter 5, Section 6.2, YChapter 15, Section 2.1) and the heterobifunctional crosslinker SMCC (succinimidyl-4-(norte-maleimidometil)ciclohexan-1-carboxilato) (Chapter 6, Section 1.3). A two-step protocol using glutaraldehyde is commonly used to try to limit the extent of oligomer formation. However, even using the most controlled reactions, this method often causes unacceptable amounts of conjugate to precipitate. Despite this drawback, glutaraldehyde conjugation is still used routinely, particularly in the preparation of some antibody reagents and enzymes that feed established diagnostic assays. the use of thenorte- The hydroxysuccinimide ester (NHS) maleimide crosslinker, SMCC, offers much better control over the conjugation process. Generally, SMCC is first reacted with HRP to produce a derivative containing sulfhydryl-reactive maleimide groups. HRP activation of the native enzyme should result in the modification of a maximum of about two amine groups in the protein since 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 carbodiimide-EDC according to the methods described in US Pat.chapter 19for the production of cationized bovine serum albumin (cBSA). EDA-modified HRP is also more stable than the unmodified version, so cationization may have advantages in retention of 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 production of antibody-enzyme conjugates since the total size of the complex can also be made small. Relatively small molecular weight conjugates can penetrate cell structures better than large polymeric complexes. For this reason, HRP conjugates are often the best choice for immunohistochemical (IHC) and immunocytochemical staining techniques. The small size of the conjugate means better accessibility to antigenic structures within tissue sections.

Another key benefit of HRP is its strength 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 oxidized to periodate.they form aldehyde groups on their polysaccharide chains. Depending on the methods used for cross-linking, HRP conjugates can be designed to have either a high enzyme/antibody ratio or a low ratio, both of which retain high specific activity.

The disadvantages associated with HRP are multiple. The enzyme contains only two available primary ε-amine groups (extraordinarily few for most proteins), limiting its ability to be activated by amine-reactive heterobifunctionals. HRP is sensitive to the presence of many antibacterial agents, particularly 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 about an hour of substrate renewal, its activity can drop drastically in some situations.

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