Figure 1. Recent developments in diagnostic tools and bioanalytical methods for analysis of snake venom.

According to statistics from the World Health Organization, disability and death from venomous snake bites are still common phenomena in developing countries. Before the use of antivenom, there is still a general lack of feasible experimental methods for snake venom detection. At present, the clinical identification of snake bites mainly relies on the patient’s description of the snake’s morphology, the manifestations of the bite site and systemic symptoms, and simple tests such as coagulation, blood routine, and urine routine. However, the appearance of some non-venomous snakes is similar to that of venomous snakes. Clinically, it is also encountered that venomous snakes “dry bite” without detoxifying without poisoning. In addition, the limitations of these diagnostic methods include errors in patient description, or the time it takes for symptoms to appear after a venomous snake bite, and the lack of specificity in blood biochemistry and urine tests. Snake venom is a highly complex mixture. There are still certain differences in the composition and toxicological effects of snake venom between snakes of the same family, the same genus, or even different families and genera, or a certain snake species in different regions. Therefore, identification of the species of the injuring snake is crucial to the effectiveness of early use of monovalent antivenom in the treatment of venomous snake bites. For a long time, researchers have been committed to developing a stable, reliable, fast, simple, and specific snake venom identification method. Currently, there are radioimmunoassay, agglutination assay, enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay, and proteomics techniques have been used to detect various snake venoms and toxins.

Radioimmunoassay (RIA)

RIA uses radioactive isotopes (such as 3H, 125I, 131I, etc.) to label antigens or labeled antibodies to determine the corresponding antigen, and uses special instruments to monitor the metabolism of the labeled antigen or labeled antigen-antibody complex and calculate the number of antigens to be tested. Compared with double-antibody sandwich ELISA, this method has simpler operation steps and requires less body fluid. However, this method uses radioactive materials and is expensive. In addition to problems related to the short half-life of 125I, it also requires specialized and sophisticated reading equipment to measure isotope levels. It has proven impractical and not very operable in clinical patients. Compared with double-antibody sandwich ELISA, this method has simpler operation steps and requires less body fluid. However, this method uses radioactive materials and is expensive. In addition to problems related to the short half-life of 125I, it also requires specialized and sophisticated reading equipment to measure isotope levels. It has proven impractical and not very operable in clinical patients.

Enzyme-linked Immunosorbent Assay

ELISA is an immunological diagnostic technology that uses enzyme-labeled antigens or antibodies to bind to the corresponding antibodies or antigens in the sample to be tested. The basic principle is to physically adsorb the antigen or antibody to the surface of a certain solid phase carrier while maintaining immune activity, and the protease and the corresponding antibody or antigen are coupled to form an enzyme-labeled antibody or antigen, and the enzyme-labeled antibody or antigen is simultaneously It has immunological activity and enzymatic activity. After combining with the antigen or antibody on the surface of the solid-phase carrier, the enzyme is used as the detection signal. After the enzyme reaction substrate is added, it is catalyzed by the enzyme to produce a colored product. The amount of the product is equal to the amount of the antigen or antibody to be tested. Proportional, according to the color depth, it can be qualitative or a special microplate reader can be used to detect the adsorption signal for quantitative analysis. Use biotin or avidin to amplify the signal to further increase the sensitivity of the experiment. Due to its advantages of sensitivity, specificity and rapidity, ELISA is suitable for testing large quantities of samples and is an internationally recognized standardized diagnostic method.

Liquid Chromatography-Mass Spectrometry (LC-MS)

LC-MS can effectively separate and analyze complex organic mixtures. Liquid chromatography has strong separation capabilities and can effectively separate thermally unstable and high-boiling compounds in mixed organic matter. Combined with the powerful component identification capabilities of mass spectrometers, Analyze the isolated organic compounds one by one to identify the molecular weight, structure and concentration of the organic compounds. Due to the powerful electrospray ionization technology of LC-MS, its mass spectrum is simple and intuitive, and subsequent data processing is simple. Therefore, LC-MS is an essential tool for analyzing organic compounds. Snake venom is a mixture of proteins, enzymes, small amounts of metal ions, carbohydrates and amines with different molecular weights. Its composition is complex and has a variety of biological activities and pharmacological effects. Proteomics technology can be used to study the evolution of venom proteins in different snake genera or the same snake genus in different regions, the relationship between protein structure and function, and the classification status of venomous snakes. With the maturity of the development of proteomics technology, snake venom proteomics has been deeply studied, revealing the components of different snake venom proteins.

Polymerase Chain Reaction (PCR)

PCR is a molecular biology technology used to amplify and amplify target DNA or RNA fragments. The target DNA or RNA is replicated in vitro. The biggest feature of PCR is that it can greatly increase trace amounts of gene fragments.

Fluorescent Immunoassay (FIA)

FIA is a non-radioactive labeling immunoassay technology based on enzyme-labeled immunoreactants and highly active enzyme-catalyzed substrate color or luminescence methods to achieve the purpose of quantitative analysis. Fluorescence methods were introduced into immunological analysis to improve the sensitivity of immunoassays. 4-Methylumbelliferyl phosphate is widely used as a fluorescent substrate in combination with alkaline phosphates.

Immunosensor

Immune sensors are biosensors, also known as optical immune sensors. Their key information converters use photosensitive elements and work using optical principles. Biorecognition molecules (protein antigens, etc.) are solidified on the sensor and interact with light in the optical element, causing the optical signal to change. The immune response is detected by detecting the optical signal caused by the physical change in the thickness of the molecular film on the optical silicon chip. Therefore, the test object can be analyzed and measured.

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Figure 1. Major cardiometabolic actions of GLP1.

GLP-1 Receptor Agonists Affect DCM by Regulating Metabolic Disorders

Hyperglycemia and insulin resistance are the most significant features of DCM. In addition, they are also accompanied by changes in myocardial structure and function caused by calcium imbalance, abnormal lipid metabolism, lipid deposition, and the combined effects of multiple hormone changes. The metabolic disorder of myocardial cells is mainly caused by the increase in blood sugar levels caused by insulin resistance. When diabetes occurs, there are conversion obstacles in both the anaerobic glycolysis and aerobic oxidation of glucose in the body. The body’s utilization of glucose is reduced, lipolysis is increased, and excessive free Fatty acids are absorbed by cardiomyocytes, reducing the heart’s ability to use glucose. At this time, the heart mainly relies on fatty acid oxidation to provide energy, but high oxygen consumption is accompanied by low energy metabolism efficiency. The accumulation of free fatty acids in the form of triglycerides promotes fatty degeneration of the myocardium. Therefore, long-term hyperglycemic environment, abnormal lipid metabolism, and lipid deposition lead to the occurrence and development of DCM in diabetic patients. Multiple studies have shown that GLP-1 receptor agonists can directly or indirectly improve the function of cardiomyocytes by controlling the above multiple metabolic factors and related signaling pathways.

​GLP-1 Receptor Agonists Affect DCM by Improving ERS

In diabetic patients, the endoplasmic reticulum dysfunction caused by cardiac oxidative stress, abnormal lipid metabolism and inflammatory response is called ERS when the body is in a high glucose state. Pathophysiological factors such as ischemia, hypoxia, oxidative stress, or calcium homeostasis disorders can disrupt endoplasmic reticulum homeostasis, cause endoplasmic reticulum dysfunction, and lead to the accumulation of unfolded or misfolded proteins, thus inducing ERS and triggering unfolded protein response, thereby activating the corresponding endoplasmic reticulum and calcium regulatory mechanisms and other signal transduction pathways to restore endoplasmic reticulum homeostasis. When the ERS intensity is too high or lasts for a long time, the endoplasmic reticulum cannot restore homeostasis. At this time, multiple pathways induce cell apoptosis, which together with multiple factors lead to cardiomyopathy and heart failure. Increasing evidence shows that ERS is associated with diabetes, endothelial dysfunction, and atherosclerosis. GLP-1 receptor agonists can inhibit ERS and reduce intracellular calcium concentration through indirect effects, thereby reducing myocardial damage. Their effects may be related to the inactivation of the ERS signaling pathway.

GLP-1 Receptor Agonists Affect DCM by Interfering with Inflammatory Response

Oxidative stress is an important factor driving the inflammatory response. High glucose status and insulin resistance in diabetic patients promote cell metabolism disorders and release a large amount of reactive oxygen species, which in turn activates nuclear factor κB, induces the expression of inflammatory factors, damages vascular endothelial cells, and causes abnormal proliferation of smooth muscle cells. It will affect the structure and function of the heart in the long term, leading to the occurrence and development of DCM. GLP-1 receptor agonists can inhibit the inflammatory pathway through direct or indirect effects, activate the AMP-activated protein kinase (AMPK) pathway, reduce the activity of nuclear factor κB, reduce the production and release of inflammatory mediators, inhibit the differentiation of inflammatory monocytes and macrophages, and Reduce heart and blood vessel inflammation, etc.

GLP-1 Receptor Agonists affect DCM by protecting Mitochondria and Improving Oxidative Stress

The tissue damage of DCM mainly has the following five mechanisms: (1) Increased metabolism of glucose and other sugars through the polyol pathway; (2) Formation of intracellular advanced glycation end products; (3) Advanced glycation end products Increased expression of receptors and ligands; (4) activation of protein kinase C subtype; (5) overexpression of hexosamine pathway activity. Mitochondria are one of the main sources of reactive oxygen species in cardiomyocytes, and reactive oxygen species are the upstream products of the above five pathways. In the body’s high-glucose state, due to changes in metabolic substrates, the oxidation of free fatty acids increases the transfer of reducing equivalents, which can damage the ability of the electron transport chain, increase the generation of reactive oxygen species, excessive reactive oxygen species deposition, or weaken the antioxidant mechanism. The redox imbalance leads to oxidative stress; this process and the high glucose state can simultaneously promote oxidative stress and the body’s inflammatory response, damage myocardial cells, and then lead to myocardial fibrosis, ventricular hypertrophy, coronary microvascular damage, cardiac systolic and diastolic function obstacles, eventually developing into DCM. In the entire pathogenesis of DCM, the effects of GLP-1 receptor agonists on metabolic disorders, accumulation of reactive oxygen species, ERS, and inflammatory responses all require the oxidative stress link. The oxidative stress link can be called the key to the pathogenesis of DCM. Therefore, intervening in the oxidative stress process is an important step in improving the prognosis of DCM. Studies have shown that exenatide reduces oxidative stress in cardiomyocytes and improves mitochondrial function by activating autophagy; it can also remove dysfunctional mitochondria to inhibit cardiomyocyte apoptosis, achieve anti-apoptosis and antioxidant effects, and protect cardiac function. Exenatide can also inhibit the activation of caspase-1, indirectly attenuating mitochondrial damage and reducing reactive oxygen species deposition, thereby attenuating oxidative stress.

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The role of ADE was first reported by Hawkes in the 1960s. Since then, the presence of ADE has been found in the infection of more than 40 kinds of viruses in multiple families and genera. It is often difficult to prevent and control such viral diseases with ADE effects.

Mechanism of ADE Action in Viral Infection

This intricate mechanism has been the focus of intense scrutiny, with scientists examining the involvement of various immune cells such as macrophages, monocytes, B cells, neutrophils, and granulocytes. Further studies have confirmed the involvement of FcRII-specific monoclonal antibodies in blocking ADE when West Nile virus infected macrophage-like cell lines.

Another intricate mechanism of ADE action in flavivirus infection is ADE action mediated by complement receptor (CR). Researchers discovered that the infectivity of West Nile virus to P388D1 cells with FcR was enhanced in the presence of antiviral IgM, and that the specific monoclonal antibody of complement type 3 receptor (CR3) could block this enhancement. Interestingly, FcR antibodies could not block this effect, adding another layer of complexity to the ADE action mechanism.

Figure 1. Two main ADE mechanisms in viral disease.

In vitro studies of human immunodeficiency virus infection have revealed at least five perplexing mechanisms of the ADE effect. These include: the interaction between the virus-antibody complex and FcR, which enhances adhesion to cells; the virus-antibody-complement complex interacts with the CR2 on the target cell, enhancing adhesion; the deposition of complement components on virions assists in the fusion of the viral envelope and the cell membrane; through FcR or CR, complement activation products and intracellular signal transduction have a stimulating effect on target cells, such as enhancing cellular endocytosis; and when the antibody or soluble CD4 is below the neutralizing concentration, it can bind to one subunit of the gp120 oligomer, causing changes in the configuration of other subunits, activating gp120, and promoting the fusion of the viral envelope and the cell membrane.

Despite the perplexing nature of the ADE action mechanism, it is crucial to continue studying it to develop effective treatments for viral infections. With its intricate and mind-bending complexities, ADE action mechanism remains a captivating topic for researchers to explore further.

Significance of Research on the Mechanism of Action of ADE

The salience of scrutinizing the intricacies of antibody-dependent enhancement (ADE) is rooted in its ability to exacerbate diseases and catalyze outbreaks. A prime example of this was Thailand’s horrific dengue virus infection epidemic in the 1980s, whereby young children who had previously contracted the virus often exhibited dengue hemorrhagic fever and dengue shock syndrome. Moreover, these children frequently underwent secondary infections, predominantly involving a blend of dengue virus types 1 and 2. Subsequently, researchers discovered that pre-infection with dengue virus type 4 in monkeys could intensify subsequent infections with dengue virus type 2, and inoculating immune serum into these primates could instigate high levels of viremia. Collectively, these findings revealed that while the emergence of dengue hemorrhagic fever and shock syndrome is determined by numerous factors, ADE can indeed augment the pathogenicity of dengue virus infection.

Ever since the advent of the rabies vaccine by Pasteur, it has been discovered that pre-existing antibodies can expedite disease progression and aggravate disease severity in various viruses. Although this phenomenon is not necessarily entirely attributable to ADE, it is still a formidable concern for researchers studying antiviral vaccines. As evidenced by numerous studies, the inactivated vaccine for goat arthritis not only fails to deter infection, but also fosters the occurrence of inflammatory diseases. At times, vaccines for monkey AIDS and equine infectious anemia can also incite infection. In clinical practice, vaccination with the porcine reproductive and respiratory syndrome virus (PRRSV) vaccine can occasionally exacerbate symptoms. Antibodies elicited by the PRRSV vaccine virus can heighten the replication of wild strains of the virus in pigs, and the propagation of the vaccine virus can also be augmented by maternal antibodies engendered by wild strains of the virus. Following a pig population’s infection with wild strains of PRRSV, the levels of immunity within the population fluctuate dramatically, and there are marked disparities in ADE between different individuals. Additionally, piglets that are passively immunized against PRRSV by maternal antibodies exhibit ADE activity when the antibody levels drop below protective levels (sub-neutralizing levels), which escalates the risk of animal infection and disease.

Significance of Research on the Mechanism of Action of ADE

The genetic diversity of viruses is prodigious, and this diversity is correlated with the development of vaccines, as immunity provoked by one strain may only partially resist limited infections by different strains. The virus strain displays a vast array of antigenic diversity, and there are significant disparities in ADE. Therefore, it is imperative to employ pathological models to scrutinize the position and role of ADE in the pathogenic mechanism of viruses with ADE effects, identify the antigenic determinants linked to ADE in these viruses, and modify or eliminate them from candidate vaccine viruses in order to engender safe and effective vaccines.

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Antibody’s main funcion is to bind with antigens including the outside and the inside to eliminate the microorganism and oarasite which intrude the host’s body. Antibody is also a protein produced by reaction of antigen and binding with specificity of antigen. Thus every kinds of antibody can bind with Epitope of specific antigen. These binds can inactivat antigens but it doesn’t work and even cause the Pathological damage of the host somtimes.

Antibody always has its law. The following is the reaction process:

Initial reaction to produce antibodies: when antibody enter the host, it will produce antibody after an incubation period. It produce a little antibody and antibody last a short time.
The reaction to produce antibodies again: When same antigen enter the host again, some elements of old antibody of host will bind with the new antibody at first. Then, antibody titers increase rapidly and it will grow more than several and even dozens of times than the antibody in first entry. In addition, it will be kept in a long period.
Recalls the reaction to produce antibodies: because the antibody sitmulated by antigen will appear after a period. It will increase rapidly again if it bind with disappeared antibody. If the second antigen stimulating host is same as the first one, it can be called Specific recall response and if it is different from the first one, it called Non-specific recall response. Antibody’s growth produced by non-specific recall response is temporary and it will drop in rapid speed.

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Biological Detection Method

The detection method of Staphylococcus aureus enterotoxin B initially used relevant detection methods such as animal experiments. By intraperitoneal injection and feeding of samples with enterotoxin B added to the test animals, the abnormal physiological responses and activities of the animals were observed and recorded. The existence and toxicity of enterotoxin B were qualitatively analyzed. Such experiments are intuitive in the interpretation of the results, but the shortcomings such as poor specificity limit the application of this method.

Immunoassays Method

Immunoassays are mainly based on the principle of specific binding of antigens and antibodies, including immunoagglutination assays, agarose diffusion assays and enzyme-linked immunosorbent assays. However, the detection sensitivity of these methods is only 0.1 ng/mL, and the sample preparation and detection takes a long time. In addition, this method needs to ensure that the monoclonal antibodies have a uniform structure and avoid cross-reaction with each other.

Figure 1. Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor.

Immunoagglutination Assay

The immunoagglutination test is the agglutination produced by the combination of antigen and antibody in the presence of electrolytes. These include the reverse indirect hemagglutination test and the reverse passive latex agglutination test. The former uses the antiserum of enterotoxin B to adsorb on the surface of red blood cells of animals. When the test specimen contains SEB, hemagglutination occurs. This method can interpret the results in about 2 hours, and the detection sensitivity is greatly improved compared with the previous method; the latter adsorbs the anti-SEB specific antibody on the latex particles. When the electrolyte and SEB exist at the same time, the latex particles will agglutinate phenomenon, the detection time is about 18 h.

Immunolabeling Technology

Immunolabeling technology can be divided into enzyme-linked immunosorbent assay, radioimmunoassay and immunofluorescence detection technology. It is based on the traditional antigen-antibody reaction, by modifying fluorescent groups on the antibody, such as hydroxyfluorescein, fluoresceine isothiocyanate (FITC), water-soluble indocyanine fluorescein (cyanine-5), etc., radioisotopes, horseradish peroxidase (HRP) or alkaline phosphatase. Relevant instruments and equipment are used to output the signal intensity to achieve the purpose of detection.

• Enzyme-linked immunosorbent assay (ELISA) is an antigen-antibody reaction based on a solid-phase carrier (such as polystyrene, etc.), which catalyzes the color development of the substrate by HRP or AP, thereby amplifying the detection signal. Double-antibody sandwich and indirect competition methods are usually used to detect SEB, in which the former can obtain lower detection sensitivity and wider linear range. At present, ELISA technology has been widely used in practical detection, and targeted detection kits have been developed.
• Radioimmunoassay is based on the competitive reaction of labeled antigens (labeled with radioisotopes) and unlabeled antigens to antibodies, and is used to detect the antigen concentration of the sample to be tested. This method has high sensitivity and specificity, but it is not suitable for widespread use due to the contamination of radioisotopes.
• Immunofluorescence detection technology uses fluorescent substances (FITC, Cy5, quantum dots, up-conversion particles, etc.) as signal molecules labeled on the antibody, and indirectly determines the concentration of SEB through the fluorescence intensity generated by the labeled antibody bound to SEB, usually quantitatively determined using a fluorescence spectrophotometer.

Colloidal Gold Test Strip Detection Technology

Colloidal gold-labeled immunoassay strips mainly utilize the excellent physical and chemical properties of colloidal gold. Due to the negative charge on the surface of colloidal gold, the antibody can be immobilized on the surface by physical adsorption under alkaline conditions, and the biological activity of the antibody itself will not be affected. This method can quickly interpret the results under the naked eye, and is generally used for initial screening in the field. Among them, researchers designed a lateral flow assay (LFA) to measure SEB based on a double-antibody sandwich format on a porous nitrocellulose membrane. When a sample containing SEB is applied to the LFA device, the SEB initially reacts with polyclonal antibody-coated colloidal gold particles. These reactions produce red lines in the detection zone, the intensity of which is proportional to the SEB concentration, which can be achieved within 5 min using this method. SEB was detected at 1 ng/mL with high reproducibility.

Gene Probe Method

Enterotoxin B is transcribed and translated from the toxin expression gene sequence SEB of Staphylococcus aureus. By detecting the enterotoxin B gene of Staphylococcus aureus in food, it can be used for the positive strain of Staphylococcus aureus. Specifically, this method mainly adopts nucleic acid amplification technology, which includes polymerase chain amplification and nucleic acid isothermal amplification technology. The polymerase chain reaction (PCR) technique is mainly to design a pair of upstream and downstream primers for the target gene sequence. After adding the total DNA of the object to be detected, PCR or real-time fluorescent PCR is used to amplify the sequence of the SEB target gene, which can be quantitatively monitored by electrophoretic bands or fluorescence intensity.

Instrumental Analysis Methods

With the continuous improvement of the current detection instruments and equipment, many scholars are no longer limited to traditional immunology and other related methods, but instead use modern large-scale precision instruments for qualitative and quantitative detection of enterotoxin B. Some instrumental analysis methods can directly reveal the molecular structure of toxins, such as liquid chromatography-diode array detector, electrospray ionization, matrix-assisted laser desorption ionization, and time-of-flight mass spectrometry.

Biosensing Detection Method

Biosensor connects biochemical reactions with transducers through physical and chemical instruments, and displays them in the form of electricity. It mainly uses biological components (such as enzymes, antibodies, nucleic acids, lectins), the organic combination of biological response signal amplification (cell or tissue) and biological response signal amplification has the advantages of less sample usage and fast analysis speed, and can be used for the simultaneous detection of multiple substances. At present, the biosensors used for detection mainly include electrochemical sensors, surface plasmon resonance and piezoelectric crystal sensors.

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Polyethylene glycol is the polymer with the lowest level of absorption by proteins and cells among the known polymers. Polyethylene glycol is easily soluble in water, ethanol and most common solvents at room temperature, and is non-toxic, harmless and non-irritating to the human body, has good biocompatibility, lubricity and moisture retention. Because of the above-mentioned good characteristics, polyethylene glycol has a very wide range of applications in various industries, and can be seen everywhere in daily life, such as, toothpaste, hair cream, deodorant in the cosmetics and daily necessities industry; calendered paper in the paper industry, Nylon fiber, photographic developer; ointment, suppository, tablet, etc. in the pharmaceutical industry.

According to the molecular weight, spatial conformation, end groups, etc. of polyethylene glycol derivatives, it can be divided into monomolecular polyethylene glycol derivatives, methoxy polyethylene glycol derivatives, and Y-type branched polyethylene glycol derivatives, and multi-arm polyethylene glycol derivatives etc.

Although polyethylene glycol is widely used, it is very difficult to produce high-purity polyethylene glycol raw materials for the pharmaceutical industry. As the basis for the preparation of polyethylene glycol modified drugs, the purity of polyethylene glycol raw materials also directly affects the quality of the final drug. Medical medicinal polyethylene glycol derivatives are important materials in the production and preparation of downstream APIs and polyethylene glycol gel medical device products. In addition to higher molecular weights, they have higher standards of purity, polydispersity, and impurity content.

Application of Polyethylene Glycol in the Pharmaceutical Industry

PEGylation is a chemical modification process that connects active derivatives of polyethylene glycol to therapeutic protein/peptide drugs or drug delivery systems (such as nanoparticles, nucleic acid drug delivery platforms, etc.).

Pegylation is the world’s advanced pharmaceutical molecular modification and drug delivery technology. When polyethylene glycol derivatives are coupled to the surface of drug molecules, it will significantly improve or change the hydrophilicity, volume, molecular weight, spatial conformation, and steric hindrance of molecular interaction of drug molecules, thereby changing the solubility and spatial barrier of drug molecules. , Reduce enzymatic hydrolysis, have outstanding advantages such as attenuating toxicity, reducing immunogenicity, prolonging half-life, changing tissue distribution and increasing the concentration of targeted sites. It is the mainstream solution for long-acting drugs. At the same time, due to its excellent biocompatibility and hydrophilic properties, it can improve the cell compatibility of the copolymer polymer materials, and has the potential for the controlled release of drugs and the application of new biological drugs such as proteins, peptides and oligonucleotides. In addition, because of its good gel and degradability, polyethylene glycol can also be widely used in the fields of medical devices and medical materials.

PEGylation is the earliest and most used for long-acting protein or peptide drugs. With the deepening of PEGylation research, its application boundaries are constantly widening. In addition to peptide and protein drugs, it is also applied to small molecule drugs, gene drugs, etc., which can improve the effect of drugs, extend the half-life, reduce toxicity, and change the affinity of the target site, improve the solubility, change the membrane penetration characteristics, reduce the immune response, reduce the drug metabolism rate, etc.

Although PEG is considered safe and reliable, its side effects as a drug delivery vehicle into the body still need to be monitored. Moreover, after long-term administration, the potential side effects caused by the massive accumulation of PEG in tissues and organs also need to be explored. Therefore, efficient and sensitive detection methods are also an important part of the clinical application of PEG. In order to perform sensitive measurements on PEGylated compounds, Creative Diagnostics has produced a set of specific anti-PEG monoclonal antibodies. These antibodies have been verified to be used in various types of immunoassays, and can be used as detection tools for high-quality research to develop sensitive and specific assays.

Application of Polyethylene Glycol in the Field of Medical Devices

Polyethylene glycol is a synthetic polymer material with the best biocompatibility. In addition to being widely used in medicine for long-term and synergistic effects, its applications in medical devices are also being continuously developed. Because polyethylene glycol material has the advantages of solubility, good biocompatibility, non-toxicity, low immunogenicity, etc., it can be widely used in the adhesion, hemostasis, anti-leakage, and anti-adhesion treatment of wounds in various surgical operations equipment materials of the human body. At the same time, the polyethylene glycol material can also be used as a raw material for implanted medical devices in the human body, replacing the widely used plant, animal and human sources materials, so it has a very wide range of medical applications.

The main application of medical device products is multi-arm polyethylene glycol derivatives. Multi-arm polyethylene glycol derivatives can form hydrogels due to their relatively large molecular weights, and have good water barrier properties and tissue activity. Utilizing this characteristic of polyethylene glycol material, it can be made into a gel medical device for hemostasis and tissue isolation, which can be gradually degraded in the human body and completely discharged outside the body. The above-mentioned tissue sealant or tissue isolation medical device can be widely used in surgery or tumor radiotherapy, and has a broad prospect.

The post Application of Polyethylene Glycol (PEG) in the Medical Field first appeared on Creative Diagnostics.

Single domain antibody (sdAb), also known as nanobody (nanobody). It is a special antibody composed of only two heavy chains that naturally occurs in camelids and cartilaginous fishes. It contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Single-domain antibodies bind antigens through a variable region (VHH) on the heavy chain, which can exist independently and stably in vitro, and are called camelid single-domain antibodies (SdAb) or nanobody (nanobody). Nanobody crystals are 2.5nm wide and 4nm long. The molecular weight is only 1/10 (about 15kD) of traditional intact antibodies, but they still have complete antigen recognition capabilities. The complete antibody sequence is generally obtained through phage screening. The individually cloned and expressed V_HH region has the structural stability and the same antigen binding activity as the original heavy chain antibody. VHH is the smallest unit known to bind the target antigen.

Advantages of Nano Secondary Antibodies

Nano secondary antibodies developed based on single domain antibodies provide researchers with many advantages. Unlike traditional secondary antibodies, small nanobodies will not oligomerize with the primary antibody when incubated together. On the bench, this can shorten the staining step because the primary antibody can be “labeled” by incubating the primary antibody with the labeled Nanobody. After the pre-incubation step, the first antibody-nanobody mixture can be added to your sample, thus eliminating the incubation of the second antibody. In addition, nano secondary antibodies are about 10 times smaller than conventional secondary antibodies. The small size can achieve better tissue penetration, antigen entry, and reduce the distance between the epitope and the label. Therefore, they are ideal probes for super-resolution microscopy. Similarly, Creative Diagnostics Nano secondary antibodies select only Nanobodies with the required specificity during the development process. Eliminates cross-reactivity with other commonly used species of IgG and other IgG subclasses. This combination of high specificity and extremely low background makes the immunoassay more applicable and reduces the challenge of immunoassay (such as multiple western blotting or multiple super-resolution microscopy detection, even in pre-adsorbed mouse serum Later, it is also common to see cross-reactions between mouse IgG subclasses.). Our nanosecondary has ultra-high specificity and therefore does not require any type of pre-adsorption. In addition, nanosecondary antibodies are very suitable for parallel detection of multiple primary antibodies, which come from different species, or even different subclasses of the same species.

The post Nano Secondary Antibodies first appeared on Creative Diagnostics.

A_xB_y = xA+ yB

The complex AxBy is decomposed into x A subunits and y B subunits, and the final dissociation constant can be expressed as:

KD=[A]^x[B]^y/[AxBy]

Where [A], [B] and [A_xB_y] are the equilibrium concentrations of A, B and the complex A_xB_y, respectively.

In the process of binding a biological antibody to an antigen, KD is the equilibrium dissociation constant between the antibody and its antigen, which is the calculated ratio of Koff/Kon. The association constant (K_on) is used to characterize the rate at which an antibody binds to its target. The dissociation constant (K_off) is used to measure the rate at which the antibody dissociates from its target. KD is inversely proportional to affinity, so the lower the KD value (the lower the concentration), the higher the affinity of the antibody. High-affinity interactions are characterized by low KD, rapid recognition (high K_on), and strong stability of the formed complex (low K_off). Therefore, KD can be used to evaluate antibody affinity or sensitivity. For example, when the KD value is 10^-4 to 10^-6, the antibody sensitivity is micromolar; when the KD value is 10^-7 to 10^-9, the antibody sensitivity is nanomolar; When the KD value is 10^-10 to10 ^-12, its antibody sensitivity is picomolar concentration; when KD value is 10 ^-13 to 10 ^-15, its antibody sensitivity is femtomolar concentration.

H4ow to measure KD value?
Antibody binding affinity is an important parameter that can be used for antibody verification, which refers to the strength of the binding of antibody molecules to epitopes. KD and affinity are inversely proportional, so the antibody affinity can be calculated by detecting KD.
The affinity determination of monoclonal antibodies can perform high-precision detection because they are only selective for the same epitope; but in the case of polyclonal antibodies, because they detect epitopes that are heterogeneous and have different affinities Antibody mixture. Therefore, only the average affinity can be obtained. In order to determine antibody affinity, the following methods exist. These methods include ELISA-based methods, as well as other biophysical methods, such as micro-scale thermophoresis (MST) and surface plasmon resonance (SPR).

ELISA-Based Method

ELISA is the most popular method for studying antibody affinity. They do not require the use of large amounts of antibodies and antigens, nor do they require purification of proteins. In this method, a fixed concentration of antibody is incubated with the antigen in the solution until stable. Then, the concentration of unbound antibody was measured by indirect ELISA. This method requires a preliminary experiment to determine the antibody concentration used in the linear range of the ELISA reaction.

Micro Thermophoresis (MST)

MST is a biophysical method that can detect the affinity of antibody molecules at various concentrations. It judges the affinity between molecules by measuring the movement of molecules along the microscopic temperature gradient induced by infrared lasers. This movement depends on many factors, including the hydration shell, charge, and molecular size. These molecules are initially uniformly distributed in the solution and can diffuse freely. When the infrared laser is turned on, unbound molecules usually move out of the heating spot. The binding of one molecule to another molecule (such as antibodies and antigens) causes changes in the overall temperature gradient. Then fluorescent labeling can be performed after the molecules move to obtain the affinity parameters of the antibody molecules.

Surface Plasmon Resonance (SPR)

SPR is an optical technology used to detect molecular interactions, in which one molecule is fixed in a metal film (ie a sensor chip) and the other molecule is movable. The combination of these molecules changes the refractive index of the film. Therefore, when polarized light is irradiated on the film, the extinction angle of the light changes and can be monitored by an optical detector.
In antibody affinity measurement, the antibody to be analyzed is first captured by a high-affinity anti-IgG antibody immobilized on the surface of the sensor chip. Then a series of concentrations of antigen are sequentially injected across the surface. According to the sensor graph curve, one can rank antibodies according to kinetic parameters and screen the monoclonal antibodies with the best characteristics. First, the universal anti-antibody (such as rabbit anti-mouse immunoglobulin) is covalently attached to the surface of the chip, and then the first antibody is captured by the previously attached universal antibody. After blocking the unsaturation site, the injected antigen will be bound by the first antibody to form an Ab-Ag complex. After administration of the second monoclonal antibody (used to assess binding affinity), if the second antibody binds to a different independent epitope, a unique sensorgram curve will be observed.

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For the same type of antibody, the most characteristic feature that constitutes each immunoglobulin molecule is expressed in the 110-120 residues of the -NH base end of the polypeptide chain. The amino acid sequence of this part is different from antibody protein to antibody protein. However, the rest of the antibody polypeptide chain is constant. Therefore, according to the amino acid sequence of the constant region, the light chain can be divided into two types, namely K (kappa) and λ (Lamda). Among them, K has no subtype, while the constant region of the λ type has a certain change. By analyzing the amino acid sequence at positions 1-20 of -NH2, it can be typed again. Among them, K is divided into I, II, and III, and λ is divided into I, H, I, W, and V. The K subgroup cannot be combined with the lambda type, and vice versa. Further research found that a plasma cell can only produce one type.

The heavy chain can also be classified according to the amino acid sequence of its constant region, which is divided into five categories: IgG (γ chain), IgM (μ chain), IgA (α chain), IgD (δ chain) and IgE (ε chain). Similar Ig Molecules are divided into several subclasses according to the position and number of disulfide bonds between chains. For example, IgG is divided into four subclasses, IgM is divided into two subclasses, and IgA is divided into two subclasses. There are 4 disulfide bonds in the γ and α chains, and 5 (or more) disulfide bonds in the μ, ε and δ chains. According to the characteristics of 1-20 amino acids at the base end of -NH, the heavy chain can be divided into four subgroups. The subgroups of the H chain are different from the subgroups of the L chain, and they can be combined with the constant regions of other heavy chains. For example, VHI can be combined with the constant region of α and γ.
The above classes, subtypes and types, subtypes and subgroups are all of the same genus, and the Ig specificity shared by all individuals is called isotype. The same species but different individuals, the polypeptide chain can have one or more amino acid differences, which is called allotype. This situation is very similar to the situation of ABO blood type and major histocompatibility antigen (H-LA). Generally speaking, Gm is the type of IgG. There are 30 such genotypes. The Gm factor is located at CH1-3. There is also the Am type of IgA, which is mainly determined by the α subtype, namely Am1-2. The difference lies in the presence or absence of disulfide bonds. Km is mainly different from the amino acid at position 191. For example, when it is leucine, it is called Km1; when it is valine, it is called Km3. Except the genotype of IgG is determined by the Fc segment, everything else is determined by the Fd segment. In addition, there is a so-called idiotype, which is determined by the antibody-producing cell. In the same genus, each individual produces different antibodies when stimulated by the same antigen, so there are countless idiotypes. Another notable feature of the polypeptide chains of the H and L chains is that each chain includes a homologous segment of approximately 110 residues. Its characteristics are as follows:

1) These fragments show homology in the amino acid sequence of either the H chain or the L chain constant region. CL and CH, CH2, and CH are similar in structure, that is, they have homology.

2) These fragments contain an intrachain disulfide bond between the two cysteines, which is separated by approximately 65 amino acids.

3) Each fragment is folded independently of other fragments to form a globular protein.

4) The spherical structure of different chains is a unit with unique functions generated by the interaction of non-covalent bonds. When this fragment is separated from the entire molecule, its function is naturally retained. Different functional areas have different functions, they can bind antigen, complement, or cells. Therefore, after the antibody molecule is divided by various enzymes, fragments of Fab, F(ab)2, Fc, Fabc or Fv can be produced.

secondly, antibody is the existence of hypervariable areas. As mentioned earlier, different antibodies have different variable region sequences. Both the H chain and the L chain have a special amino acid sequence region, which is more variable than the rest of the V region. These regions are called hypervariable regions of variable regions. Variability is determined by the ratio of the frequency of different amino acids at a certain position to the most common amino acid at that position. Of the 110 variable region amino acids in the light chain, 25 are hypervariable. 30 of the 120 amino acids in the heavy chain are hypervariable. There are three light chains and three heavy chains. The light chain hypervariable region and the heavy chain hypervariable region are different. Recognizing one antigenic determinant requires a set of heavy chain and light chain hypervariable regions to complement it, and another antigenic determinant requires another set. Complementary hypervariable regions. At present, more than 200 kinds of hypervariable regions have been discovered, and their different sets can recognize countless antigenic determinants. The relatively constant region between hypervariable regions is called the framework region. Because the variability of the amino acid sequence outside the hypervariable region is very small, this indicates that the hypervariable region alone forms a highly specialized surface for the binding site. This has been confirmed by different hapten-labeled affinity experiments. The amino acids in the framework region also have some mutation rates, but they are very low. Therefore, it can be divided into subgroups accordingly. The main function of the framework region is to facilitate the movement of the hypervariable region in three-dimensional space, such as forming a cavity or shallow groove to fix the antigen.

Antigen-antibody binding through non-covalent bonds is reversible but specific. Moreover, it was further discovered that not all hypervariable regions are involved in binding antigen, which is related to the number of antigenic determinants. And it proves that the role of H is greater than that of L, but they work together.
The so-called affinity (affinity) refers to the binding ability of antibody molecule Fab and antigen, and avidity refers to the ability of the entire antibody molecule to bind to the antigen. The two are different. Because IgM is a pentamer, the avidity of IgM is stronger than IgG, but their affinities are equal. It has been confirmed that the functional regions of all immunoglobulins show a very similar three-dimensional structure. In this folding, the three-dimensional size of a functional area is 40 x 25x25A, composed of two layers, which are connected by disulfide bonds. There are many non-covalent bonds between the two layers, which play a stabilizing role.

These hierarchical filamentous structures are connected by loops of variable length, and hypervariable region residues are located on some loops. Immunoglobulins are produced by B cells. The average concentration of various types of immunoglobulin in normal human serum and the concentration outside the blood vessel are different. IgG is the most common protein in serum, and it is most easily transferred through the cell membrane, so its distribution in blood vessels and outside blood vessels is almost equal. One of the main functions of IgG is to neutralize soluble antigens, such as bacterial toxins. IgM exists in blood vessels and is mainly responsible for processing particulate antigens in serum. lgM has a high agglutination effect and the role of complement binding. IgA can be present in serum, but more importantly in secretions (saliva, colostrum, tears, nose, trachea, and intestinal secretions). In these secretions, Ig A forms a barrier against microorganisms invading and mucosal surfaces. IgE is present in the serum and is distributed in the exocrine fluid at a very low concentration. It is mainly related to the body’s allergic reaction. It can connect to mast cell receptors and bind to allergens, causing the release of histamine. IgD is the only immunoglobulin that can be hydrolyzed by plasma proteases. It is produced in the plasma cells of the tonsils and other glands.

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Existing studies have shown that SARS-CoV-2 infection is triggered by the combination of viral transmembrane spike (S) glycoprotein through receptor binding motif (RBM) and angiotensin converting enzyme 2 (ACE2), resulting in membrane Fusion and enter the host cell. For all coronaviruses, SARS-CoV-2 S is the main target of neutralizing antibodies (Abs) and the focus of vaccine design and therapeutic targeting efforts. Although the vaccine development plan is very rapid, it may take several months to achieve community protection due to limitations in large-scale production and administrative management. However, preventive and/or therapeutic antiviral drugs can address this gap before safe and effective vaccines are widely available, and will continue to play a role in individuals who have not been vaccinated or who have responded poorly to vaccines.

Recently, researchers described a monoclonal antibody (mAb), which was isolated from the memory B cells of SARS survivors 10 years later, and the study found that this antibody can recognize the S receptor binding domain (RBD) to Neutralizes SARS-CoV-2 and SARS-CoV, but does not prevent ACE2 from binding. The optimized version of this mAb (named S309) is currently being evaluated in Phase 2/3 clinical trials. Many other targeting RBD neutralizing antibodies were isolated from COVID-19 recovery patients and proved that they provide in vivo protection against SARS-CoV-2 challenges in small animals and non-human primates, indicating that RBD is neutralizing natural The main target of CoV-infected Abs. The clinical evaluation of therapeutic Abs that directly interfere with ACE2 binding is ongoing. Compared with the existing mAb, its mAb with abnormally high neutralizing power and a unique and complementary mechanism of action can enable the formulation of mAb mixtures with enhanced efficacy to control the spread of viruses and prevent drug resistance.

The researchers used ultrafiltration membranes to isolate and analyze human neutralizing antibodies against SARS-CoV-2 and found that antibodies such as S2E12 and S2M11 can help hamsters resist the challenge of SARS-CoV-2. The structure of cryo-electron microscopy shows that S2E12 and S2M11 can competitively block the connection of angiotensin-converting enzyme 2 (ACE2). S2M11 also locks one closed conformation spikes by recognizing a quaternary epitope spanning two adjacent receptor binding regions. The final result found that the antibody cocktail consisting of S2M11, S2E12 or previously identified S309 antibodies can broadly neutralize a group of circulating SARS-CoV-2 isolates and activate effector functions. The results of this study paved the way for the use of antibody cocktails to prevent or treat, circumvent or limit the emergence of virus escape mutations.

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