Terms about structure and function of antibodies

  1. Antibody

An antibody refers to a protective protein produced by the body due to stimulation of an antigen. It is a large Y-shaped protein secreted by plasma cells (effector B cells) that is used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, etc. Antibodies can recognize a unique feature of a particular foreign object (an antigen).

  1. Antibody engineering

Antibody engineering refers to the process of transforming and reassembling antibody genes by using recombinant DNA and protein engineering techniques, expressing antibody molecules after transfecting appropriate recipient cells, or modifying antibody molecules by cell fusion or chemical modification. These antibody engineered antibody molecules are novel antibody molecules that are reassembled according to human design, which can retain (or increase) the specificity and major biological activity of natural antibodies, remove (or reduce or replace) irrelevant structures.

  1. Immunoglobulin (lg)

An immunoglobulin has an antibody activity and similar chemical structure to an antibody.

  1. Functional region

The H chain and L chain of the lg molecule can be folded into several spherical structures by intrachain disulfide bonds, and each spherical structure consists of 110 amino acids, which has a certain physiological function and is called a functional region, also called the domain.

  1. Variability

The variability of a position is the ratio of the number of different amino acids present at that position to the frequency of occurrence of the most frequently occurring amino acid residues at that position.

  1. Immunoglobulin superfamily (IGSF)

IGSF is a collection of molecules containing immunoglobulin folded structurally-like functional region. Most members of the lg molecular superfamily are cell membrane surface molecules.

  1. Classification according to the antigen characteristics of immunoglobulin

1) Isotype: refers to the antigen specificity of antibodies shared by all individuals in the same species, present in the constant region. Isotype antigen specificity varies by species, mainly including lg class, industry, type and business type.

2) Allotype: refers to the different antigen specificity of lg molecules between individuals of the same genus. As a genetic marker, there is a constant region of lg.

3) Unique type: each antibody forms an antigen specificity specific to the antibody molecule produced by the cell clone. It is determined by the difference in the amino acid sequence of the VH or VL variable region. It is closely related to the specificity of the antibody-binding antigen.

  1. CDR epitopes

VH and VL are tightly bound together to form a dense globular structure, which becomes the Fv segment. The six CDRs are located at the N-terminus of the Pv segment and are called CDR surfaces, ie, antigen binding sites.

  1. Binding force

The combination of antigen and antibody depends on electrostatic attraction, hydrogen bonding, van der Waals force, etc., and requires suitable temperature, pH, ionic strength and intact antibody. This combination is reversible.

  1. Antibody-dependent cell cytotoxicity (ADCC)

The antibody molecule binds to the surface of the target cell and can be combined with the Fc receptor on the surface of the killer cell to promote the pair. The killing effect of target cells is called ADCC. Mononuclear macrophages, neutrophils and NK cells have ADCC effect.

An overview to the third generation of antibody-drug conjugates

Antibody-drug conjugates (ADCs) can be defined as prodrugs, which recognize targets that express tumor antigens and that are coupled to cytotoxins by “linkers” to form targeted delivery system for tumor cells. Under ideal conditions, the prodrug is not toxic when it is administered systemically, and when the antibody in the ADC drug binds to a target cell expressing a tumor antigen, and the entire ADC drug is endocytosed by the tumor cell, the small molecule cytotoxin component will be released in a sufficient amount and a highly active form to kill the tumor cells.

Therefore, an ideal ADC needs a perfect combination of four parts:

  1. Selection of antibodies(targets): for tumor-specific (related) antigens, higher expression levels, good endocytic efficiency, good antigen affinity, no immunogenicity, drug accessibility;
  2. Selection of linkers: stable in the blood circulation, rapid disintegration in the cells, and efficient release of poisons;
  3. Coupling method: site-directed coupling, a uniform antibody conjugate;
  4. Cytotoxic agents: generally, the toxicity of the agent is required to be strong enough to achieve IC50 <1 nmol for single use; sufficient water solubility and stability in serum; the target of the toxin is located in the cell.

The third-generation of antibody–drug conjugates (ADCs)

A large number of methods have been developed by researchers to conjugate cytotoxic drugs with defined sites and stoichiometric antibodies. Here we listed the main ways as follows:

  1. Thiomab technology: Thiomab technology was first reported by Genentech’s Junutula et al., using genetic engineering techniques to insert cysteine ​​residues at specific positions in the light chain V110C and heavy chain A114C of trastuzumab, and then a site-specific antibody-drug conjugate was synthesized by coupling a thiol group on cysteine ​​with monomethyl auristatin E (MMAE), with a Drug-to-Antibody ratio (DAR) of 2 up to 92.1% (shows as the figure 1).

Figure 1. Thiomab technology

  1. ThioBridge technology: the disulfide bond of the mAb is reduced, and the dibromo (or disulfonate) reagent is reacted with the reduced interchain disulfide to provide a re-bridged mAb, and the DAR is mainly 4.

Figure 2. ThioBridge technology

  1. Introduction of the non-natural amino acid method: This method uses an evolved tyrosyl-t RNA/aminoacyl-t RNA synthetase that specifically recognizes unnatural amino acids, transfected by Chinese hamster ovary cells (CHO) instead of the 21st amino acid of the amber codon, the resulting cells can be used to synthesize various antibodies with a gene-decoding para-acetylphenylalanine residue, and then the hydroxylamine is deuterated. The main DAR is 2.

Figure 3. Introduction of the non-natural amino acid

  1. Enzymatic catalysis: for example, sortase A (Srt A), an enzyme with membrane-bound thiol transpeptidase catalyzing function, recognizes the main sequence of protein LPETG, lyses the peptide bond of threonine and glycine, forms a stable intermediate, and attaches the thiol group in Srt A to the threonine carboxyl group via a thioester bond.

Figure 4. Enzymatic catalysis

In my opinion, ADCs are actually designed to improve chemotherapy drugs. Because traditional chemotherapeutic drugs do not have the ability to recognize tumor specifically, while the targeted delivery of cytotoxic drugs to cancer cells by antibodies increases the percentage of drug molecules that reach the tumor, thereby reducing the minimum effective dose of the chemotherapeutic drug and increasing the maximum tolerated dose. However, the first and second generation of ADCs still have off-target toxicity and heterogeneous antibody conjugates due to linkers and ligation methods. Nowadays, scientists have developed a variety of site-directed coupling methods, linkers, and cytotoxic agents. And the therapeutic applications of ADCs have been greatly improved.

13 successful tips for ELISA troubleshooting (part one)

As a classic immunoassay, ELISA seems pretty usual, but it is not easy to do. What are the secrets to make a successful ELISA assay? In this article, we collect 11 ELISA tips for you.

  1. Make sure your sample type and kit are compatible

Common sample types include serum, plasma, and cell culture supernatant, if it is verified by the manufacturer’s experiment, it can be purchased according to the product description.

Note: Anticoagulants used in plasma preparation include EDTA, citrate, heparin, etc., and the corresponding plasma properties are different, and compatibility must be confirmed separately.

Special samples, such as urine, pleural effusion, cerebrospinal fluid, lavage fluid, tears, tissue homogenate, etc., may lack the support of manufacturer data. Therefore, you’d better to consult relevant expert or make a self-experimental verification of feasibility.

  1. The kit detection range needs to match the concentration of the marker in the sample.

The quantitative detection range of the ELISA kit needs to refer to the standard curve, which is a linear range in the lowest and highest concentration range of the standard curve, and the values ​​are credible and quantifiable. Samples with concentrations above the linear range are diluted to a linear range for measurement, while samples with concentrations below the linear range cannot be used to calculate concentrations and can only be used as a reference. A common situation is that the concentration of many markers in healthy human samples is low and the measured value is lower than the lowest value of the standard curve.

Many companies’ product data sheet provides you with a comparison of the results of a healthy person sample to confirm the reliability of your results. It should be noted that due to differences in race, disease and preservation methods, even if similar samples in the reference documents such as the manual can be detected, the testability of your sample cannot be guaranteed. This sample concentration below the measurement range can be identified by ND (not detectable), or <” lowest point concentration of standard curve ” (eg <3.1pg/ml, IL-6 Quantikine) and statistically analyzed.

  1. Five useful tricks to sample collection

Take serum as an example, the process can refer to the kit instructions, but pay attention to the following points.

  1. Samples should be collected in sterile tubes as much as possible to avoid the effects of bacterial enzymes and metabolic products on the results.
  2. Hemolysis should be avoided during the collection process because the active substances released by red blood cell lysis may affect the detection.
  3. If there is a precipitate during the preservation process, the supernatant should be taken after centrifugation.
  4. If the sample is not measured immediately, it is recommended to store it at -20 °C according to the amount of each use.
  5. One tube per test is used to avoid cross-contamination and repeated freezing and thawing. It can ensure the parallel comparability of the test results.
  6. Prepare the appropriate reagents before the experiment

All reagents are equilibrated to room temperature in advance, which takes about half an hour. The washing liquid is a concentrated liquid, and if it is crystallized, it needs to be completely dissolved and then diluted. A and B chromogenic substrate were mixed in a ratio of 1:1 before 15 minutes of use. In order to ensure the quality of the protein standard solution preparation, the protein standard is lyophilized powder. Before reconstitution, the tube should be centrifuged, and the buffer specified in the instruction manual should be added. Shake it slowly on the shaker or mix it by inversion for at least 15 minutes. If it needs to be stored after dissolving, it should be stored according to the temperature required by the instructions after being packed according to the amount of each use. When using a gradient-diluted protein standard, it is recommended to use a polypropylene tube (low adsorption for protein), mix thoroughly at each step and replace the new tip to ensure gradient accuracy.

  1. Preparation of equipment is also essential

Related equipment includes pipettes, washers/shakers, and microplate readers. The accuracy of the pipette is critical to the reliability of the ELISA results and requires regular calibration maintenance. The use of a shaker is to allow the reaction system to quickly reach equilibrium and achieve stable, repeatable results. The usual effect of not using a shaker on the experiment is that the OD value is low and the CV is large. Equipment such as microplate readers are preheated 15 minutes before use.

  1. Pre-experiment: Usually the pre-experiment includes all standard curves and small samples.

Pre-experiment has two main purposes, one is to familiarize with the experimental process and find problems that may be ignored. The second is to test whether the sample and the kit match, and if there is a mismatch, to make sure no waste of too many samples. In addition, a general understanding of the abundance of the analyte of interest in the sample is made to determine the appropriate dilution.

…to be continued in PART TWO.

Guide for antibody selection and antibody application in cancers (part four)

An overview of several commonly used muscle tissue and tumor markers

  1. Desmin (DM)

In the fine structure of the striated muscle, there is a distinction between light and dark bands, and a film in the center of the light and dark band is called a film. In the myocardial fiber structure, there is also a similar film. The main component of these structures is desmin. In addition, the myocardium of the myocardium, smooth muscle also contains intermuscular protein.

Desmin is an intermediate filament protein that is normally distributed in smooth muscle cells, cardiomyocyte bone cells and myoepithelial cells. It is commonly used in clinical applications to detect such cell-derived tumors, such as uterine fibroids and sarcomas, stomach, intestinal muscle-derived tumors, rhabdomyosarcoma and myoepithelial tumors.

The antibody is adapted to various tissue sections and results are better after antigen retrieval.

  1. Myosin

The microscopic structure of myeloid muscle can be seen under electron microscope. Myofibril is composed of 1-2 thousand parallel muscle filaments. The myofilament is divided into two types: thick muscle and thin muscle. The thick filaments are composed of many bean sprouts of myosin. In the middle stage of myocardial and smooth muscle development and differentiation, myosin is also seen.

Currently, there are two antibodies of myosin for clinical use.

(1). Myosin (skeletal)

This antibody is used to detect myeloid muscle and its derived tumors. It is suitable for various sections and has better results after antigen retrieval.

(2). Myosin (smooth muscle)

This antibody is used to detect myoepithelial and smooth muscle cells, as well as tumors derived from this. It is suitable for various tissue sections and better results can be obtained after antigen retrieval.

  1. Actin

Myofibril consists of two components, thin filaments and thick filaments. The thin filaments are composed of actin, tropomyosin and myogenic protein. The actin, its molecules are spherical, they are connected to each other to form long chains, and each thin filament is made up of two actin molecules.

The antibodies currently used to detect actin are as follows:

(1). Monoclonal mouse anti-human actin, clone HHF35. It is a broad-spectrum labeled antibody that can be used to detect tumors derived from myeloid and myeloid muscles. It can also be used to detect smooth muscle, vascular smooth muscle, myocardium, myoepithelial cells and tumors derived from it. It is suitable for various tissue sections. Good results can be obtained without microwave antigen retrieval. The dilution is 1:100.

(2). Monoclonal mouse anti-human actin (smooth muscle), clone 1A4. The antibody is mainly used for detecting smooth muscle, myocardium, vascular smooth muscle, myoepithelial cells and various tumors derived therefrom, and experiments have shown that capillaries can also be clearly displayed. It is suitable for a variety of sections with a dilution of 1:100.

How Important Is the Role of the Lentiviral Vector?

Abstract: Transgenic technology makes it possible for humans to change the trait phenotype of organisms according to their subjective wishes. The type and quality of vectors are directly related to the efficiency of subsequent transgenes. Therefore, the construction of expression vectors has become one of the key links in transgenic research. Lentivirus, also known as retrovirus, belongs to the family of retroviruses and is an RNA virus. Since this virus contains reverse transcriptase, it is called retrovirus. Lentivirus in the host cell, using RNA as a template, synthesizing cDNA under the action of self-reverse transcriptase, and then synthesizing double-stranded DNA using cDNA as a template, and cyclizing and integrating the chromosome of the host cell by viral integrase Expressed on the long term. The lentiviral vector (LV) is constructed by the lentivirus to remove part of the gene based on its genome and is constructed with the desired gene and marker of interest. Compared with other vectors, the lentiviral vector has a large capacity to carry the gene fragment. The dyeing efficiency is high, it can infect dividing cells and non-dividing cells, and the target gene can be stably expressed in the host cell for a long time and has good safety. It has become an ideal carrier for transferring the target gene. This article will focus on thelentiviral vectorconstruction and their applications in genetics.

Keywords: lentiviral vector, construction, application

The development of lentiviral vectors

In the early days of the development of lentiviral vectors in the early 1990s, HIV-1-based lentiviral vector research was mainly used for the study of the biological characteristics of HIV and the treatment of AIDS. The expression vector is mainly constructed for target cells expressing CD4, and most of the packaging systems retain all HIV genes as a control, and a series of viral genomes such as mutation or deletions of a certain sequence are modified. The purpose was to observe the effects on viral packaging, replication, infection, etc., and to study the genome composition of lentivirus HIV-1. This laid a solid foundation for the subsequent research of lentiviral expression vectors.

In the mid-1990s, the three-plasmid system of Didier Trono of the University of Geneva and Luigi Naldini and Inder Verma of the Sack Biological Research Institute of California marked the birth of the first generation of the lentiviral vector multi-plasmid system. The system consists of a vector plasmid, a packaging plasmid, and an envelope plasmid. The vector plasmid is responsible for loading the foreign gene, deleting genes such as gag, pol, env, but retaining 5′ and 3′, LTR, Psi sites, and Rev Response elements, thus improving the carrier loading while ensuring its efficiency. On the basis of this, Zufferey et al designed the second generation lentiviral vector, and deleted three auxiliary genes of vif, vpr and nef on the packaging plasmid, and obtained the infection of non-dividing cells, mononuclear macrophages and nerves. Cell-capable lentiviral vector and did not affect viral yield. Since these helper genes can express virulence viral proteins, their deletion not only increases the safety of lentiviral vectors but also increases the stability of the packaging cell line. The third generation of lentiviral vectors subsequently has higher biosafety. The vector plasmid contains only the cis-acting site and does not express any HIV protein. The packaging plasmid contains only three HIV functional genes, gag, pol and rev, and all other helper genes are deleted.

Construction of lentiviral vector

Ø The capacity and characteristics of lentiviral vectors

Due to the restriction of the size of the inserted gene in the packaging capacity of the viral capsid, the possible loading capacity of the target gene in the vector should be considered first when constructing the vector. The length of the HIV proviral genome is about 9.8 kb, and the length of the plasmid vector can be subtracted. The maximum length of the fragment of interest is loaded, which is the biggest disadvantage of lentiviral vectors compared to micromanipulation. At the same time, the packaging systems for the 2nd and 3rd generation lentiviral vectors are not universal because the second generation vector plasmid is Tat-dependent and the third generation packaging system does not contain the Tat gene.

Ø Optimization of lentiviral vectors

The expression efficiency can be increased by inserting a regulatory sequence. For example, by inserting the woodchuck hepatitis virus post-transcriptional regulatory element into the three untranslated region of the target gene, the expression level of the target gene can be increased by 5 to 8 times.

Ø Double gene transfer vector design

In the transgenic research, the strategy of double gene co-transfer is often used. The most common method for constructing lentiviral double gene transfer vector is to design bicistronics using internal ribosome entry site (IRES). The disadvantage of this strategy is downstream. The gene expression is low, and even the expression of downstream genes is not detected at all, and the use of two transcription units in tandem on the lentiviral vector effectively overcomes this shortcoming, enabling the downstream gene to be efficiently expressed.

Application of lentiviral vector

Treating HIV infection

With the surge in AIDS patients worldwide, the way to treat AIDS as soon as possible has become the most important issue. Lentiviral vectors have many advantages in the treatment of HIV-1 infection. First, some lentiviral vectors from HIV-1 and HIV-2 compete with viral RNA for packaging and competition for viral proteins for viral replication, inhibiting their replication, thereby controlling HIV infection. Second, lentiviral vectors can stably transfect dividing cells and non-dividing cells, especially those associated with HIV-1 replication and immune recovery, such as dendritic cells, T cells, and the like. Third, lentiviral vectors can be designed to express genes with therapeutic anti-HIV-1, specifically controlling the various stages of viral replication.

Treatment of tumor

For a long time, the treatment of tumors has been based on surgery, radiotherapy and drugs. Tumor gene therapy is a new treatment method after these traditional treatment methods. The lentiviral vector can safely and effectively implant the tumor gene into the human body for long-term effective expression. The use of lentiviral vectors increases the sensitivity of tumor cells to traditional chemotherapeutic drugs. RNAi technology is also used in tumor gene therapy. The occurrence of cancer is mainly due to the mutation and abnormal activation of proto-oncogenes. Lentivirus can specifically reduce the expression of RNAi against proto-oncogenes in various cells, thereby inhibiting canceration. Cell proliferation; shRNA capable of efficiently transmitting tumor-associated genes, silencing after transcription, inhibiting the expression of the target gene, reducing its promotion in tumor development and tumor invasion, thereby playing a therapeutic role.

Treatment of transplant rejection

In recent years, organ transplantation has been widely used to treat severe organ failure, but how to reduce transplant rejection and graft-versus-host disease (GVHD) after organ transplantation has become the most urgent problem to be solved. Activation, differentiation and proliferation of T lymphocytes play a key role in the immune response to transplantation. Genetic modification of T lymphocytes to reduce rejection and GVHD is one of the important directions in the field of transplantation. Li Zhenyu et al. observed the expression of the lentiviral vector in mouse lymphocytes by constructing a lentiviral three-plasmid system containing the green fluorescent protein gene, and found that the lentiviral vector has high infection efficiency on mouse T lymphocytes and can be rapidly stabilized. Transfer foreign genes to T cells.


[1] SUN Ke-ning, ZHU Hua-bin, LIN Feng, Wang Dong, HAO Hai-sheng, DU Wei-hua, ZHAO Xue-ming. Development on Lentiviral Vector Construction and Transgentic Animals by Lentiviral Vector.

[2] Buchschacher G L J, Panganiban A T. Human immunodeficiency virus vectors for inducible expression of foreign genes [J]. J  Virol, 1992, 66(5):2731-2740

The current situation and development trend of cancer vaccine

The cancer vaccine uses the patient’s own tumor cells, tumor-specific antigens and other immune-regulatory cells to treat and prevent cancer. The difference between the cancer vaccine and the mechanism of action of the pathogenic vaccine is that the former mainly achieves therapeutic purposes by stimulating the body’s specific immune response to cancer antigens. Thecancer vaccine is mainly obtained from the host and most macromolecules, is a normal autoantigen present in normal cells, has the specificity of the immune system, and can accurately identify cancer cell antigens from the host cells. The development of cancer vaccines is based on the premise that tumor cells are fundamentally different from normal cells, and that the immune system is able to identify and impart (by immunization), these distinctions to the goal of identifying malignant cells and regulating tumor rejection.

1. Pancreatic cancer vaccine

Pancreatic cancer is the fourth most common malignant tumor in developed countries, with a 5-year survival rate of only 7%. Due to the special anatomical location and physiological characteristics of pancreatic cancer, the onset of pancreatic cancer is insidious, there is no obvious symptoms in the early stage of the disease, the diagnosis is difficult, and it is highly invasive, so the mortality rate is very high.Diagnosis of pancreatic cancer can detect cancer antigen 19-9 levels. Cancer antigen 19-9 can be used as pancreatic cancer. An auxiliary diagnostic indicator for malignant tumors such as gallbladder cancer. In the embryonic stage, the pancreas, gallbladder, liver, intestine and other tissues have such antigens, and the normal human tissue content is very low; in the digestive tract malignant tumors, especially in the serum of patients with gallbladder cancer and pancreatic cancer, the content of cancer antigen 19-9 is significantly increased. However, the early diagnosis is of little value, mainly as a monitoring indicator and an indicator of recurrence. In addition, differential diagnosis of digestive tract diseases (such as pancreatic cancer and pancreatitis, gastric cancer and gastric ulcer) also has certain value.

Pancreatic cancer is highly immunosuppressive, and tumor-associated reactive T cell infiltration in the microenvironment is associated with a good prognosis for pancreatic cancer. Studies have shown that pancreatic cancer has multiple mechanisms to evade surveillance by the immune system, such as recruitment of negative regulatory T cells, secretion of transforming growth factor beta (TGF-beta) and interleukin 10 to suppress the immune response of the immune system, and down-regulation Expression of major histocompatibility complex I (MHC I), and the like. Recent studies have shown that necrotic apoptosis in pancreatic cancer induces chemokine ligand and macrophage-induced C-type lectin receptor signaling, promotes macrophage-induced adaptive immunosuppression, and accelerates pancreatic cancer progression.

Breaking through the immunosuppression of pancreatic cancer, enhancing the recognition of tumor-associated antigen (TAA) and activation of tumor-specific T cell responses are the core issues in the treatment of pancreatic cancer tumor vaccines. In recent years, with the development of omics, more and more relatively specific TAAs have been discovered. For example, exon sequencing can be specifically focused on a certain gene coding part, so most of the oncogene changes can be identified.There are an average of 63 genes in pancreatic cancer. These genes are mainly concentrated in 12 core signaling pathways such as Kras signal, TGF-β signal, SHH signaling pathway, etc. These changes are important for tumor tissue growth and differentiation, suggesting pancreas Cancer is the result of multiple gene mutations, and it also provides a basis for the target of pancreatic cancer tumor vaccines.The pancreatic cancer genotypes are divided into four subtypes: squamous, pancreatic progenitor, immunogenic and aberrantly differentiated endocrine exocrine (ADEX). The immunogenic gene program is associated with B cell signaling pathway, antigen presentation, CD4+ T cells, CD8+ T cells, and Toll-like receptor signaling pathways, and its acquired immune signaling pathways such as CTLA-4 and PD1 are up-regulated, indicating specificity. There may be breakthroughs in the study of tumor vaccines for immunogenic genotypes, suggesting that the treatment of pancreatic cancer needs to be personalized and precise.

1.2. Pancreatic cancer vaccine type

1.2.1. Peptide/gene vaccine KRAS peptide vaccine

The KRAS gene belongs to a member of the Ras gene family and plays an important role in regulating cell proliferation and differentiation. More than 90% of pancreatic cancers have KRAS mutations and are among the earliest genetic alterations in precancerous lesions. When the KRAS gene is mutated, the conformational change of the Ras protein promotes invasion and metastasis of pancreatic cancer through its downstream RAF/MEK/ERK protein kinase cascade. Telomerase vaccine

Telomerase maintains chromosome stability and plays an important role in cell senescence and carcinogenesis. When telomerase is activated, tumor cells are prevented from telomere-mediated cell death. Telomerase is a key molecule in inducing cell carcinogenesis. In general, telomerase is activated in more than 85% of tumor cells. Vascular endothelial growth factor receptor 2 protein vaccine

Vascular endothelial growth factor (VEGF) signaling pathway plays a crucial role in tumor angiogenesis, in which VEGF receptor 2 (VEGFR2) mediates vascular endothelial proliferation and chemotaxis. Cells, which increase the permeability of blood vessels, are the main functional receptors of VEGF. In pancreatic cancer, VEGF/VEGFR2 is closely related to tumor growth and infiltration by regulating angiogenesis. Mucin vaccine

Mucin1 (MUC1) is a high molecular weight type I transmembrane glycoprotein, which is normally expressed in the proximal luminal or glandular surface of epithelial cells in various tissues and organs, but in 90% of patients with pancreatic cancer. Overexpression. MUC1 interferes with cell-cell and cell-matrix linkages and plays a role in tumor signal transduction, invasion, and distant metastasis. WT1 epitope peptide vaccine

Wilm’s tumor protein, (WT1) is a type of TAA, which is expressed in solid tumors such as lung cancer, breast cancer, thyroid cancer, and pancreatic cancer, in addition to high expression in various types of leukemia.

1.2 .2. Cell vaccine Tumor cell vaccine

Injection of a radiation-irradiated tumor cell culture vaccine is the most primitive tumor vaccine, which utilizes all relevant tumor antigen expression expressed by tumor cells to produce a specific anti-tumor immune response. GVAX is an allogeneic whole-cell vaccine derived from two tumor cells and genetically engineered to express granulocyte colony-stimulating factor (GM-CSF). Α-galactosyl (α-GAL) is not synthesized in normal human cells, and serum contains a large amount of anti-α-GAL antibody, but tumor cells can synthesize α-GAL, so α-GAL can be used as an antigen. Induction of an anti-tumor response. Algenpantucel-L is an allogeneic tumor vaccine produced by NEWLink, which is produced using two human pancreatic ductal cancer cells genetically modified to express α-GAL. Dendritic cell vaccine

Dendritic cell (DC) is a professional antigen presenting cell (APC), which can efficiently present and activate MHC I and MHC II to CD8+ and CD4+ T cells. Stimulating memory T cells and memory B cells produce specific anti-tumor responses and play an important role in primary and secondary immune responses against tumors.

1.2.3. Vaccine combination therapy Immunological checkpoint treatment and vaccine combination therapy

Immunological checkpoints are key to maintaining immune tolerance to chronic antigen exposure and preventing tissue damage. T cell activation is dependent on the interaction between co-stimulatory, co-inhibitory receptors and their ligand complexes. Usually, co-stimulatory receptors have CD40, CD28, OX40 and 4-1BB, while inhibitory receptors have CTLA-4, PD-1, B7 family receptors and their ligands CD80, CD86, PD-L1 and PD-L2. . Immunological checkpoint therapy is an antibody based on the inhibitory receptors CTLA-4, PD-1 and its ligands. CAR-T Cell Therapy

The chimeric antigen receptor (CAR) is a type of genetically engineered transmembrane fusion receptor that binds to primitive cell surface antigens and transmits specific T cell activation signals. CAR-T cells (CAR-T) are a type of T-cells that are genetically engineered to encode tumor-specific antigen receptor genes, which allow T cells to express related antigen receptors and restore T cells. Immune surveillance can identify tumor surface antigens, so that a large number of tumor-associated antigens released by tumor cell rupture are presented, which can trigger the recognition and complete killing effect of the body’s immune system on tumors, which can be regarded as a special kind of cell vaccine. The key to developing CAR-T cell therapy is to select the appropriate targeting antigen and immune receptor.

2.Colon cancer vaccine

Colon cancer is one of the high-grade malignant tumors of the digestive system. The incidence rate is the third in the world for malignant tumors and has risen to the second place in economically developed areas. The emergence of coloncancervaccine will definitely bring new hope to the treatment of colon cancer.

Tumor vaccines use tumor cells or tumor antigens to induce the body to produce immune responses against tumor cells, inhibit their growth, and prevent recurrence and metastasis. Tumor antigens have been found on the surface of spontaneous tumors and human tumor cells in animals. Tumor antigens are generally classified, and two anti-tumor antigen classification methods including tumor-specific antigen (TSA) and tumor-associated antigen (TAA) are introduced. TSA is only present on the surface of tumor cells and is an antigen unique to a certain tumor cell. TAA is unique to non-tumor cells and is an antigen that can be expressed by normal cells. However, when cells are cancerous, their content is significantly increased, and such antigens only show quantitative changes without

Strict tumor specificity, embryonic antigen is a typical representative of it. Embryonic antigen refers to the normal component produced by embryonic cells during embryonic development. It is reduced in the late stage of embryonic development, gradually disappears after birth or remains extremely small, and such antigens regenerate when the cells become cancerous. At present, there are two kinds of embryo antigens that are the most intensive: 1) Alpha-fetoprotein (AFP): a glycoprotein synthesized by fetal liver cells, which inhibits maternal immune rejection. Adults are almost undetectable, and hepatocellular carcinoma is abundantly expressed when it is cancerous. 2) Carcinoembryonic antigen (CEA): It is an antigen that loosely binds to the cell membrane and is easily detached, such as carcinoembryonic antigen produced by intestinal cancer cells. AFP and CEA are weakly immune, as they have emerged during the embryonic period, and the body’s immune system has been immune to it and does not produce an immune response. However, AFP and CEA can be used as tumor markers to detect the early diagnosis of primary liver cancer and colon cancer by detecting the levels of AFP and CEA in the serum of patients.

At present, the main research tumor vaccines include the following:

2.1. Inactivate tumor cell vaccine

The tumor cell vaccine extracts tumor cells from the tumor tissues of the body, and inactivates the tumor cells that have lost the tumorigenicity but still maintains their immunogenicity, thereby inducing the body’s active immune response. In theory, such vaccines can provide tumor antigens, including TSA and TAA, to induce the body to produce an anti-tumor immune response.

2.2. Dendritic cell vaccine

Dendritic cells (DC) vaccine (referred to as DC vaccine) is the most active and fruitful biotherapeutic topic in research today. DC can be used as an important component of tumor immunotherapy. The mechanism of DC vaccine for malignant tumors is as follows: 1) Dendritic cells are special antigen-presenting cells, which help the immune system recognize tumor cells; 2) DCs carrying tumor antigens will antigen The information is presented to and activated by T cells, which induces the body to produce a large number of T lymphocytes with specific cytotoxic functions, which have specific killing effects on tumor cells. Dendritic cell therapy is a very cutting-edge new technology, and the application of DC vaccine has brought good news to the treatment of cancer patients. Numerous studies have shown that DC vaccines are safe, easy to handle, and immunosuppressive for a range of tumor types. The safety of the DC vaccine is also very good, and there have been no reports of serious adverse reactions. The successful development of dendritic cell vaccine treatment has brought new hopes to countless tumor patients and opened up a new way for the treatment of cancer.

2.3. Protein or polysaccharide vaccine

Such vaccines are obtained by mixing or linking tumor-associated proteins or polysaccharides and adjuvant molecules into the human body to induce humoral or cellular immunity, thereby achieving the purpose of killing tumor cells. The reason why tumors cannot be recognized by the immune system is mainly due to the weak immunogenicity of tumors. Therefore, the use of immunoadjuvants to enhance the immunogenicity of tumors is a hallmark of early tumor vaccines. The protein or polysaccharide vaccine is composed of an adjuvant such as Corynebacterium, alum, BCG, Freund’s complete adjuvant, etc. in the lysate of autologous or allogeneic tumor cells or tumor cells. Its mechanism of action may be related to the activation of antigen presenting cells (APC) by the inflammatory response at the injection site, the production of cytokines and the accumulation of B and T cells around the antigen.

2.4. Gene vaccine

Gene therapy is a hot research area in current medicine and biology. Gene vaccines, also known as nucleic acid vaccines or DNA vaccines, are often referred to as “naked” DNA vaccines. It contains no peptide, protein or viral vector, but consists of an antigen-encoding gene derived from the pathogen and plasmid DNA as its carrier. The birth of the genetic vaccine has revolutionized the treatment of colon cancer patients. : It is easy to operate and can be easily controlled by increasing or decreasing the amount of DNA injected. It does not require complex processes such as separation and purification of proteins. One or two weeks after DNA vaccine injection, an immune response is produced. After 14 days, the expression in the muscle is reached. The peak, then gradually decline, remains at low levels for months or even 1 year. In recent years, many researchers have actively developed tumor-related gene vaccines. The preliminary experiments have also proved that genetic vaccines have good curative effect and high immune performance, which makes people have a strong interest and expectation for the development of genetic vaccines.

The use of tumor vaccines can cause specific immune responses, thereby inhibiting tumor growth. Although tumor vaccines use the patient’s own tumor cells, tumor-specific antigens and other immune-regulatory cells to treat and prevent tumors, opening up a modern way to safely and effectively treat tumors, but at present, there are some difficulties in the development of tumor vaccines, such as further regulation. Enhance immune effector cells, etc. Once these problems are resolved, the tumor vaccine can be used on a large scale in the clinic. In addition, due to the complex composition of human tumors and the heterogeneous expression of tumor antigens, it may be necessary to immunize with a variety of tumor antigens in order to induce an effective immune response in patients.


[1] Keilholz U, Weber J, Finke J H, et al. Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy.[J]. Journal of Immunotherapy, 2002, 25(2):97.

[2] Maron D F. Cancer Vaccine.[J]. Scientific American, 2017, 317(5):16-16.

[3] Li X, Min M, Du N, et al. Chitin, Chitosan, and Glycated Chitosan Regulate Immune Responses: The Novel Adjuvants for Cancer Vaccine[J]. Clinical & Developmental Immunology, 2015, 2013(7378):387023.

[4] Shindo Y, Hazama S, Nakamura Y, et al. miR-196b, miR-378a and miR-486 are predictive biomarkers for the efficacy of vaccine treatment in colorectal cancer[J]. Oncology Letters, 2017, 14(2):1355-1362.

[5] Berry J, Vreeland T, Trappey A, et al. Cancer vaccines in colon and rectal cancer over the last decade: lessons learned and future directions[J]. Expert Review of Clinical Immunology, 2017, 13(3):1.

Notoginseng folium saponins is a traditional Chinese medicine

Notoginseng folium saponins is a traditional Chinese medicine that has the effect of shortening sleep time, prolonging sleep time, reducing the number of awakenings, improving headache, dizziness, palpitations, fatigue, and treating neurasthenia.


In addition to the special effects of treating bruises, Notoginseng folium has the effect of nourishing and strengthening.  Modern pharmacological studies have shown that Notoginseng folium has obvious preventive and therapeutic effects on hyperlipidemia, hyperviscosity, hypertension, and arrhythmia.

The main pharmacological effects of the main active constituents of Panax notoginseng, Panax notoginseng, are calming, soothing, analgesic and lipid-lowering.

The role of the central nervous system: only 100-200mg / kg dose of notoginsenoside can significantly reduce the spontaneous activity of mice, enhance the sedative and hypnotic effects of thiopental, pentobarbital sodium, etc., and can fight the central excitement. The excitatory effect caused by caffeine indicates that this product has significant central inhibition.  Panax notoginseng saponins can improve the blood supply of the brain, nourish and regulate the nerves, restore the coordination functions of the nervous system, thereby restoring physiological sleep to treat neurasthenia, and strengthen the inhibition of the cerebral cortex and make the cortex  The rise of excitability is reduced and it is resistant to anxiety and can be used to treat generalized anxiety disorder.

Panax notoginseng saponins are better for shortening sleep time, prolonging sleep time, reducing the number of awakenings, and improving headache, dizziness, palpitations and fatigue.  The total effective rate of treatment of neurasthenia was significantly higher than that of the gastrodin control group.

Effect of lowering blood fat: Panax notoginseng saponins can significantly reduce serum total cholesterol (TC) and serum triglyceride (TG) levels in rats with high-fat models, which are related to the ginseng diol contained therein.  The type of saponin is related.  According to reports, TC decreased by an average of 22.4%, TG decreased by an average of 37.7%, and high-density lipoprotein increased by an average of 18.8%.  Using self-control method, taking 100 mg of esculin in patients diagnosed with hyperlipidemia, 3 times a day, 60 days for a course of treatment, the total effective rate was 81.6%.

Content ratio

Panax notoginseng leaves usually refers to the dried stems of the stems and leaves of the aboveground parts of Panax notoginseng. It has high medicinal value. The main pharmacological effects of the extracted total saponins of Panax notoginseng are calming and soothing, analgesic and lipid-lowering.  notoginseng has the characteristics of convenient eating, moistening mouth and thirst quenching. The main function is similar to that of Sanqi stem. It is also raw and cooked.  Rawnotoginseng can lower blood pressure, lower blood sugar, lower blood fat, etc.; notoginseng can also be soaked like drinking tea, drink a few cups a day!  The monomeric saponins contained in Panax notoginseng are mainly 20(s)-protopanaxadiol saponins, and contain almost no original ginseng triol saponins, which is the biggest difference between Panax notoginseng saponins and Panax notoginseng saponins.  The total saponin content of Panax notoginseng leaves is 4% to 6%, mainly containing ginsenoside Rb3, Rb1

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A Highly Sensitive Method for Protein Product

Simple Introduction

First of all, let’s start with its definition: Peptide mapping is the main method for the analysis and identification of protein products and preparations. In addition, there are some other core techniques such as peptide mass mapping and peptide mapping ms.


The following are functions of Peptide mapping:

To confirm that a protein primary structure (amino acid sequence) including the N and C terminals.

  • To provide protein loci and the proportion of modified groups, such as glycosylation, acetylation, sulphates a00nd phosphorylation.
  • To provide qualitative and quantitative information on protein degradation products
  • To get information on protein oxidation and deamination directly.

According to these useful features above, peptide mapping has already gained in popularity in biological field. After getting numerous data from peptide mapping, the next step is deeper analysis.


A sample graph of peptide mapping

Peptide Mapping Analysis

Based on graphs of peptide mapping and also peptide mass mapping, scientists can get a lot of useful information. According to the size of molecular weight of proteins, peptides and amino acid composition characteristics, the use of strong specificity of proteolytic enzyme [is commonly endopeptidase] on the special peptide chain site will peptide fragment into smaller fragments, through the separation of a certain form characteristic fingerprint detection methods. eolytic enzyme [is commonly endopeptidase] on the special peptide chain site will peptide fragment into smaller fragments, through th

The picture below shows the steps of peptide mapping method:

Step 1:

Protein digestion: Immobilized trypsin provides rapid and simple protein digestion with high reproducibility, high sensitivity and excellent data quality in a format that is compatible with automated operations.

Step 2:

Peptide separation: this technique provides a complete set of chromatographic tools for all development applications requiring peptide separation and analysis

Step 3:

Biological mass spectrometer: It provides a fast and easy to solve multicomponent analysis method, Used for sequence determination, structural analysis, molecular weight determination and component content determination of polypeptides. What’s more, it has the characteristics of high sensitivity, strong selectivity and good accuracy.

Step 4:

Peptide analysis software: Integrated software can save time and identify more materials. Simple software workflows can guide biotherapy, characterization pathways, and provide comprehensive coverage, including peptide sequence validation and identification of all variants and modifications.


So peptide mapping analysis is quite effective and important in some specific aspect of research. Let’s take an example here:

Study on quality control of impurities and harmful substances in Genetic Recombinant Drug is a crucial project., in particular, when the recombinants are used in the production of genetically engineered drugs undergo mutations. Probably, they would bring some mutations into drugs. Besides strengthen control of original material and process of production, peptide mapping analysis is necessary method to ensure safety and consistency.


As a result, peptide mapping analysis has given rise to a number of new things. For instance, HPCE, A new electrical technology for separation which emerged in 1980s. Because of its relatively high resolution, HPCE has started a wider road for structural analysis and quality control of protein drugs.


At present, in addition to the routine analysis of amino acid sequence of some small peptides, the peptide graph analysis is one of the important conventional indicators to control the consistency of most gene engineering products. All in all, technologies on peptide mapping have already penetrated into every corner of our everyday life.


New antibody screening technology – protein chip

What is an autoantibody?

Autoantibodies are antibodies that target tissues, organs, cells, and cellular components. The growth, development and survival of the human body have the maintenance of a complete autoimmune tolerance mechanism. The normal immune response has a protective defense effect, that is, it does not react to its own tissues and components. Once the integrity of self-tolerance is destroyed, the body regards its own tissues and components as “foreign substances”, and an autoimmune reaction occurs to produce autoantibodies. Normal human blood may have low titers of autoantibodies, but no disease occurs. However, if the titer of autoantibodies exceeds a certain level, it may cause damage to the body and induce disease.

There are many kinds of autoimmune diseases antibodies , the most important of which are antinuclear antibodies. In addition, anticardiolipin antibodies, neutrophil cytoplasmic antibodies, anti-mitochondrial antibodies, anti-erythrocyte antibodies, anti-platelet antibodies, anti-endothelial cells antibodies, anti-neurovirus antibodies, rheumatoid factor, anti-thyroglobulin antibodies, anti-insulin bodies Antibodies and the like are also autoantibodies.

Reasons for autoantibody production:

Antibodies are generally produced by the immune system by foreign proteins or other substances (especially pathogenic bacteria) that enter the body and are used in immune reactions to eliminate harmful foreign substances. Usually, the immune system can recognize and ignore the body’s own cells, and does not produce antibodies to it; at the same time, the immune system does not overreact to substances (such as food) that are not threatened in the environment. However, under certain circumstances, the immune system recognizes the body’s own substances and treats them as foreign invaders, thereby producing antibodies (ie, autoantibodies) against these substances, triggering autoimmunity. These autoantibodies attack the cells, tissues, and organs of the body, causing an inflammatory reaction and causing damage to the body.

The production of autoantibodies may be due to the presence of some of the same molecular structures between pathogenic antigens (bacteria, viruses, etc.) and their own components: an immune response that cross-reacts with autoantigens; or some infectious agents that cause autoantigens Denatured, the immune system produces autoantibodies to these exposed new antigens.

The pathogenic effect of autoantibodies is still unclear. Whether it is the “cause” or “consequence” of autoimmune diseases has different opinions. For patients with high titers of autoantibodies, those without clinical symptoms may not need treatment, but should go to the hospital regularly. Review and review.


In tumors, inflammation, autoimmune diseases (such as lupus erythematosus, genital warts, Crohn’s disease, multiple sclerosis), neurodegenerative diseases, infectious diseases, etc., a large number of autoantibodies are produced and accumulated in patients. Some autoantibodies have emerged in the early stages of a specific disease, even before the onset of symptoms of the disease, providing a reliable disease biomarker for the early diagnosis; some autoantibodies are the body’s own protection against disease. Sexual antibodies, which provide new ideas for the treatment of the disease, as data from the world-renowned pharmaceutical giants show that 60% of the profits of large pharmaceutical companies have come from drugs that belong to antibodies. So how do you discover these potential autoantibodies?

Methods for screening for autoantibodies:

At present, the most suitable method for screening autoantibodies is the protein chip method. A protein chip often has thousands of protein spots, which can screen one autoantibody that can interact with these proteins at one time, and then pass fluorescent These autoantibodies can be found by incubation of the marker against the anti-Human IgG secondary antibody and fluorescence detection.

The principle is simple, but it is very difficult to do, why? This has to say about the binding process of antibodies to antigens. In short, the corresponding antibody recognizes a specific epitope on the antigen and then binds it. The epitopes are divided into two types, linear epitopes and non-continuous epitopes. A linear epitope consists of a contiguous sequence of amino acids. An antibody that recognizes a linear epitope recognizes this amino acid sequence and produces an antigen-antibody binding reaction; a non-contiguous epitope is composed of a discontinuous amino acid, which is correct by the antigenic protein. After folding, they are close together and recognized by the corresponding antibody, producing an antigen-antibody binding reaction.


In the body, most autoantibodies are non-continuous epitopes that recognize antigens. Correct identification and screening of these autoantibodies requires that the proteins on the protein chip be full-length, correctly folded, and biologically functional. However, the traditional protein chip can only guarantee that the protein synthesized on the chip is full-length (some can not be guaranteed), can not guarantee the correct folding of the protein, and can not guarantee the normal biological function of the corresponding protein. Other problems affecting the screening of autoantibodies by traditional protein chips include high CV values (>30%), poor reproducibility, low resolution, high background signal, and inability to distinguish autoantibodies with low expression levels. Screening for autoantibodies with such protein chips is like fishing with a large network full of loopholes. Screening of autoantibodies with such protein chips has brought great resistance to researchers and companies in the research of antibody screen. Sengenics’ ImmunomeTM Protein Chip Research Platform, invented by Professor Jonathan Blackburn at the University of Cambridge in 1996, is a collaboration between Oxford and Cambridge. It is the only one in the world that is fully-length, correctly folded and functionally validated. Protein chip platform. Autoantibodies that recognize non-contiguous epitopes can be screened for advantages that cannot be replaced by other protein chip products.

The ImmunomeTM Protein Microarray Research Platform contains 1631 full-length, correctly folded and functionally validated human proteins, covering cancer antigens, transcription factors, kinases, signaling pathway molecules, etc., to meet the research needs of users in different directions. Daban’s low autoantibodies provide strong technical support as biomarkers. Compared to traditional protein chip products in the screening of autoantibody applications, Sengenics ImmunomeTM protein chip products can be described as “Skynet is restored, not leaking”, full-length, correct folding, functional verification, wide coverage, low coverage CV low background signal, high-resolution full-generation protein chip platform technology helps researchers and business users to “capture big fish” in the field of autoantibody research, and return home.