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Polyclonal antibody preparation process

2020-07-06
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principle

Antibody: An important effector molecule that mediates humoral immunity. It is a type of immunoglobulin that is synthesized and secreted by B cells that proliferate and differentiate into plasma cells after being activated by antigen.

Clone: refers to asexual reproduction cell line, which is a cluster of pure cells formed by the division and reproduction of a single ancestor cell. In all members of this family, if no mutation occurs, the genes are identical.

Polyclonal antibody (polyclonal antibody, pAb): immunizing animals with an antigen containing multiple epitopes can stimulate multiple B cell clones in the body to produce different antibodies against multiple epitopes. The obtained immune serum is actually a mixture containing multiple antibodies, namely polyclonal antibodies.

Polyclonal antibody preparation process

Significance of polyclonal antibody preparation

Immune serum: Antibodies produced by injecting antigen into the body are mixed antibodies against multiple antigenic determinants, and serum containing this specific antibody.

The role of immune serum: It is of great significance for the diagnosis, prevention and treatment of infectious diseases, but also for organ transplantation, tumors and certain scientific research work.

Pros and cons of polyclonal antibodies

   Advantages: comprehensive effect, neutralizing antigen, immune conditioning, complement-mediated cytotoxicity, ADCC and other effects, a wide range of sources, easy to prepare.

   Disadvantages: The specificity is not high, and cross-reactions are easy to occur, which limits its application.

Polyclonal antibody preparation process

(1) Preparation of immunogen

(2) Immunized animals

(3) Collection of immune serum

(4) Identification of immune serum

(5) Preservation of immune serum

Polyclonal antibody procedure

1. Preparation of immunogen

Immunogen refers to the antigen that can stimulate the body’s immune system to produce specific antibodies or sensitize lymphocytes. The most important property is immunogenicity

Immunogens—natural antigens, synthetic antigens; protein, polysaccharide, lipid, nucleic acid antigens;

(1) Preparation of cellular antigens:

   Sheep red blood cells (SRBC)-hemolysin;

   Bacteria-antibacterial antibody (animal immune serum)

(2) Preparation of soluble antigen: refers to protein, polysaccharide and lipid.

1) Cell disruption (physical method, chemical method)

2) Extraction and purification:

①Ultra-speed centrifugal separation is a method of separating antigen particles with different molecular sizes by differential or gradient density centrifugation according to the specific gravity characteristics of the separated substances, which is only suitable for the separation of a few large molecular substances.

②Selective precipitation—Select an anti-principle characteristic and use the corresponding precipitant or solution environment.

③Ultrafiltration method—Using special membranes with different pore sizes to filter antigens with different molecular sizes.

④ Chromatography-gel filtration, ion exchange, affinity chromatography

⑤Electrophoresis——

3) Identification: identify the content of purified protein, relative molecular mass, purity and immunological activity.

(3) Preparation of hapten immunogen

1. Carrier selection

Commonly used carriers: proteins, peptide polymers, macromolecular polymers.

(1) Proteins: commonly used are human serum albumin, bovine serum albumin, and blood protein, among which bovine serum albumin is the most commonly used.

(2) Peptide polymer: It is a synthetic peptide polymer and a good carrier. Poly-lysine is commonly used.

(3) Macromolecular polymer: Polyvinylpyrrolidone (PVP), carboxymethyl cellulose (CMC), etc. can be combined with hapten, adding Freund’s complete adjuvant can induce animals to produce good antibodies.

2. Connection method

         There are physical and chemical methods for the connection of hapten and carrier.

The physical method is to connect the carrier and the hapten by physical adsorption. The principle is to absorb the hapten through the charge and micropores. The adsorption carrier mainly includes PVP and CMC.

The chemical method uses certain functional groups to connect the hapten to the white matter or polypeptide polymer carrier. Different haptens should use different methods to connect.

(4) Immune adjuvant

Some substances are injected into the body together with or before the antigen, which can enhance the body’s specific immune response to the antigen or change the type of immune response. Such substances are called immunoadjuvant (adjuvant).

2. Animal immunity

(1) Selection of immunized animals

The following factors should be considered when selecting animals:

(1) Selection of animals (the following factors should be considered when selecting animals)

① The relationship between antigen source and animal species. The further the difference between the source of the antigen and the species of the immunized animal, the stronger the immunogenicity and the better the immune effect.

② Selection of individual animals. Normal animals of appropriate age, health, and weight (males are preferred); when the antibody requirement is low, choose small animals such as rabbits, guinea pigs, and chickens; when the antibody requirement is large, you can choose sheep, goats, horses, donkeys, etc. Big animal.

③ Antibodies used in general laboratories usually use rabbits and sheep;

(2) Adjuvant

Concept: Pre-injected into the body at the same time as the antigen, can enhance the body’s immune response to the antigen or change the type of immune response of non-specific immune enhancing substances.

Type: Adjuvants generally include the following categories:

①Inorganic adjuvant: such as aluminum hydroxide, bright vanadium, etc.

②Biological adjuvants: such as Mycobacterium tuberculosis, BCG, Corynebacterium parvum, Pertussis bacteria, Gram-negative bacilli endotoxin, B subunit of cholera toxin, muramyl dipeptide and cytokines;

③Synthetic adjuvant: such as double-stranded polyinosinic acid: cytidylic acid (poly I: C), double-stranded poly adenosine acid: uridine (poly A: U);

④Oil: such as Freund’s complete adjuvant, peanut oil emulsion, etc.;

⑤ Nano adjuvant

The main mechanism of action of adjuvant:

① Change the physical properties of the antigen, delay the degradation and elimination of the antigen, and prolong the retention time of the antigen in the body

② Stimulate the mononuclear-macrophage system to enhance its ability to process and present antigen

③ Stimulate the proliferation and differentiation of lymphocytes, thereby enhancing and expanding the ability of immune response.

(3) Preparation of immune emulsion

(4) Immunized animals

3. Collection of immune serum

Before collecting immune serum, it is necessary to test the antibody titer determination in advance. If the titer meets the requirements, blood should be collected in a week after the last immunization, otherwise the antibody titer will decrease.

①Carotid blood sampling

②Heart blood collection method

③ Venous blood sampling

4. Identification of immune serum

1. Determination of potency: Agglutination test can be used for granular antigens, soluble antigens are commonly used in two-way immunodiffusion test, ELISA and other methods.

2. Specific identification: antibody specific identification commonly used two-way immunodiffusion method, immunoelectrophoresis method

3. Purity identification: The purity of antibody can be identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-way diffusion test, immunoelectrophoresis and other methods. IgG contains heavy and light chains. SDS-PAGE results of pure IgG should have two protein electrophoresis bands (molecular weight about 53kD and 22kD). If there are multiple electrophoresis bands, it indicates that the prepared antibody is mixed with miscellaneous proteins and needs further purification.

4. Identification of affinity: There are many methods for determining the affinity of antibodies, such as equilibrium dialysis, ELISA or RIA competitive binding test.

5. Preservation of immune serum

After taking the blood, put it in 37℃, and after 30min, let the serum be analyzed (can not be frozen, otherwise hemolysis will occur), the serum is separated at 3000rpm/5min, and the rest is stored in the refrigerator at -20℃.

The above is about the preparation of polyclonal antibodies, which is from the official website of Medicilon.

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