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Method for preparing polyclonal antibody against mouse nerve growth factor (NGF) and application thereof

A technology of nerve growth factor and polyclonal antibody, which is applied to the preparation method of peptide, anti-growth factor immunoglobulin, chemical instruments and methods, etc.

Active Publication Date: 2012-08-29
SINOBIOWAY BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the weak immunogenicity of NGF, it is difficult to obtain antibodies, and it is still blank to obtain high-titer antibodies to NGF in China

Method used

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  • Method for preparing polyclonal antibody against mouse nerve growth factor (NGF) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Preparation of polyclonal antibodies

[0055] prepare

[0056] 1. After the purified NGF is polymerized by glutaraldehyde, at 150μg per animal, it is fully mixed and emulsified with an equal volume of complete Freund's adjuvant, and injected into normal rabbits subcutaneously at multiple points;

[0057] 2. One month later, the same amount of antigen and incomplete Freund's adjuvant were fully mixed and emulsified, and subcutaneously injected again at multiple points;

[0058] 3. After one month, repeat step 2;

[0059] 4. One week after the last immunization, ear blood was collected, and the titer was measured by indirect ELISA, and the titer was greater than 1:1000;

[0060] 5. Blood was collected from the marginal ear vein, 50ml / time, collected twice, serum was collected, and stored at -20°C.

Embodiment 2

[0061] Example 2 Purification of polyclonal antibodies

[0062] 1. Preparation of antigen affinity column: The purified antigen (mouse submandibular gland nerve growth factor NGF) was dialyzed in coupling buffer (0.1mol / L NaHCO) 3 , 0.5mol / LNaCl, pH8.3), pre-cooled 0.5mM ~ 50mM HCl wash CNBr-activated sepharose 4fast flow gel several times, negative pressure ultrafiltration, and replaced in coupling buffer; Mix the colloidal particles with 100% gelatin, and stir overnight at 4°C; wash the incompletely bound antigens with coupling buffer; block the activation sites that are not saturated with antigens by blocking the activation site with the blocking buffer (0.2mol / L glycine) for more than 0.5 hours at room temperature; Buffer and acetate buffer 0.1mol / LCH 3 COO·Na and 0.5mol / L NaCl, pH 4.0) were alternately washed for more than 2 times, and the micelles coupled with antigen were stored in 20% alcohol.

[0063] 2. Binding buffer (PBS) fully equilibrates the antigen-conjugated...

Embodiment 3

[0068] Embodiment 3 ammonia sulfate-n-octanoic acid method purification method:

[0069] Take a certain volume of serum and dilute it by 2 or 4 times with 0.06mol / L HAc-NaAc buffer at pH 4.8; add n-octanoic acid dropwise to make the final concentration 75 μl / ml (n-octanoic acid / rabbit serum), and stir 30min, centrifuge at 12000r / min for 20min at room temperature and take the supernatant; add 0.1mol / L pH7.4 PBS solution with a volume of 1 / 10 of the supernatant, let stand for 2 hours or overnight at 4°C; centrifuge at 12000r / min at 4°C for 30min , discard the supernatant; dissolve the precipitate in a certain volume of 0.01 mol / L pH 7.4 PBS, and dialyze with 50-100 times the volume of 0.01 mol / L pH 7.4 PBS at 4°C overnight.

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Abstract

The invention discloses a method for preparing polyclonal antibody against mouse nerve growth factor (NGF) and application thereof in an enzyme-linked immunoassay method. The polyclonal antibody which is prepared by using mouse submandibular gland NGF as an immunogen can be used for quantitatively detecting the mouse growth factor. Therefore, an assembled kit can determine fixed position and expression of different tissue cell nerve growth factors and detect NGF contents in different samples such as blood, cells, tissues, culture medium supernatant, and the like.

Description

technical field [0001] The invention relates to a preparation method of a polyclonal antibody against mouse nerve growth factor (NGF), and an ELISA kit for detecting the nerve growth factor (NGF). Background technique [0002] Nerve growth factor (NGF) is a neurotrophic factor discovered by Italian scientist Levi-Montalcini in 1953. It is the most thoroughly researched nerve cell growth factor with dual biological functions of neuron nutrition and synaptic growth promotion. It is a regulatory factor that plays an important regulatory role in the development, differentiation, growth, regeneration and expression of functional properties of central and peripheral neurons. In 1960, American scientist Cohen extracted and purified NGF and proved its biological activity. In 1986, Montalcini and Cohen were awarded the Nobel Prize in Physiology or Medicine for their outstanding research on NGF. Since the 1990s, many pharmaceutical companies and drug research institutions at home a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/22C07K1/14G01N33/68
Inventor 任宏伟邹宇飞熊玲媛马彬云孙朗杨幼瑶马凌燕付永超吴敏华
Owner SINOBIOWAY BIOMEDICINE
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