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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 peptides, anti-growth factor immunoglobulin, chemical instruments and methods, etc., and can solve the problems of difficulty in obtaining antibodies and weak immunogenicity

Active Publication Date: 2010-01-27
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

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Experimental program
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Effect test

Embodiment 1

[0052] The preparation of embodiment 1 polyclonal antibody

[0053] preparation

[0054] 1. The purified NGF was polymerized with glutaraldehyde at 150 μg / rat, fully mixed with an equal volume of complete Freund’s adjuvant, emulsified, and subcutaneously injected into normal rabbits at multiple points;

[0055] 2. One month later, the same amount of antigen was fully mixed and emulsified with incomplete Freund's adjuvant, and subcutaneously injected again at multiple points;

[0056] 3. After one month, repeat step 2;

[0057] 4. Ear blood was collected one week after the last immunization, and the titer was measured by indirect ELISA method, which should be greater than 1:1000;

[0058] 5. Blood was collected from the ear vein, 50ml / time, twice, and the serum was collected and stored at -20°C.

Embodiment 2

[0059] The purification of embodiment 2 polyclonal antibody

[0060] 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), the CNBr-activated sepharose 4fast flow gel was washed several times with pre-cooled 0.5mM~50mM HCl, negative pressure ultrafiltered, and replaced in the coupling buffer; the antigen was combined with the activated The micelles were mixed and stirred overnight at 4°C; the incompletely bound antigen was washed with coupling buffer; the blocking buffer (0.2mol / L glycine) was reacted at room temperature for more than 0.5 hours to block the activation site that was not saturated by the antigen; coupling buffer and acetate buffer 0.1mol / LCH 3 COO·Na and 0.5mol / L NaCl, pH 4.0) were washed alternately for more than 2 times, and the micelle coupled with the antigen was stored in 20% alcohol.

[0061] 2. Binding buffer (PBS) full...

Embodiment 3

[0066] Embodiment 3 ammonium sulfate-n-octanoic acid method purification method:

[0067] Take a certain volume of serum, dilute 2 times or 4 times with 0.06mol / L pH4.8 HAc-NaAc buffer; add n-octanoic acid dropwise to make the final concentration 75μl / ml (n-octanoic acid / rabbit serum), and stir 30min, centrifuge at 12000r / min at room temperature for 20min and take the supernatant; add 0.1mol / L PBS solution with pH7.4 whose volume is 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 0.01mol / LpH7.4 PBS, dialyze overnight at 4°C with 50-100 times the volume of 0.01mol / LpH7.4 PBS.

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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 an...

Claims

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Application Information

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Patent Type & Authority Applications(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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