AU2020104287A4

AU2020104287A4 - A BDNF enzyme-linked immunodetection kit and its preparation method

Info

Publication number: AU2020104287A4
Application number: AU2020104287A
Authority: AU
Inventors: Jiannan CHEN; Jiarong Guo; Zhigang Guo; Ziyu ZHANG
Original assignee: Nanjing Kanghe Institute of Cell Genetic Engineering Co Ltd
Current assignee: Nanjing Kanghe Institute of Cell Genetic Engineering Co Ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2021-03-11
Anticipated expiration: 2028-12-23

Abstract

The invention discloses a BDNF enzyme-linked immunodetection kit and its preparation method, which comprises an ELISA plate coated with coating antigen, 6 bottles of BDNF standard product solution, a BDNF monoclonal antibody working liquid, a goat anti-mouse antibody labeled with horseradish peroxidase, a substrate chromogenic solution, a termination liquid, a concentrated scrubbing solution and a concentrated reconstitution fluid. The invention also discloses a method for detecting BDNF by the enzyme-linked immunoreagent kit, which comprises the following steps: first, pre-treating the sample, then detecting with the reagent kit, and finally analyzing the detection result. The main reagents of the enzyme-linked immunosorbent kit provided by the invention are in the form of working solution, with high specificity, high sensitivity, high accuracy, which is capable of on-site monitoring and suitable for screening of a large number of samples. C3 4:1l 7:1 00 -L U UU A 141 Standard concentration (mgf'L) Figure 1.

Description

C3 4:1l

7:1

00

-L

U UU A 141

Standard concentration (mgf'L)

Figure 1.

A BDNF enzyme-linked immunodetection kit and its preparation method

TECHNICAL FIELD

The invention relates to the technical field of enzyme-linked immunosorbent assay, in

particular to a BDNF enzyme-linked immunosorbent assay kit and its preparation method.

BACKGROUND

Brain derived neurotrophic factor (BDNF) is the second neurotrophic factor to be

found after nerve growth factor. It is a kind of basic protein with molecular weight of 12.3

kD. It is composed of 119 amino acid residues and 3 pairs of disulfide bonds. BDNF plays

an important role not only in the development, differentiation, growth and regeneration of

various types of neurons, but also in the regeneration and repair of injured neurons and the

prevention of neurodegeneration.

In recent years, researchers at home and abroad have found a close relationship

between BDNF and many diseases such as depression, atopic dermatitis, schizophrenia,

stroke, Alzheimer's disease, tinnitus and tumor.

At present, the common methods used to detect BDNF have some problems such as

strict test conditions, high cost, cumbersome sample pretreatment and long test period.

Enzyme-linked immunosorbent assay (ELISA) is a solid-phase immunoassay technique,

which utilizes the characteristic of specific bond binding between antigen and antibody to

detect the body. Since the antigen or antibody on the solid carrier is still immunoreactive,

the binding mechanism is designed to show whether the specific antigen or antibody exists, and it can be quantitatively analyzed by color depth. Compared with other detection methods, ELISA has high sensitivity, high specificity, simple pretreatment and short analysis time, so it is more suitable for field monitoring and screening of a large number of samples.

SUMMARY

The present invention aims to provide a BDNF enzyme-linked immunoassay kit and

its preparation method to solve the problems existing in the prior art.

To achieve the above purpose, the invention provides a preparation method of BDNF

enzyme-linked immunoassay kit, which includes the following content:

(1) Preparation of ELISA plates coated with BDNF coupled antigens; (2) Preparation

of BDNF standard product solution in 6 bottles with concentrations of 0 mg/L, 0.2 mg/L,

0.6 mg/L, 1.8 mg/L, 5.4 mg/L, 16.2 mg/L, respectively; (3) Preparation of BDNF

monoclonal antibody working solution; (4) Goat anti-mouse antibody labeled with

horseradish peroxidase; (5) Preparation of substrate chromogenic solution, which is

composed of liquid A and liquid B, liquid A is urea peroxide, and liquid B is

tetramethylbenzidine; (6) Preparation of of termination liquid of 2 mol/L hydrochloric acid;

(7) Preparation of concentrated washing solution with a pH value of 7.2-7.6, which contains

0.5%-1.0% Tween-20, 0.1-0.3%o thimerosal preservative, 0.01-0.03mol/L phosphate

buffer, and the percentage is the weight volume percentage; (8) Preparation of concentrated

reconstitution fluid, which contains 5%-8% ovalbumin, 0.1-0.4 mol/L phosphate buffer,

and the percentage is weight-volume percentage.

The invention also provides an enzyme-linked immunoreagent kit for detecting

BDNF, and the kit comprises an ELISA plate coated with coating antigen, BDNF standard

product solutions, an enzyme marker, a substrate chromogenic solution, a termination

liquid, a concentrated scrubbing solution and a concentrated reconstitution fluid.

Further, the coating antigen is BDNF antigen, antibody or anti-antibody.

Further, the enzyme marker is an enzyme labeled BDNF antigen, an enzyme labeled

BDNF antibody or an enzyme labeled anti-antibody.

Further, the BDNF antibody is a monoclonal antibody.

Further, the labelled enzyme of the enzyme marker is horseradish peroxidase.

The invention discloses the following technical effects:

(1) The BDNF enzyme-linked immunoassay kit of the invention has good specificity

and it can accurately determine the concentration of BDNF in plasma;

(2) The BDNF enzyme-linked immunoassay kit of the invention has high sensitivity

for differential diagnosis and can guarantee a good linear relationship;

(3) The BDNF enzyme-linked immunoassay kit of the invention can be kept for 12

months at-20°C.

BRIEF DESCRIPTION OF THE FIGURES

In order to explain more clearly the embodiment of the invention or the technical

scheme in the prior art, a brief introduction will be given below to the drawings required

in the embodiment. Obviously, the drawings described below are only some embodiments of the invention. For ordinary technicians in the field, additional drawings may be obtained without creative labour.

Figure 1 shows the standard curve of BDNF as a kit containing the coating antigen

and the enzyme labeled antibody as an enzyme marker.

DESCRIPTION OF THE INVENTION

The technical scheme in the embodiment of the present invention is described clearly

and completely in conjunction with the figure in the embodiment of the present invention.

Obviously, the embodiments described are only part of the embodiment of the present

invention, not all of them. Based on the embodiments of the present invention, all other

embodiments obtained by ordinary technical personnel in the field without creative labour

fall within the scope of the protection of the present invention.

For the purposes, characteristics and advantages of the present invention to be more

clearly understood, the invention is described in further detail below in conjunction with

the drawing and specific embodiments.

Embodiment 1. Preparation of kit components

1.1 Preparation of BDNF monoclonal antibody

Injecting BDNF into the mice with the immune dose of 100 g/piece to produce the

polyclonal antibody serum. After high serum assay results, taking mouse splenocytes

which is fused with myeloma cells in the ratio of 9:1, and cell supernatants are determined

by indirect competition ELISA. Screening positive wells, cloning positive wells by limited

dilution method, until hybridoma cell lines secreting monoclonal antibodies are obtained.

The monoclonal hybridoma cell line of BDNF obtained by screening can produce unlimited

chloramphenicol specific antibody, and the antibody specificity is for BDNF, its sensitivity

can reach 0.025 g/L.

The monoclonal hybridoma cell line of BDNF is prepared by freezing liquid into cell

suspensions of 1 x 106 /ml, which are preserved in liquid nitrogen for medium-long term.

The cryopreservation tube is taken out during the resuscitation, and immediately dissolved

in water bath at 37C. Balb/c mice are injected with 0.5 ml of sterile paraffin oil/piece.

After 7 days, 1 x106BDNF monoclonal hybridoma cells are injected intraperitoneally, and

ascites are collected 7 days later. The ascites are purified by octanoic acid saturated

ammonium sulfate method, then they are stored at - 20 °C.

1.2 Preparation of Polyclonal Antibody

Rabbits are immunized with BDNF as immunogen and immunized with 1.5 mg/kg.

Mixing Immunogen with the same amount of Freund's complete adjuvant to make

emulsifier, which is injected into the neck an the back subcutaneously at multiple points

every 3 weeks. Taking the same dose of immunogen and the same amount of Freund's

incomplete adjuvant every 3 weeks to make the emulsifier. Performing booster

immunization for five times, and blood is collected 10 days after the last immunization, so

as to obtain purified polyclonal antibodies by fractional precipitation with ammonium

sulfate.

1.3 Preparation of anti-mouse antibody of goat

Goat anti-mouse antibody is obtained by immunizing goat with mouse antibody as

immunogen.

1.4 Preparation of goat anti-rabbit anti-antibody

Goat anti-rabbit antibody is obtained by immunizing goat with rabbit antibody as

immunogen.

1.5 Preparation of ELISA plate

The BDNF antigen is diluted to 0.2 g/ml with the coated buffer, adding 100d1to each

well, carrying out incubation at 37°C for 2h. Removing the coating solution, which is

washed twice with 20 times diluted concentrated washing solution, 30s each time. Patting

them dry, then adding 200dblocking solution to each well for incubation at 37°C 2h,

removing the liquid in the holes. After drying, storing the antigens in vacuum sealed with

aluminum film.

1.6 Preparation of anti-mouse antibody of goat labeled with enzyme

The modified sodium periodate method is used to coupling the anti-antibody with

horseradish peroxidase (HRP). The traditional sodium periodate method requires that the

molar ratio of enzyme to antibody in the reaction system be 4: 1.

Embodiment 2 Construction of ELISA kit for detecting BDNF

The enzyme-linked immunosorbent kit for the detection of BDNF is constructed from

the components in embodiment 1 which contains the following components:

0.6 mg/L, 1.8 mg/L, 5.4 mg/L, 16.2 mg/L, respectively; (3) Preparation of BDNF

monoclonal antibody working solution; (4) Goat anti-mouse antibody labeled with horseradish peroxidase; (5) Preparation of substrate chromogenic solution, which is composed of liquid A and liquid B, liquid A is urea peroxide, and liquid B is tetramethylbenzidine; (6) Preparation of of termination liquid of 2 mol/L hydrochloric acid;

0.5%-1.0% Tween-20, 0.1-0.3%o thimerosal preservative, 0.01-0.03mol/L phosphate

and the percentage is weight-volume percentage.

Embodiment 3 Sample Pretreatment

Taking 20 mg of pig plasma protein powder. After dissolving, shaking and mixing

well, then the solution is mixed with the diluted sample compound solution in the ratio of

1:49, and taking 50[ 1of which for analysis.

Embodiment 4 Sample detection with kit

Adding 50 L of the BDNF standard product solution or sample solution to the

micropore of the ELISA plate coated with BDNF coupled antigen, 50 L of the BDNF

monoclonal antibody working fluid, and working solution of horseradish peroxidase

labeled goat anti-mouse anti-antibody with 50 L/hole. After covering the plate with the

cover film, placing the solutions in a constant temperature oven for 30 minutes at 37C.

Pouring out the liquid in the well, adding 250d1of washing solution to each well, taking

out the liquid in the well after 30 seconds, and the operation of washing the plate is repeated

for 5 times, then which is patted with absorbent paper for drying; Adding 50[ 1of urea

peroxide solution A and 50[ 1of tetramethylbenzidine (TMB) solution B of substrate color development solution to each well. Gently shaking and mixing, covering the plate with a cover film, and the solutions are placeedt in a 37°C thermostat for 15 minutes. Adding 50[d of 2mol/L stop solution hydrochloric acid to each well, gently shaking and mixing. Setting the wavelength at 450nm with a microplate reader, and measuring the absorbance value

(OD value) of each well.

Dividing the absorbance value (Bo) of the first standard solution (0 standard) by the

obtained average absorbance value (B) of the standard solution of each concentration and

multiply it by 100% to obtain the percent absorbance value.Taking the half logarithm value

of BDNF standard concentration as X axis and the absorbance value as Y axis to draw the

standard curve (Figure. 1). The concentration of each sample ( g / L) and the content of

BDNF can be read from the standard curve.

Embodiment 5 Precision experiment of standard product

Drawing a batch of ELISA plates from the ELISA plates prepared in three different

time periods, with 10 kits from each batch. Drawing 20 microwells from each plate, and

measuring the absorbance value of 1.8mg/L standard solution. Calculating the coefficient

of variation, and the results are shown in Table 1.

Table 1 Standard repeatability test (CV %)

1 2 3 4 5 6 7 8 9 10

No.1 3.9 9.3 4.5 4.1 3.4 5.5 3.1 7.7 5.8 6.4

CV% No.2 3.3 6.0 8.2 7.6 3.3 4.6 6.0 3.7 4.2 6.3 No.3 5.3 4.9 3.6 4.5 7.2 8.0 2.5 3.5 5.6 7.2

The results show that the variation coefficient of 10 standard products per batch is

between 2. 5% - 9. 3%, which is in accordance with the requirements.

Embodiment 6 Sample precision test

Adding BDNF standard to negative pig plasma protein powder without BDNF, so that

the content of BDNF in pig plasma protein powder is 30%(30mg/g). Taking two kits from

three different batches, with each concentration repeated for 3 times. Calculating the

coefficient of variation respectively. The results are shown in Table 2.

Batch Measured value pg/kg Coefficient of Number variation CVo

1 28.0 27.4 32.6 9.1

31.5 30.7 32.5 7.1

2 28.6 29.8 31.5 9.7

29.8 27.4 32.7 9.1

3 30.5 28.1 31.9 7.0 32.0 28.6 30.5 8.2 The results show that the coefficient of variation of serum protein powder is between

7.0%- 9.7%.

Embodiment 7 Kit stability test

The storage condition of the kit is 2-8°C. After 6 months of determination, the

maximum absorbance value (zero standard), 50% inhibitory concentration, and actual

measured value of BDNF addition of the kit are all within the normal range. Considering

that there will be abnormal storage conditions during transportation and use, the kit is

stored at 37°C for 6 days, and accelerated aging experiments are performed. The results

showed that the indicators of the kit fully met the requirements. Taking into account the

occurrence of freezing of the kit, the kit is placed in the refrigerator at -20°C for 5 days,

and the measurement results also showed that the indicators of the kit are completely normal. From the above results, it can be concluded that the kit can be stored at 2-8°C for at least 12 months.

The above-mentioned embodiments only describe the preferred mode of the present

invention, and do not limit the scope of the present invention. Without departing from the

design spirit of the present invention, those of ordinary skill in the art have made various

contributions to the technical solutions of the present invention. Variations and

improvements should fall within the protection scope determined by the claims of the

present invention.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

The method for preparing BDNF enzyme-linked immunoassay kits, which includes

the following steps:

(1) Preparation of ELISA plates coated with BDNF coupled antigens; (2) Preparation

of BDNF standard product solution in 6 bottles with concentrations of 0 mg/L, 0.2 mg/L,

0.6 mg/L, 1.8 mg/L, 5.4 mg/L, 16.2 mg/L, respectively; (3) Preparation of BDNF

monoclonal antibody working solution; (4) Goat anti-mouse antibody labeled with

horseradish peroxidase; (5) Preparation of substrate chromogenic solution, which is

composed of liquid A and liquid B, liquid A is urea peroxide, and liquid B is

tetramethylbenzidine; (6) Preparation of of termination liquid of 2 mol/L hydrochloric acid;

(7) Preparation of concentrated washing solution with a pH value of 7.2-7.6, which contains

0.5%-1.0% Tween-20, 0.1-0.3%o thimerosal preservative, 0.01-0.03mol/L phosphate

buffer, and the percentage is the weight volume percentage; (8) Preparation of concentrated

reconstitution fluid, which contains 5%-8% ovalbumin, 0.1-0.4 mol/L phosphate buffer,

and the percentage is weight-volume percentage.
2. The enzyme-linked immunoassay kit for detecting BDNF prepared according to the

preparation method of claim 1, is characterized in that the kit comprises an ELISA plate

coated with coating antigen, BDNF standard product solutions, an enzyme marker, a

substrate chromogenic solution, a termination liquid, a concentrated scrubbing solution and

a concentrated reconstitution fluid.
3. According to claim 2, the kit is characterized in that the package is proto-BDNF

antigen, antibody or anti-antibody.
4. According to claim 2, the kit is characterized in that the enzyme marker is an

enzyme labeled BDNF antigen, an enzyme labeled BDNF antibody, or an enzyme labeled

anti-antibody.
5. According to claim 3, the kit is characterized in that the BDNF antibody is a

monoclonal antibody.
6. According to the kit described in claim 2 or claim 4, it is characterized in that the

marked enzyme of the enzyme marker is horseradish peroxidase.
7. The application of the kit in the detection of BDNF content in accordance with any

of the claims 2-6.

Figure 1.