CN117268877A

CN117268877A - Method for detecting nerve growth factor in human tear and method for treating tear

Info

Publication number: CN117268877A
Application number: CN202311557896.9A
Authority: CN
Inventors: 王雪莉; 张红梅; 李弯弯; 王变珍
Original assignee: United Power Pharma Tech Co ltd
Current assignee: United Power Pharma Tech Co ltd
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2023-12-22
Anticipated expiration: 2043-11-21
Also published as: CN117268877B

Abstract

The present application provides methods for detecting nerve growth factor NGF in human tears and methods for treating tears thereof. The method comprises the following steps: a) Providing a tear sample for NGF detection on a matrix material; and b) extracting NGF in a tear sample provided on the matrix material using the extraction solution. The application establishes the detection method of NGF in human tears for the first time, and the method has the advantages of good specificity, high sensitivity, high recovery rate, simple operation and good repeatability; and the interference of tear substances on NGF detection can be well solved by adopting the sample extraction method.

Description

Method for detecting nerve growth factor in human tear and method for treating tear

Technical Field

The present application relates to the field of detection, and in particular, to a method for detecting nerve growth factor in human tears and a method for processing tear samples thereof.

Background

Neurotrophic Keratitis (NK) is a rare and problematic disease caused by damage to the trigeminal nerve, resulting in sensory and dominating dysfunction of the corneal nerve, 300-400 times the nerve endings of the skin, originating from the ciliary long and short nerves of the trigeminal ocular branch. The main reason for the strong corneal sensitivity is the large number of demyelinated trigeminal nerve endings in the corneal epithelium. The disease is characterized by a reduced or absent perception of the cornea, the appearance of dry eye, corneal epithelial defects and corneal ulcers, and eventually the dissolution and perforation of the corneal stroma. NK is common in patients with recurrent episodes of viral keratitis, surgical injuries, craniocerebral tumors, craniocerebral trauma, diabetes, and the like.

NGF is a neurotrophic factor that promotes the growth and survival of sensory and sympathetic neurons, and restores neuronal function, primarily through the high affinity receptor TrkA, thus promoting healing of NK corneal lesions and improving corneal perception and tear production.

During the development of new drugs, the therapeutic goal of NK as a whole is to prevent disease progression and avoid severe injury. The treatment method comprises using autologous serum, umbilical cord serum and nerve growth factor. The nerve growth factor has effects of promoting cornea nerve fiber regeneration, promoting cornea epithelial growth and differentiation, promoting tear secretion, and relieving inflammation.

There is no method available to detect NGF concentration in tears after administration of nerve growth factor eye drops to the eye. The blood concentration of the eye drops of the nerve growth factor is very low after the eye drops are given to the human body, and the eye drops are basically undetectable. Thus, there is an urgent need to establish a general method for detecting NGF concentration in human tears, which not only can provide NGF drug concentration data in local target organs, but also can help to study NGF pharmacokinetics in tears.

Disclosure of Invention

The inventor of the application finds that the pretreatment of the tear sample by a specific method and then the measurement of NGF in the pretreated tear sample can well solve the interference of tear matrix on NGF detection, thereby realizing the accurate measurement of the concentration of NGF in tear.

Specifically, the application provides the following technical scheme.

In a first aspect, the present application provides a method of processing a tear sample for detecting neurotrophic factor NGF, comprising:

a) Providing a tear sample for NGF detection on a matrix material; and

b) NGF in a tear sample provided on the matrix material is extracted using an extraction solution.

In a second aspect, the present application provides a method for detecting NGF concentration in a tear sample, comprising:

a) Providing a tear sample treated by the method of the first aspect; and

b) And measuring the concentration of NGF in the treated tear sample.

The application establishes the detection method of NGF in human tears for the first time, and the method has the advantages of good specificity, high sensitivity (100 pg/mL can be reached), high recovery rate, simple operation and good repeatability; the biological matrix in the tears has large interference on NGF detection and strong matrix effect, but the sample extraction method can well solve the interference of the tear matrix on NGF detection.

Detailed Description

The inventor of the application finds that the pretreatment of the tear sample by a specific method and then the measurement of NGF in the pretreated tear sample can well solve the interference of tear matrix on NGF detection, thereby realizing the accurate measurement of the concentration of NGF in tear. Such a method may not only provide data on the concentration of NGF drug in a local target organ, but also aid in studying the pharmacokinetics of NGF in tears.

Specifically, the application provides the following technical scheme.

a) Providing a tear sample for NGF detection on a matrix material; and

Nerve growth factor (nerve growth factor, NGF) can promote the growth, development, differentiation and maturation of central and peripheral neurons, maintain the normal functions of the nervous system, and accelerate the repair after the damage of the nervous system. NGF is widely distributed in various tissues and organs of the body (including the brain), and the concentration in target tissues is related to the density of branches of sympathetic nerves and sensory nerves in target areas and the mRNA content. The nerve growth factor family includes: nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, neurotrophin-6, neurotrophin-7, etc., mainly the first 4. NGF is the predominant class of nerve growth factor family, exists predominantly in the form of precursors in tissues, and is processed to form mature NGF in the submandibular glands. The biological effects of nerve growth factor mainly comprise functions of nourishing nerve, protecting nerve, promoting nerve regrowth, etc.

"matrix material" in the present application generally refers to a solid support for supporting a collected tear sample, such as filter paper, paper towels, tear extraction strips, and the like, preferably Schimer's strip.

In particular embodiments, the extract used to extract NGF in a tear sample provided on the matrix material may be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., ascites in the testes), vaginal rinse, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage, drainage fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), intraocular fluid (e.g., aqueous humor), and the like. The body fluid may be diluted with a buffer to form a body fluid dilution.

Preferably, the extract is blood, plasma or serum. More preferably, the extract is serum. Most preferably, the extract is a serum dilution. The serum may be prepared from a mixture of blood obtained from a human collection.

In a specific embodiment, the serum dilution is prepared by diluting serum with 1.25-20 volumes, preferably 2-18 volumes, more preferably 4-16 volumes of buffer. Preferably, the serum dilution is prepared by diluting serum with about 5 volumes of buffer.

The buffer solution is prepared from a buffer pair consisting of weak acid and salts of conjugate base thereof or weak base and salts of conjugate acid thereof, and can slow down pH change when a certain amount of other substances are added. The buffers used herein may be selected from phosphate, citrate, carbonate, acetate, barbituric acid, tris (Tris) hydroxymethyl aminomethane), borate, MOPS buffer, HEPES buffer, tricine buffer, and the like. Preferably, the buffer is a PBS buffer, for example a PBS buffer containing 1% BSA.

The serum may be derived from mammals, including but not limited to primates, cows, horses, pigs, sheep, goats, dogs, cats, rodents such as rats and mice. Preferably, the mammal is a non-human primate or human. Particularly preferred mammals are humans.

In a specific embodiment, the tear sample is derived from a human.

In particular embodiments, the tear sample is provided in an amount of about 20 mL to about 30 mL.

In a specific embodiment, the extract is in an amount of about 700 mL to about 900 mL.

The term "about" as used in this application refers to +/-10% of the indicated value. For example, with respect to the number "100," about 100 "may refer to a range of 90 to 110.

a) Providing a tear sample treated by the method of the first aspect; and

b) And measuring the concentration of NGF in the treated tear sample.

The concentration of NGF may be measured by methods known in the art, such as ELISA methods, high Performance Liquid Chromatography (HPLC), mass spectrometry, and the like.

In this specification and claims, the words "comprise", "comprising" and "include" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.

It should be understood that features, characteristics, components or steps described in particular aspects, embodiments or examples of the present application may be applied to any other aspects, embodiments or examples described herein unless contradicted by context.

The foregoing disclosure generally describes the present application, examples are further illustrative of the present application and are not to be construed as limiting the present application.

Examples

Example 1 method for detecting nerve growth factor in human tear fluid

The principle of the detection method is briefly described as follows:

coating reagent (mouse anti-human beta-NGF capture antibody, R & D, cat# 840366) in an ELISA plate, adding the coated sample, and capturing the object to be detected in the sample by the coating reagent. Then adding detection reagent (biotinylated goat anti-human beta-NGF detection antibody, R & D, cat. No. 840367) to form a "coating reagent-analyte-detection reagent" complex, adding enzyme-labeled reagent (streptavidin-HRP), and finally adding HRP substrate (TMB). After the termination reaction of the termination solution, reading the response value (OD value) of the instrument at the detection wavelength 450 nm and the reference wavelength 570 nm, wherein the absorbance and the NGF content are in a functional relation, and calculating the NGF concentration in the sample by using the functional relation, thereby realizing the detection purpose of quantitatively detecting the NGF in tear.

Pretreatment method of human tear sample:

20-mL-30 mL tear sample is sucked into a centrifuge tube (2 ml capacity), tear extraction test paper such as Schimer test paper (Tianjin's crystal, cat# AztdkaWF) is put into the centrifuge tube, the tear sample is automatically soaked in the test paper, then 700-900 mL extract (healthy human mixed serum: 1% bsa PBS volume ratio=1:1.25-1:20) is sucked into the centrifuge tube, and vortex mixing is performed to extract NGF on the test paper.

The test method comprises the following experimental steps:

coating: coating reagent working solution (mouse anti-human beta-NGF capture antibody: PBS=1:100-1:150) is added into the ELISA plate at 90-110 mu L/hole, and the plate is sealed and covered for 16-18 hr at 2-8 ℃.

Washing the plate: the ELISA plate was removed, plate washing solution (pH 7.2-7.4, 1. Times. PBST) was added at 300-400 mL/well, the plates were washed 3-6 times, and the plates were dried on paper.

Closing: blocking solution (PBS containing 1% BSA) is added into the ELISA plate at 250-300 mu L/hole, membrane sealing plates are closed, and the plates are incubated for 1-2 hr in a biochemical incubator at 25+ -3 ℃.

Washing the plate: taking out the ELISA plate, adding the plate washing liquid into the ELISA plate at a ratio of 300-mL-400 mL/hole, washing the plate for 3-6 times, and beating the plate on paper.

Sample adding: human tear standard curve samples, quality control samples, or NGF samples after the above extraction were added at 90-110 mL/well, membrane sealed plates were closed, and incubated in a biochemical incubator at 25+ -3deg.C for 2hr + -20 min (about 450-600 rpm) with shaking plates, according to the plate diagrams shown in tables 1 and 2 below.

Washing the plate: taking out the ELISA plate, adding the plate washing liquid into the ELISA plate at a rate of 300-mL-400 mL/hole, washing the plate for 3-6 times, and beating the plate on paper to dry.

Adding a detection reagent working solution: detection reagent working solution (biotinylated goat anti-human β -NGF detection antibody: pbs=1:60-1:100 with 1% bsa) was added to the elisa plate at 90-110mL per well, the plates were sealed, and incubated for 2hr ±20min (about 450-600 rpm) in biochemical incubator shake plates at 25±3 ℃.

Washing the plate: taking out the ELISA plate, adding the plate washing liquid into the ELISA plate at 300-400/mL/hole, washing the ELISA plate for 3-6 times, and beating the ELISA plate on paper to dry.

And (3) adding an enzyme-labeled reagent working solution: an enzyme-labeled reagent working solution (streptavidin-HRP: pbs=1:20-1:50 with 1% bsa) was added to the elisa plate at 90-110 mL/well, the plates were sealed, and incubated in a biochemical incubator at 25±3 ℃ for 30min±15min (about 450-600 rpm) with shaking.

Adding a substrate: the substrate (Thermo, cat# 34029) was added at 90-110mL per well and reacted at room temperature for 30 min.+ -. 10min away from light.

Stopping adding a stopping solution: adding stop solution (1M H) at 40-60 μl/well ₂ SO ₄ ) The method comprises the steps of carrying out a first treatment on the surface of the Slight shaking ensures that the edges of each well are free of blue-green phenomena.

Reading a plate: OD values were read at detection wavelength 450 nm and reference wavelength 570 nm within 20min after addition of stop solution, and data were processed with Molecular Device inc. Reader SoftMax Pro GxP version 6.4.4.

Standard curve, preparation of quality control samples and validation samples:

and (5) absorbing Recombinant Human Beta Nerve Growth Factor (beta-NGF) (source: R & D, batch number: 1544729) into a centrifuge tube containing human mixed tears to prepare stock solution of a standard curve and a quality control sample, wherein the concentration is 7500pg/mL, and gradually diluting the stock solution with the human mixed tears according to a required proportion to obtain standard curve samples STD 01-STD 07, BLK and the quality control sample. And then obtaining a pretreated marked curve sample and a pretreated quality control sample from each obtained linear concentration point sample and each quality control concentration sample according to the pretreatment method of the human tear sample (without using a test strip), and verifying that the sample is the human tear sample extracted by the test strip according to the pretreatment method of the human tear sample.

And regression is carried out on the read OD value and the concentration of the object to be detected through MD plate reader software to generate a standard curve, and the concentrations of the quality control sample and the verification sample are calculated by the following standard curve.

Table 1: arrangement sequence of samples in ELISA plate

Remarks: STD01-07: standard curve samples, ULOQ-LLOQ: a tear quality control sample;

BLK: blank;

pretreatment of each standard curve sample and quality control sample in table 1 was performed using an extract (healthy human mixed serum: 1% bsa PBS volume ratio=1:5).

Table 2: arrangement sequence of samples in ELISA plate

Remarks: STD01-07: standard curve sample, ULOQ-after extraction-LLOQ-after extraction: the corresponding sample is extracted by a test strip;

BLK: blank;

pretreatment of each standard curve sample and validation sample in table 2 was performed using an extract (healthy human mixed serum: 1% bsa PBS volume ratio=1:5).

Table 3: raw OD values corresponding to each sample shown in table 1

Table 4: raw OD values corresponding to each sample shown in table 2

Table 5: standard curve fitting results for each standard curve sample shown in table 1

Table 6: quality control sample fitting results for each quality control sample shown in table 1

Table 7: standard curve fitting results for each standard curve sample shown in table 2

Table 8: quality control sample fitting results for each quality control sample shown in table 2

The biological matrix in the tears has great interference on NGF detection in the tears and has strong matrix effect, but from the results, the NGF concentration as low as 100 pg/mL can be detected even by adopting the method in the application, and the recovery rate of the extraction method in the application reaches more than 90 percent, so that the interference problem of the tear matrix on NGF detection can be well solved.

While the foregoing has been with a general description and specific embodiments, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the invention as claimed.

Claims

1. A method for processing a tear sample to detect neurotrophic factor NGF in the tear sample, comprising:

a) Providing the tear sample on a matrix material; and

2. The method of claim 1, wherein the matrix material is filter paper, tissue, or tear-extraction test paper.

3. The method of claim 1 or 2, wherein the extraction solution is a body fluid.

4. A method according to claim 3, wherein the body fluid is diluted with 1.25-20 volumes, preferably 2-18, more preferably 4-16, most preferably about 5 volumes of buffer.

5. The method of claim 4, wherein the buffer is a PBS buffer or a PBS buffer containing 1% bsa.

6. A method according to claim 3, wherein the body fluid is of human origin and is selected from blood, plasma or serum.

7. The method of claim 1 or 2, wherein the tear sample is of human origin.

8. The method of claim 1 or 2, wherein the tear sample is provided in an amount of 20 mL to 30 mL.

9. The method of claim 1 or 2, wherein the extract is in an amount of 700 mL to 900 mL.

10. A method for detecting NGF concentration in a tear sample, comprising:

a) Providing a tear sample treated by the method of any one of claims 1 to 9; and

b) The concentration of NGF in the treated tear sample is measured, for example by ELISA methods.