CN115060907A - ELISA kit for detecting Nogo-B protein expression level - Google Patents

Abstract

An ELISA kit for detecting Nogo-B protein expression level comprises a Nogo-B antibody, a coating solution, a confining solution, an enzyme-labeled secondary antibody, positive control serum and negative control serum. The ELISA kit prepared by the invention can detect the degree of illness of fatty liver patients by detecting the expression level of Nogo-B in a serum sample, has high detection success rate, good technical reproducibility, less material consumption, low cost, simple operation and convenient and quick use, greatly improves the detection efficiency and the diagnosis efficiency of fatty liver patients, and has good application prospect.

Description

ELISA kit for detecting Nogo-B protein expression level

Technical Field

The invention relates to an ELISA kit for detecting Nogo-B protein expression level, belonging to the technical field of biological medicine.

Background

nogo genes were an unknown gene discovered in 2000, and were named nogo because their transcriptionally encoded proteins inhibited the regeneration of Central Nervous System (CNS) axons. It is a large group of transmembrane proteins mainly localized in endoplasmic reticulum and cell membrane of eukaryotic cells, and is also called reticulin family 4 (RTN 4) because the C-terminal of the protein contains a reticulin homeodomain. The nogo gene can encode three proteins: Nogo-A, Nogo-B, Nogo-C.

Nogo-B (also known as human myelin-associated growth inhibitor B) consists of 373 amino acid residues and has a molecular weight of about 37 kDa. Exon 2 of Nogo-B isoform Nogo-B2 is 58 bp more abundant at the DNA level than Nogo-B, making it 1.9kDa larger in protein molecular weight than Nogo-B. The C-terminus of the Nogo-B protein is highly homologous to the Nogo-a/C protein, consists of 188 amino acid residues, contains two highly conserved transmembrane hydrophobic groups, 35 and 36 amino acid residues in size, respectively, and is long enough to span the membrane twice, with an extracellular loop of 66 amino acid residues (i.e., Nogo-66) joining between these two putative transmembrane regions, which can bind to the Nogo receptor subunit NgR. The 200 amino acid residues at the Nogo-B N-terminus are essentially identical to Nogo-A, the N-terminus is directed extracellularly, and there is no specific secondary or tertiary conformation, presumably it is a functional region of Nogo-B. In normal individuals, Nogo-B mRNA is expressed in the optic nerve, spinal cord, brain, sciatic nerve, lung and kidney, and its expression was not detected in skeletal muscle. The protein is expressed in oligodendrocyte, testis, myocardial cell of brain and spinal cord, vascular endothelial cell of cardiovascular system, vascular smooth muscle cell, airway epithelial cell of lung, airway smooth muscle cell, pulmonary alveolar epithelial cell, liver cell of liver, liver and gall bladder endothelial cell, hepatic stellate cell, fibroblast, etc. Nogo-B is an important member of Nogo, is widely distributed in vivo and can be positioned in various tissues and organs to play different functions, and the existing research shows that the Nogo-B mainly participates in various pathophysiological processes such as tissue injury repair, inflammation regulation and control, tumor inhibition, apoptosis, nervous system regeneration inhibition, vascular intimal injury repair and the like. The research on Nogo-B from the initial central nervous system to the current fields of cardiovascular diseases, liver diseases, malignant tumors, tissue repair, inflammatory diseases and the like, the importance of Nogo-B is further clarified, and the relationship between Nogo-B and various diseases is gradually revealed with the deepening of mechanism research.

Nogo-B subcellular localization is mainly on endoplasmic reticulum and cell membrane, and under pathological conditions, Nogo-B protein expression is correspondingly changed. Nogo-B expression is known to be reduced in disease states such as vascular mechanical or ischemic injury, tissue acute chemical inflammation, acute and chronic asthma, and to be expressed at a low level in atherosclerotic plaque and aneurysm; high expression of Nogo-B in serum of patients with coronary heart disease, eye tissues of patients with proliferative diabetic retinopathy, human retinal endothelial cells in high sugar environment, and the like; the 'non-alcoholic fatty liver disease' is up to 75 percent in the insulin resistant people such as obesity, diabetes and the like, can develop liver fibrosis and cirrhosis even liver cancer from simple liver steatosis and steatohepatitis, and seriously threatens the health of Chinese people. The pathogenesis of the fatty liver disease is complex, at present, the clinical thinking that obesity, diabetes, long-term hormone use, improper diet and life style, insufficient exercise and the like are all the pathogenic causes, and researches show that in a chronic hepatitis microenvironment in a fatty liver patient, LDL can be oxidized into OXLDL and absorbed into the liver through a CD36 receptor on the surface of a liver cell, fat deposition and cell degeneration in the liver are caused, the expression of Nogo-B protein on endoplasmic reticulum is activated, Nogo-B is mainly expressed in nonparenchymal cells of the liver, and the transformation growth factor-beta 1 signal channel is inhibited through inducing interleukin-6/signal transduction and transcriptional activator 3 signal channels, so that the proliferation and regeneration of the liver cell are promoted, the fibrosis development of the liver is induced, and the severity degree of the liver disease is aggravated. Thus, elevated plasma Nogo-B expression levels following fatty liver or liver fibrosis and cirrhosis are positively correlated with liver lesion severity, whereas inhibition of Nogo-B protein expression may alleviate liver injury by down-regulating endoplasmic reticulum stress. Therefore, the research team believes that the expression level of Nogo-B protein can be used as a novel marker for detecting fatty liver patients.

At present, there are two general methods for detecting the expression level of Nogo-B protein, one is the common method is protein immunoblotting (Western Blot), which is a protein detection technique that transfers the total protein of cells or tissues after electrophoretic separation from gel to NC membrane or PVDF membrane as a solid support, and then detects a specific antigen with a specific antibody. This method can only be used for detecting protein samples with antibodies and is often used only for qualitative analysis, generally for semi-quantitative analysis; the other is immunohistochemical method, which is to determine the antigens (polypeptides and proteins) in tissue cells by using the principle of immunological basic principle-antigen-antibody reaction, namely the principle of specific binding of antigen and antibody, and developing color development agents (fluorescein, enzyme, metal ions and isotopes) for labeling the antibody through chemical reaction, and to perform research on positioning, qualitative and quantitative (mainly for positioning) of the antigens and the antibody. The two detection methods have the defects of complicated detection process, time waste, incapability of accurately carrying out quantitative analysis on the protein expression amount and the like.

Disclosure of Invention

The invention aims to provide an ELISA kit for detecting Nogo-B protein expression level.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: an ELISA kit for detecting Nogo-B protein expression level comprises a Nogo-B antibody, a coating solution, a confining solution, an enzyme-labeled secondary antibody, positive control serum and negative control serum.

The preferable technical scheme is as follows: the coating solution is carbonate buffer solution with pH value of 9.6 and concentration of 50 mM; the blocking solution is PBST buffer solution containing 2% (W/V) BSA; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled anti-Nogo-B protein; the positive control serum is serum of a fatty liver patient; the negative control serum was the serum of a normal person who did not suffer from fatty liver.

The preferable technical scheme is as follows: the kit also comprises an enzyme label plate, a sample diluent, a second antibody diluent, a washing solution, a developing solution A and a developing solution B.

The preferable technical scheme is as follows: both the sample diluent and the second antibody diluent were PBST buffer containing 1% (W/V) BSA; the washing solution is 0.01M PBST buffer solution containing 0.05% Tween 20 and having a pH value of 7.4.

The preferable technical scheme is as follows: the color developing solution A is 0.02% (W/V) TMB, and the preparation method comprises the following steps: dissolving 0.005g of methylbenzidine in 25ml of deionized water; the color developing liquid B is 0.006% (W/V) peroxideThe urea chemical comprises the following components in parts by weight: taking 4.665g of citric acid and Na ₂ HPO ₄ 18.40g of the extract is dissolved in 400ml of deionized water, 3.2ml of 0.75 percent of urea hydrogen peroxide is added, the pH value is adjusted to 5.0-5.5, the deionized water is added to the solution until the volume reaches 500ml, and the mixture is uniformly mixed and stored at 4 ℃.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

1. the invention uses specific marker (Nogo-B antibody) to prepare an ELISA kit which can accurately measure the expression level of Nogo-B protein. Compared with the prior art, the kit has the following advantages: one is that the sample to be tested is more of a variety such as serum, plasma, urine, cells or tissue culture supernatant; secondly, the method is mainly used for quantitative analysis, and the sensitivity is particularly high; and the detection process is simple, rapid, stable and easy to operate automatically, time and experiment expenses are saved, and the method has the characteristics of high specificity and sensitivity.

2. The invention firstly proposes that fatty liver and severity thereof can be effectively detected by detecting the expression level of Nogo-B protein in human serum, and the detection sensitivity is high and the specificity is strong. The detection rate and the accuracy of the early fatty liver patients are improved to a great extent, the misdiagnosis rate of the early fatty liver is greatly reduced, and the accuracy of the early fatty liver diagnosis is greatly improved.

3. The ELISA kit prepared by the invention can detect the degree of illness of fatty liver patients by detecting the expression level of Nogo-B in a serum sample, has high detection success rate, good technical reproducibility, less material consumption, low cost, simple operation and convenient and quick use, greatly improves the detection efficiency and the diagnosis efficiency of fatty liver patients, and has good application prospect.

5. The ELISA kit prepared by the invention can simultaneously detect Nogo-B protein expression in a plurality of sample serums, has application prospect in experiments or clinics, and saves a large amount of time and detection cost.

Drawings

FIG. 1 is a layout diagram of a 48-well microplate in the ELISA kit of the present invention (wherein sample represents that a sample serum to be tested is added to the well for detecting the expression level of the corresponding antigen in the serum to be tested, "+" represents a positive control well to which a positive control serum is added, "-" represents a negative control well to which a negative control serum is added, and "blank" represents a blank control well to which a sample diluent without serum is added, and the concentrations of the calibrator are 32, 16, 8, 4, 2, and 1 ng/mL in this order.

FIG. 2 is a schematic diagram of a double antibody sandwich ELISA experiment.

FIG. 3 is a statistical table showing the expression levels of Nogo-B protein in the serum of healthy people and fatty liver patients, which were determined by randomly extracting 24 from healthy people with different BMI indices and 14 from fatty liver patients.

FIG. 4 is a graph of Nogo-B expression profile in a healthy human group and a fatty liver patient group.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-4. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are provided for a better understanding of the present invention, and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1: ELISA kit for detecting Nogo-B protein expression level and application thereof

Firstly, preparation of a kit:

the invention prepares an ELISA kit for rapidly detecting Nogo-B protein expression according to the principle of a double-antibody sandwich enzyme-linked immunosorbent assay. The principle of the double-antibody sandwich enzyme-linked immunosorbent assay is that a specific antibody is connected to a solid phase carrier, an antigen to be detected in a sample is combined with the specific antibody to form a solid phase antibody-detected antigen complex, an enzyme-labeled secondary antibody is combined with the antigen in the solid phase antibody-detected antigen complex to form the solid phase antibody-detected antigen-enzyme-labeled secondary antibody complex, and then the chromogenic degree of the mixture after adding a substrate is measured to determine the content of the antigen to be detected (see figure 2).

1. Experimental materials and reagents:

(1) a Nogo-B antibody;

(2) 48-hole enzyme label plate: 3590(costar. us);

(3) coating liquid: 50mM carbonate buffer, pH 9.6;

(4) sealing liquid: PBST buffer containing 2% (W/V) BSA;

(5) sample diluent: PBST buffer containing 1% (W/V) BSA;

(6) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;

(7) enzyme-labeled secondary antibody: horseradish peroxidase-labeled anti-Nogo-B protein;

(8) washing liquid: 0.01M PBST (phosphate Tween) buffer pH7.4 containing 0.05% Tween 20;

(9) positive control serum: the positive control serum of Nogo-B, namely a fatty liver patient of which the Nogo-B protein expression is higher than the average expression quantity in the serum of a normal human population is detected by using an indirect ELISA method and a Western blot method;

(10) negative control serum: Nogo-B negative control serum, namely the serum of normal people with Nogo-B protein expression level being the average content in the serum of normal people by indirect ELISA and Western blot method;

(11) color developing solution A: 0.02% (W/V) TMB, preparation: dissolving 0.005g of methylbenzidine (TMB) in 25ml of deionized water;

(12) color developing solution B: 0.006% (W/V) of urea peroxide, formulation: taking 4.665g of citric acid and 418.40 g of Na 2 HPO, fully dissolving the citric acid and the 418.40 g of Na 2 HPO in 400ml of deionized water, adding 3.2ml of 0.75 percent urea hydrogen peroxide, adjusting the pH value to 5.0-5.5, adding deionized water to a constant volume to a final volume of 500ml, uniformly mixing and storing at 4 ℃;

(13) stopping liquid: 10% sulfuric acid;

(14) an enzyme-labeling instrument: star Fax 2100 (aware. us).

2. Preparing an antibody-coated ELISA plate:

(1) preparation of Nogo-B antibody solution:

and respectively dissolving Nogo-B antibody protein into the coating solution, and fully and uniformly mixing to prepare an antibody solution with a certain degree.

(2) Coating an enzyme label plate:

adding the prepared Nogo-B antibody solution into a sample application hole of a 48-hole enzyme label plate, wherein the sample addition amount is 100 mu l/hole; incubating at 37 deg.C for 1h, removing coating solution at 4 deg.C overnight, and washing with washing solution for 3 times, each for 3 min.

(3) And (3) sealing:

blocking solution (the amount of the blocking solution added is 300. mu.l/well) is added to the spotting wells of the coated 48-well microplate, incubated at room temperature for 2 hours, and then removed.

(4) And (3) drying and packaging:

and (3) placing the 48-hole elisa plate subjected to sealing treatment in a drying box at 37 ℃, drying, and packaging to obtain the antibody-coated 48-hole elisa plate, and storing at 4 ℃ for later use.

3. The kit comprises the following components:

(1) the 48-hole enzyme label plate coated by the antibody prepared in the step 2;

(2) sample diluent: PBST buffer containing 1% (W/V) BSA;

(3) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;

(4) enzyme-labeled secondary antibody: horseradish peroxidase-labeled anti-Nogo-B antibody;

(5) color development liquid: the color development liquid consists of a color development liquid A and a color development liquid B, wherein the color development liquid A is 0.02% (W/V) TMB, and the color development liquid B is 0.006% (W/V) urea peroxide; when in use, the color development liquid A and the color development liquid B are uniformly mixed in equal volume according to the ratio of 1: 1;

(6) stopping liquid: 10% sulfuric acid;

(7) washing liquid: 0.01M PBST (phosphate Tween) buffer pH7.4 containing 0.05% Tween 20;

(8) positive control serum: Nogo-B positive control serum;

(9) negative control serum: Nogo-B negative control serum;

and the reagents (2) - (9) are packaged respectively and then form a kit with a 48-hole enzyme label plate coated by the antibody.

Second, using method of kit

1. Incubation of serum samples:

diluting a serum sample to be detected by using a sample diluent according to the proportion of 1:10, adding the diluted serum sample into a reaction hole of a 48-hole enzyme label plate coated with an antibody, wherein the sample adding amount is 100 mu l/hole, placing the diluted serum sample in a constant-temperature incubator at 37 ℃ for incubation for 1h, then discarding the liquid in the reaction hole, and washing the serum sample for 3 times by using a washing solution, wherein the washing time is 3min each time.

2. Incubation with enzyme-labeled secondary antibody:

diluting the horseradish peroxidase-labeled anti-Nogo-B antibody with a second antibody diluent according to the proportion of 1:80000, adding the diluted horseradish peroxidase-labeled anti-Nogo-B antibody protein into a reaction hole of a 48-hole enzyme label plate, wherein the sample adding amount is 100 mu l/hole, placing the reaction hole in a constant-temperature incubator at 37 ℃ for incubation for 50min, then discarding the liquid in the sample hole, washing 3 times with a washing solution, and washing 3min each time.

3. Color development and termination reaction:

and (3) uniformly mixing the color development solution A and the color development solution B in an equal volume according to a ratio of 1:1, then quickly adding the mixed color development solution into reaction holes of a 48-hole enzyme label plate, wherein the sample addition amount is 100 mu l/hole, placing the plate at 37 ℃ in a dark place for reaction for 15min, then adding 50 mu l of stop solution into each reaction hole, stopping the color development reaction, reading the OD450 optical density value by an enzyme label instrument within 20 min, and zeroing by using a blank control hole.

4. And (4) judging a result:

and adding two standard deviations (Mean +2SD) to the average number of the OD values measured by the negative control wells to obtain a cut-off value (cut-off value), judging that the OD value reading in the reaction wells is greater than or equal to the cut-off value is positive, and judging that the OD value reading in the reaction wells is less than the cut-off value is negative.

5. Data processing:

and (3) taking the concentration of the calibrator as a vertical coordinate (a logarithmic coordinate) and the corresponding absorbance (OD) value as a horizontal coordinate, utilizing computer software, adopting four-parameter Logistic curve fitting, creating a standard curve equation, and calculating the concentration value of the sample by utilizing the equation according to the absorbance (OD value) of the sample. If the sample is diluted before detection, the sample is multiplied by a corresponding dilution multiple during final calculation, and the actual concentration of the sample is obtained.

Thirdly, the diagnostic value analysis of the kit of the invention

The kit provided by the embodiment 1 of the invention is used for detecting serum samples of fatty liver patients and normal persons so as to evaluate and analyze the value of the kit provided by the invention for early fatty liver screening.

1. Sample source

Serum samples were collected from the laboratory of the first subsidiary hospital of the university of science and technology of china, in which 24 parts of healthy human serum (control group) and 14 parts of serum from patients with fatty liver were collected. All serum from patients with fatty liver was collected at the time when the patients were initially diagnosed with fatty liver and had not received any treatment, from 1 month at 2021 to 12 months at 2021. The normal human serum comes from the outpatient clinic and is confirmed to be healthy people of patients with non-fatty liver through B-ultrasonic examination.

2. Serum preparation

5ml of fasting venous blood is extracted into a centrifuge tube, kept stand for 30 minutes at room temperature, centrifuged (2000 rpm), and the upper serum is sucked and subpackaged, wherein each tube is 100 mu l, and the tubes are stored in a refrigerator at minus 80 ℃.

3. Experimental methods

The content of Nogo-B in the serum of 14 patients with fatty liver (fatty liver group) and the serum of 24 normal human sera (control group) was examined using the kit prepared in example 1 of the present invention and the method of using the kit described in example 2 (see FIG. 3 for the results). And (3) using GraphyPad Prism software to draw expression level distribution maps of Nogo-B in the fatty liver group and the control group (the result is shown in figure 4), performing statistical test, comparing the expression of Nogo-B in the fatty liver group and the normal human group by using a two-independent sample chi-square test method, wherein the test level alpha is 0.05, and when P is less than 0.05, the result has statistical significance.

4. Analysis of results

FIG. 4 is a graph showing the profile of Nogo-B expression in the serum of a fatty liver group and a control healthy human group, from which it can be seen that the mean expression of Nogo-B in the serum of fatty liver patients is significantly higher than that in the normal human group.

Nogo-B antibodies and horseradish peroxidase-labeled anti-Nogo-B proteins were purchased from NOVUS, NBP 2-82531.

The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.

Claims (5)

1. An ELISA kit for detecting Nogo-B protein expression level is characterized in that: comprises Nogo-B antibody, coating liquid, confining liquid, enzyme labeled secondary antibody, positive control serum and negative control serum.

2. The ELISA kit for detecting Nogo-B protein expression level according to claim 1, characterized in that: the coating solution is carbonate buffer solution with pH value of 9.6 and concentration of 50 mM; the blocking solution is PBST buffer solution containing 2% (W/V) BSA; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled anti-Nogo-B protein; the positive control serum is serum of a fatty liver patient; the negative control serum was the serum of a normal person who did not suffer from fatty liver.

3. The ELISA kit for detecting Nogo-B protein expression level according to claim 1, characterized in that: the kit also comprises an enzyme label plate, a sample diluent, a second antibody diluent, a washing solution, a developing solution A and a developing solution B.

4. The ELISA kit for detecting Nogo-B protein expression level according to claim 3, wherein: both the sample diluent and the second antibody diluent were PBST buffer containing 1% (W/V) BSA; the washing solution is 0.01M PBST buffer solution containing 0.05% Tween 20 and having a pH value of 7.4.

5. The ELISA kit for detecting Nogo-B protein expression level according to claim 3, wherein: what is needed isThe color developing solution A is 0.02% (W/V) TMB, and the preparation method comprises the following steps: dissolving 0.005g of methylbenzidine in 25ml of deionized water; the color developing solution B is 0.006 percent (W/V) urea peroxide, and the preparation method comprises the following steps: taking 4.665g of citric acid and Na ₂ HPO ₄ 18.40g of the extract is dissolved in 400ml of deionized water, 3.2ml of 0.75 percent of urea hydrogen peroxide is added, the pH value is adjusted to 5.0-5.5, the deionized water is added to the solution until the volume reaches 500ml, and the mixture is uniformly mixed and stored at 4 ℃.

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