Kit for detecting hepatitis C virus core protein binding protein 6 and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to a kit for detecting hepatitis C virus core protein binding protein 6 and application. The invention provides an HCBP6 enzyme-linked immunosorbent assay kit, the kit is obtained through antibody screening and reagent optimization, the kit is used for detecting the content of HCBP6 in human serum, the detection sensitivity is high, the linear range is wide, the precision is good, the accuracy is high, the operation is simple, convenient and rapid, the use place is wide, the detection cost is relatively low, and the kit is suitable for large-scale popularization and application. And clinical diagnosis and treatment of fatty liver can be guided.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a kit for detecting hepatitis C virus core protein binding protein 6 and its application. Background Art

[0002] Fatty liver disease was named by Rokitansky based on the large amount of lipid deposits in the hepatocytes of the liver tissue of obese patients. It is divided into alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) according to whether the patient has a history of long-term heavy drinking. NAFLD refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes in patients without a history of heavy drinking (equivalent ethanol volume <30g / d for men and <20g / d for women) and other clear liver damage factors (hepatitis virus, autoimmunity, drugs, etc.).

[0003] In recent years, due to changes in people's diet and lifestyle, the global incidence of fatty liver has continued to increase, but the diagnosis of fatty liver is mainly through imaging examinations and blood routine, liver function, kidney function and other tests. Studies have shown that hepatitis C virus core protein binding protein 6 (HCBP6) plays an important role in inhibiting the occurrence of fatty liver disease or lipid metabolism. The level of HCBP6 in human serum can be used as one of the diagnostic criteria for fatty liver and can also guide subsequent treatment.

[0004] Enzyme-linked immunosorbent assay (ELISA), also known as enzyme-linked immunosorbent assay (ELISA), is a common biomolecular detection technology that can detect low antigen concentrations. It is an immunological technique that achieves very sensitive detection results through the special reaction of specific antibodies and enzymes. However, at present, there is a lack of relevant kits for HCBP6 detection based on ELISA and the detection effect is poor, so it is very important to develop an ELISA-based HCBP6 detection kit with better detection effect. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a kit for detecting hepatitis C virus core protein binding protein 6 and its application.

[0006] The present invention provides a kit for detecting HCBP6, which comprises an enzyme-labeled antibody 1, an antibody 2, a coating solution, a cleaning solution, an antigen diluent, an enzyme-labeled antibody diluent, a blocking solution and / or a substrate buffer solution;

[0007] The enzyme-labeled antibody 1 comprises an enzyme marker and an antibody 1;

[0008] The heavy chain variable region of antibody 1 has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2;

[0009] The heavy chain variable region of antibody 2 has the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:4.

[0010] The heavy chain constant region of the antibody 1 is of the mouse IgG1 subtype; the light chain constant region is of the mouse κ1 type;

[0011] The heavy chain constant region of antibody 2 is of mouse IgG1 subtype; the light chain constant region is of mouse κ1 type;

[0012] The enzyme label of the enzyme-labeled antibody 1 includes any one of horseradish peroxidase and alkaline phosphatase.

[0013] Furthermore,

[0014] The enzyme marker of the enzyme-labeled antibody 1 is horseradish peroxidase;

[0015] The preparation method of the enzyme-labeled antibody 1 comprises the following steps:

[0016] Step 1, the enzyme marker solution and the sodium periodate solution are subjected to a first reaction to obtain an activated enzyme marker;

[0017] Step 2, the activated enzyme marker and antibody 1 undergo a second reaction to obtain the enzyme-labeled antibody 1;

[0018] The concentration ratio of the enzyme marker in the enzyme marker to the sodium periodate in the sodium periodate solution is 1:1;

[0019] The concentration ratio of the activated enzyme marker to antibody 1 is 1:2.

[0020] The conditions of the first reaction are 4°C, protected from light, for 15 min to 25 min;

[0021] The conditions of the second reaction are 4° C., protected from light, for 3 to 5 hours.

[0022] In the method for preparing the enzyme-labeled antibody 1 of the present invention,

[0023] In the step 2, the activated enzyme marker and the antibody 1 further include a step of dialyzing the antibody 1 before the second reaction, and the dialysis buffer is CB buffer;

[0024] The first activation also includes a first termination;

[0025] The first termination reagent is ethylene glycol, and the volume fraction of the ethylene glycol in the reaction solution obtained by the first reaction between the enzyme marker solution and the sodium periodate solution is 15% to 20%;

[0026] The second activation further includes a second termination, the reagent for the second termination is sodium borohydride, the concentration of the sodium borohydride is, and the concentration ratio of the sodium borohydride to the enzyme-labeled antibody 1 is 1:50.

[0027] In the kit of the present invention,

[0028] The coating solution is selected from at least one of CB buffer, PB buffer or Tris-hcl;

[0029] The cleaning solution includes water, sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate and / or Twenn-20;

[0030] The antigen diluent includes water, sodium chloride, potassium chloride, disodium hydrogen phosphate and / or dipotassium hydrogen phosphate;

[0031] The enzyme-labeled antibody diluent includes newborn calf serum and a cleaning solution;

[0032] The blocking solution includes BSA and antigen diluent;

[0033] The substrate buffer comprises sodium acetate, citric acid and hydrogen peroxide.

[0034] Furthermore, in the kit of the present invention,

[0035] The coating solution is CB buffer;

[0036] The cleaning solution comprises water, 6g / L to 10g / L sodium chloride, 0.1g / L to 0.3g / L potassium chloride, 0.1g / L to 0.3g / L dipotassium hydrogen phosphate, 3g / L to 4g / L disodium hydrogen phosphate dodecahydrate and / or 0.4% to 0.6% Twenn-20 by volume;

[0037] The antigen diluent comprises water, 6g / L to 10g / L sodium chloride, 0.1g / L to 0.3g / L potassium chloride, 0.1g / L to 0.3g / L dipotassium hydrogen phosphate and 3g / L to 4g / L disodium hydrogen phosphate dodecahydrate;

[0038] The enzyme-labeled antibody diluent comprises 5% to 10% newborn calf serum and a cleaning solution;

[0039] The blocking solution includes 0.5% to 1.5% BSA by mass and an antigen diluent;

[0040] The substrate buffer comprises 12g / L to 14g / L sodium acetate, 1g / L to 2g / L citric acid and hydrogen peroxide.

[0041] Furthermore, in the kit of the present invention,

[0042] The cleaning solution includes water, 8g / L sodium chloride, 0.2g / L potassium chloride, 0.24g / L dipotassium hydrogen phosphate, 3.6g / L disodium hydrogen phosphate dodecahydrate and / or 0.5% Twenn-20 by volume;

[0043] The antigen diluent comprises water, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.24 g / L dipotassium hydrogen phosphate and 3.6 g / L disodium hydrogen phosphate dodecahydrate;

[0044] The enzyme-labeled antibody diluent includes 10% newborn calf serum and a cleaning solution;

[0045] The blocking solution includes 1% BSA by mass and an antigen diluent;

[0046] The substrate buffer comprises 13.6 g / L sodium acetate, 1.6 g / L citric acid and hydrogen peroxide.

[0047] The kit of the present invention further comprises TMB color developing solution, positive control substance and / or negative control substance.

[0048] The present invention screens the antibodies in the kit, labels 38 different HCBP6 antibodies with enzyme markers, and then coats the 38 antibodies on an ELISA plate respectively. By pairing the antibodies in pairs, an antibody combination is obtained by screening, in which antibody No. 38 (antibody 1) is enzyme-labeled and antibody No. 9 (antibody 2) is used to coat the ELISA plate. The antibody combination is used for the detection of HCBP6, and has the highest sensitivity and the best accuracy compared with other antibody combinations.

[0049] The present invention optimizes the reagent components and preparation method of the kit; experimental results show that when the coating solution selects CB buffer solution, the color development is better; when the concentration ratio of HRP solution to sodium periodate solution is 1:1, the color development reaction is better, and raw materials can be saved and costs can be reduced.

[0050] The present invention provides the use of the kit in detecting HCBP6 and / or preparing a fatty liver diagnosis product.

[0051] The present invention provides a method for detecting HCBP6 for non-diagnostic purposes, which comprises detecting a sample using the kit of the present invention.

[0052] The sample includes serum and / or plasma.

[0053] Furthermore, the detection method comprises the following steps:

[0054] Step 1, adding antibody 2 to the ELISA plate, coating and blocking the plate to obtain an ELISA plate coated with antibody 2;

[0055] Step 2: adding the sample and the enzyme-labeled antibody 1 to the enzyme-labeled plate coated with the antibody 2, and performing a first incubation and a second incubation in sequence, and then performing a color reaction in the presence of TMB color developing solution, and measuring the absorbance value at 450 nm.

[0056] In a specific embodiment of the present invention, the coating and sealing comprises the following steps:

[0057] Antibody 2 (antibody #9) was diluted with CB buffer to a final concentration of 2 μg / mL, and 0.1 mL / well was added to a 96-well ELISA plate. After reacting at 4°C overnight, the plate was washed twice with 1× PBST, and 0.2 mL / well of blocking solution was added, and the plate was blocked at 37°C for 1 hour.

[0058] The step 2 is specifically as follows: after adding 0.1 mL / well of the diluted reference substance or serum sample to the reaction plate and incubating at 37°C for 1 hour, the plate is washed 6 times with 1×PBST, patted dry, 0.1 mL / well of the diluted horseradish peroxide-labeled antibody is added, incubated at 37°C for 1 hour, the plate is washed 6 times with 1×PBST, a solution of a substrate buffer and TMB color development solution mixed in a ratio of 1:1 is added, 0.1 mL / well is used for color development at 37°C for 10 minutes, and then the stop solution is added for termination. The absorbance value at 450 nm is measured.

[0059] In the present invention, the detection method includes detection methods for diagnostic purposes and non-diagnostic purposes. Furthermore, the detection method described in the present invention is a detection method for non-diagnostic purposes, which is not intended to directly obtain health conditions. The detection method for non-diagnostic purposes includes detection of environmental samples or detection for scientific research purposes, wherein the environmental samples include but are not limited to: food, drinking water, domestic water, domestic wastewater or swabs from the surface of objects. The detection method for diagnostic purposes includes detection of human or animal bodies or in vitro samples thereof.

[0060] The present invention provides an HCBP6 enzyme-linked immunosorbent assay kit, which is obtained through antibody screening and reagent optimization. The kit of the present invention is used for detecting the HCBP6 content in human serum, has high detection sensitivity, wide linear range, good precision, high accuracy, simple and fast operation, wide application sites, relatively low detection cost, and is conducive to guiding the diagnosis and treatment of clinical fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is a linear fitting curve graph;

[0062] Figure 2 is the level of HCBP6 in different clinical samples;

[0063] Figure 3 It is the antibody screening effect;

[0064] Figure 4 These are the effects of three types of coating solutions;

[0065] Figure 5 This is a diagram of the optimization of labeling conditions for several different enzyme-labeled antibodies. DETAILED DESCRIPTION

[0066] The present invention provides a kit and application for detecting hepatitis C virus core protein binding protein 6. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0067] The basic principle of the present invention is to use two different antibodies, one fixed on the detection plate and the other labeled with an enzyme, to detect the target molecule in the sample. The sample to be tested is added to the detection plate hole covered with the fixed antibody. After the target molecule binds to the fixed antibody, the sample is washed and the enzyme-labeled antibody is added. The enzyme and the target molecule bind again to form a "sandwich" complex. Under the action of the substrate, the enzyme can convert the substrate into a color product. Only the enzyme-labeled antibody that binds to the target molecule can be clamped, so the number of target molecules in the sample can be determined, and the qualitative or quantitative detection of the target molecule can be achieved.

[0068] The amino acid sequence of the heavy chain variable region of antibody 1 (antibody #38) is: MDWLWNLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTN YGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAY LQINNLKNEDMATYFCVRLDYWGQGTTLTVSS (SEQ ID NO: 1);

[0069] The amino acid sequence of the light chain variable region of antibody 1 (antibody #38) is: MDFQVQIFSFLLISASVIISRGQIVLTQSPAIMSASPGEKVTMTCSASSSVSY MHWYQQKSGTSPKRWIYDTSKLAYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGSNPWTFGGGTKLEIK (SEQ ID NO: 2);

[0070] The amino acid sequence of the heavy chain variable region of antibody 2 (antibody #9) is: MNFGLSLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFSSYG MSWVRQTPDKRLEWVASISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQM SSLKSEDTAMYYCASHYDYDYWGQGTSVTVSS (SEQ ID NO: 3);

[0071] The amino acid sequence of the light chain variable region of antibody 2 (antibody #9) is: MRTPAQFLGILLLWFPGIRCDINMTQSPSSMYASLGERLTITCKASQDINTYL SWFQQKPGKSPKSLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPWTFGGGTKLEIK (SEQ ID NO: 4);

[0072] The reagents and consumables used in the present invention are all common commercial products and can be purchased in the market.

[0073] The present invention will be further described below in conjunction with embodiments:

[0074] Example 1 Kit for determining hepatitis C virus core protein binding protein 6 in human serum

[0075] 1. Preparation method

[0076] 1. Preparation of reagents

[0077] Coating solution (CB buffer): weigh 1.69±0.01 g of sodium carbonate and 2.86±0.01 g of sodium bicarbonate and make up to 1 L. The pH value was tested to be 9.51±0.01.

[0078] Cleaning solution: weigh 8.00g±0.01g sodium chloride, 0.20±0.01g potassium chloride, 0.24±0.01g potassium dihydrogen phosphate, 3.60±0.01g sodium dihydrogen phosphate dodecahydrate, add 5mL Twenn-20 to 1L, and adjust the pH to 7.40±0.01 with hydrochloric acid or sodium hydroxide.

[0079] Antigen diluent: weigh 8.00 g ± 0.01 g sodium chloride, 0.20 ± 0.01 g potassium chloride, 0.24 ± 0.01 g potassium dihydrogen phosphate, and 3.60 ± 0.01 g sodium dihydrogen phosphate dodecahydrate, and make up to 1 L. Adjust the pH to 7.40 ± 0.01 with hydrochloric acid or sodium hydroxide.

[0080] Enzyme-labeled antibody diluent: a cleaning solution containing 10% newborn calf serum by volume.

[0081] Blocking solution: Antigen dilution solution containing 1% BSA.

[0082] Substrate buffer: weigh 13.60 g ± 0.01 g sodium acetate, 1.60 ± 0.01 g citric acid, and add 0.3 mL of 30% hydrogen peroxide to make up to 500 mL.

[0083] TMB colorimetric solution: weigh 0.20 g ± 0.01 g of disodium EDTA, 0.95 ± 0.01 g of citric acid, and 60 mL of glycerol, then add a certain amount of ultrapure water to dissolve them. Then weigh 0.20 g ± 0.01 g of tetramethylbenzidine and dissolve it in 1 mL of DMSO. Mix the two solutions under ultrasonic conditions to a volume of 500 mL.

[0084] Stop solution: Add 1 mL of concentrated solution dropwise into 17 mL of ultrapure water.

[0085] 2. Preparation of enzyme-labeled antibodies

[0086] Antibody 1 (antibody #38) was diluted to 2.0 mg / mL and dialyzed with CB buffer overnight. HRP solution with a concentration of 20 mg / mL and sodium periodate solution with a concentration of 20 mg / mL were prepared, and the two solutions were mixed in a ratio of 1:1. After reacting at 4°C in the dark for 20 minutes, 15% to 20% ethylene glycol was added to terminate the reaction for 30 minutes. Activated HRP (horseradish peroxidase) was added to the dialyzed monoclonal antibody in a certain ratio (mass ratio HRP / antibody = 1:2), and after reacting at 4°C in the dark for 4 hours, sodium borohydride (sodium borohydride concentration is 5 mg / ml, 1 ml of reaction system, 5 mg / mL is added 8 μl) to terminate the reaction for 1 hour, and dialyzed at 4°C overnight.

[0087] 3. Detection steps

[0088] ELISA plate coating: dilute antibody 2 (antibody #9) with the above CB buffer to a final concentration of 2 μg / mL, add 0.1 mL / well to a 96-well ELISA plate, and react overnight at 4°C. The next day, wash the ELISA plate twice with 1×PBST, pat dry, add 0.2 mL / well of blocking solution, block at 37°C for 1 hour, and pat dry the reaction plate for detection.

[0089] ELISA steps: Add 0.1 mL / well of diluted reference or serum sample to the reaction plate and incubate at 37°C for 1 hour, wash the plate 6 times with 1×PBST, pat dry, add 0.1 mL / well of diluted horseradish peroxide-labeled antibody, incubate at 37°C for 1 hour, wash the plate 6 times with 1×PBST, add a solution of substrate buffer and TMB color development solution in a ratio of 1:1, color development at 37°C for 10 minutes at 0.1 mL / well, and then add stop solution (1M sulfuric acid) to terminate. Measure the absorbance at 450 nm.

[0090] 2. Experimental Data Results and Analysis

[0091] 1. Detection limit

[0092] The blank sample was tested 20 times according to the ELISA procedure at A450nm of the ELISA instrument as shown in Table 1:

[0093] Table 1. Blank sample absorbance results

[0094] Repetitions 1 2 3 4 5 6 7 8 9 10 Absorbance / A 0.185 0.149 0.166 0.175 0.151 0.161 0.174 0.183 0.178 0.179 Repetitions 11 12 13 14 15 16 17 18 19 20 Absorbance / A 0.157 0.166 0.179 0.165 0.190 0.157 0.164 0.183 0.181 0.139

[0095] The detection limit XL and minimum detection concentration CL were calculated according to the following formula.

[0096]

[0097] CL=KSb1 / S

[0098] Where, K: confidence factor, set to 3;

[0099] Blank Mean

[0100] Sb1: Blank value standard deviation

[0101] S: Sensitivity at low concentration, that is, the slope of the standard working curve in the low concentration range is 0.158.

[0102] Table 2. Detection limit and minimum detection concentration test results

[0103] average value 0.1691 Standard Deviation 0.012 Detection limit 0.2051 Minimum detection concentration 0.2278

[0104] As shown in Table 2, the detection limit XL was 0.2051 and the minimum detection concentration was 0.2278.

[0105] 2. Linear range

[0106] Dilute the antigen with the above antigen diluent to the final concentrations of 0.05ng / mL, 0.1ng / mL, 0.2ng / mL, 0.4ng / mL, 0.8ng / mL, 1ng / mL, 2ng / mL, 4ng / mL, 8ng / mL, 16ng / mL, 32ng / mL, 64ng / mL, 128ng / mL, 512ng / mL, and 1024ng / mL for testing.

[0107] Table 3. Linearity test results

[0108]

[0109] With concentration as the horizontal axis and absorbance as the vertical axis, ELISACalc was used to fit the four-parameter logistic curve. Figure 1 shown.

[0110] The linear equation is: y = (AD) / [1 + (x / C) ^ B] + D,

[0111] Where A = 1.22093, B = -1.19440, C = 95.80041, D = 0.21917, r^2 = 0.99553;

[0112] Within the reference interval, the linear correlation coefficient r≥0.990.

[0113] According to the calculation in this article, the absorbance of the lowest detection concentration is 0.2278, so the detection linear range is between 0.4ng / mL and 1μg / mL.

[0114] 3. Repeatability

[0115] For the same batch of reagents, three urine samples with different concentrations of low, medium and high were selected and tested 10 times. The test results are shown in Table 4:

[0116] Table 4. Repeatability test results

[0117]

[0118] The reference samples of low, medium and high concentrations were measured 10 times, and the coefficient of variation (CV) of the measured values ​​was ≤30%. Therefore, the accuracy and tightness experiments were successfully verified and met the expected acceptance criteria.

[0119] 4. Clinical sample testing

[0120] A total of 22 serum specimens with clinical diagnosis of fatty liver were collected, including 3 cases with fatty liver, 6 cases without fatty liver, and the remaining 13 cases were diagnosed with liver diseases of varying degrees, such as hepatitis B, hepatitis C, liver cancer and cirrhosis. The levels of HCBP6 in these samples were detected, such as Figure 2 shown.

[0121] For the detection of clinical samples, it can be concluded that the color of HCBP6 in serum samples with fatty liver and without fatty liver is obviously different. Fatty liver samples do not show color, indicating low HCBP6 content; normal samples show obvious color, indicating high HCBP6 content. The level of HCBP6 in different degrees of liver disease and the reference range of HCBP6 need further verification with a large number of samples.

[0122] The present invention can qualitatively or quantitatively detect the level of HCBP6 in human serum, and is expected to provide a new basis for the diagnosis of fatty liver. At the same time, the antigen and antibody used in the present invention can be used to develop other platforms such as chemiluminescence detection kits.

[0123] Example 2 Optimization comparison of antibodies and reagents

[0124] 1. Screening of 38 antibody pairs

[0125] The 38 developed antibodies were coated with CB buffer at 2 μg / mL, and 38 enzyme-labeled antibodies were prepared and cross-matched. Figure 3 , Figure 3 Only some of the pairs with good test results (9# wrapped with 38# label) were placed, among which the best pair was 9# wrapped with 38# label, followed by 11# wrapped with 1# label. Subsequent experiments used 9# and 38# pairings for experimental research.

[0126] 2. Optimization of coating solution

[0127] CB buffer, 20mM PB (pH=8.0) buffer and 20mM Tris-Hcl (pH=8.5) buffer were respectively prepared to coat the preferred antibody 9# antibody in this experiment at a concentration of 2μg / mL, and the enzyme-labeled antibody was 38# with a working concentration of 2K. The detection data are shown in Table 5:

[0128] Table 5. Absorbance values ​​of different coating solutions at 450nm

[0129]

[0130] With concentration as the horizontal axis and the average absorbance minus the blank value as the vertical axis, the effect curve is as follows: Figure 4 The above three coating solutions were used to detect different concentrations of HCBP6 recombinant protein. Figure 4 It can be clearly seen that the color development using CB as the coating solution is significantly higher than that of PB and Tris-Hcl.

[0131] 3. Optimization of enzyme-labeled antibody labeling method

[0132] Optimization condition 1: Prepare 5 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a 1:1 ratio, and react at 4°C in the dark for 20 min;

[0133] Optimization condition 2: Prepare HRP solution and sodium periodate solution with a concentration of 10 mg / mL, mix the two solutions in a ratio of 1:1, and react at 4°C for 20 min;

[0134] Optimized condition 3: Prepare 20 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a 1:1 ratio, and react at 4°C in the dark for 20 min.

[0135] Optimized condition 4: Prepare 30 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a 1:1 ratio, and react at 4°C in the dark for 20 min.

[0136] Optimized condition 5: Prepare 20 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a ratio of 1:2, and react at 4°C in the dark for 20 min.

[0137] Optimized condition 6: Prepare 20 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a ratio of 1:3, and react at 4°C in the dark for 20 min;

[0138] Optimized condition 7: Prepare 20 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a 1:1 ratio, and react at 4°C in the dark for 10 min.

[0139] Optimization condition 8: Prepare 20 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a 1:1 ratio, and react at 4°C in the dark for 30 min;

[0140] Optimized condition 9: Prepare 20 mg / mL HRP solution and sodium periodate solution, mix the two solutions in a 1:1 ratio, and react at 4°C in the dark for 40 min.

[0141] The enzyme-labeled antibody was prepared according to the above conditions, and 4 replicates of blank wells and 4 replicates of recombinant HCBP6 protein at a concentration of 40 ng / mL were detected. The absorbance values ​​obtained are shown in Table 6:

[0142] With the labeling optimization conditions as the horizontal axis and the average absorbance as the vertical axis, the effect curve is obtained as follows Figure 5 . Combined with the color development and blank value background of 40ng / mL, Figure 5The bar graph intuitively reflects that conditions 3, 5, and 8 are better, but considering saving raw materials and time costs, condition 3 is comprehensively selected as the optimal solution for enzyme-labeled antibody labeling in this study.

[0143] Table 6. Comparison of absorbance at 450nm for optimized enzyme-labeled antibody labeling conditions

[0144]

[0145]

[0146] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Kit for detecting HCBP6, It is characterized in that It includes enzyme-labeled antibody 1, antibody 2, coating solution, cleaning solution, antigen diluent, enzyme-labeled antibody diluent, blocking solution and / or substrate buffer; The enzyme-labeled antibody 1 comprises an enzyme marker and an antibody 1; The heavy chain variable region of antibody 1 has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2; The heavy chain variable region of antibody 2 has the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

4.

2. The kit according to claim 1, It is characterized in that The heavy chain constant region of the antibody 1 is of the mouse IgG1 subtype; the light chain constant region is of the mouse κ1 type; The heavy chain constant region of antibody 2 is of mouse IgG1 subtype; the light chain constant region is of mouse κ1 type; The enzyme label of the enzyme-labeled antibody 1 includes any one of horseradish peroxidase and alkaline phosphatase.

3. The kit according to claim 1, It is characterized in that The enzyme marker of the enzyme-labeled antibody 1 is horseradish peroxidase; The preparation method of the enzyme-labeled antibody 1 comprises the following steps: Step 1, the enzyme marker solution and the sodium periodate solution are subjected to a first reaction to obtain an activated enzyme marker; Step 2, the activated enzyme marker and antibody 1 undergo a second reaction to obtain the enzyme-labeled antibody 1; The concentration ratio of the enzyme marker in the enzyme marker to the sodium periodate in the sodium periodate solution is 1:1; The concentration ratio of the activated enzyme marker to antibody 1 is 1:2; The conditions of the first reaction are 4°C, protected from light, for 15 min to 25 min; The conditions of the second reaction are 4° C., protected from light, for 3 to 5 hours.

4. The kit according to any one of claims 1 to 3, It is characterized in that The coating solution is selected from at least one of CB buffer, PB buffer or Tris-hcl; The cleaning solution includes water, sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate and / or Twenn-20; The antigen diluent includes water, sodium chloride, potassium chloride, disodium hydrogen phosphate and / or dipotassium hydrogen phosphate; The enzyme-labeled antibody diluent includes newborn calf serum and a cleaning solution; The blocking solution includes BSA and antigen diluent; The substrate buffer comprises sodium acetate, citric acid and hydrogen peroxide.

5. The kit according to claim 4, It is characterized in that The coating solution is CB buffer; The cleaning solution comprises water, 6g / L to 10g / L sodium chloride, 0.1g / L to 0.3g / L potassium chloride, 0.1g / L to 0.3g / L dipotassium hydrogen phosphate, 3g / L to 4g / L disodium hydrogen phosphate dodecahydrate and / or 0.4% to 0.6% Twenn-20 by volume; The antigen diluent comprises water, 6g / L to 10g / L sodium chloride, 0.1g / L to 0.3g / L potassium chloride, 0.1g / L to 0.3g / L dipotassium hydrogen phosphate and 3g / L to 4g / L disodium hydrogen phosphate dodecahydrate; The enzyme-labeled antibody diluent comprises 5% to 10% newborn calf serum and a cleaning solution; The blocking solution includes 0.5% to 1.5% BSA by mass and an antigen diluent; The substrate buffer comprises 12g / L to 14g / L sodium acetate, 1g / L to 2g / L citric acid and hydrogen peroxide.

6. The kit according to claim 5, It is characterized in that The cleaning solution comprises water, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.24 g / L dipotassium hydrogen phosphate, 3.6 g / L disodium hydrogen phosphate dodecahydrate and / or 0.5% Twenn-20 by volume; The antigen diluent comprises water, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.24 g / L dipotassium hydrogen phosphate and 3.6 g / L disodium hydrogen phosphate dodecahydrate; The enzyme-labeled antibody diluent includes 10% newborn calf serum and a cleaning solution; The blocking solution includes 1% BSA by mass and an antigen diluent; The substrate buffer comprises 13.6 g / L sodium acetate, 1.6 g / L citric acid and hydrogen peroxide.

7. The kit according to any one of claims 1 to 6, It is characterized in that The kit also includes TMB color developing solution, positive control substance and / or negative control substance.

8. Use of the kit according to any one of claims 1 to 7 in the detection of HCBP6 and / or the preparation of a fatty liver diagnosis product.

9. Methods for detecting HCBP6 for non-diagnostic purposes, It is characterized in that The invention relates to detecting a sample using the kit described in any one of claims 1 to 7.

10. The detection method according to claim 9, It is characterized in that The sample includes serum and / or plasma.

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