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

Abstract

The present application 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 thereof. The present application provides an HCBP6 enzyme-linked immunoassay kit, which is obtained by antibody screening and reagent optimization. The kit is used for detecting the content of HCBP6 in human serum, and has the advantages of high detection sensitivity, wide linear range, good precision, high accuracy, simple and rapid operation, wide application site, relatively low detection cost, and the like, and is conducive to guiding the diagnosis and treatment of fatty liver in clinic.

Description

TECHNICAL FIELD

[0001] The present application 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 thereof. BACKGROUND

[0002] Fatty liver disease is named by Rokitansky according to a large amount of lipid deposition in the liver parenchymal cells of the liver tissue of obese patients. According to whether the patient has a long-term and large amount of drinking history, it is divided into alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). NAFLD refers to a clinical and pathological syndrome characterized by excessive deposition of fat in hepatocytes without a large amount of drinking history (equivalent to alcohol amount < 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 the change of people's dietary structure and lifestyle, the global incidence of fatty liver continues to rise, but the diagnosis of fatty liver is mainly through imaging examination and blood routine, liver function, kidney function and other examinations. 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 glycolipid metabolism. The level of HCBP6 in human serum can be used as one of the diagnostic criteria for fatty liver and can guide the treatment in the later stage.

[0004] Enzyme-linked immunoassay, also known as enzyme-linked immunosorbent assay (ELISA), is a common biomolecule detection technology that can detect low antigen concentrations. It is an immunological technique that uses specific antibodies and enzymes to achieve very sensitive detection. However, at present, there is a lack of related kits for HCBP6 detection based on enzyme-linked immunoassay, and the detection effect is not good, so it is crucial to develop an HCBP6 detection kit based on enzyme-linked immunoassay with good detection effect. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a kit for detecting hepatitis C virus core protein binding protein 6 and application thereof.

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

[0007] The enzyme-labeled antibody 1 comprises an enzyme label and an antibody 1.

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

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

[0010] The heavy chain constant region of the antibody 1 is a mouse IgG1 subtype, and the constant region of the light chain is a mouse kappa 1 type;

[0011] The heavy chain constant region of the antibody 2 is a mouse IgG1 subtype, and the constant region of the light chain is a mouse kappa 1 type;

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

[0013] Further,

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

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

[0016] Step 1, the activated enzyme label is obtained by the first reaction of the enzyme label solution and the sodium periodate solution;

[0017] Step 2, the enzyme-labeled antibody 1 is obtained by the second reaction of the activated enzyme label and the antibody 1;

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

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

[0020] The condition of the first reaction is 4℃, light shielding, 15min-25min;

[0021] The condition of the second reaction is 4℃, light shielding, 3-5h.

[0022] In the preparation method of the enzyme-labeled antibody 1,

[0023] In step 2, the activated enzyme label and the antibody 1 are further subjected to the second reaction, and the antibody 1 is subjected to a dialysis step before the second reaction, and the buffer for the dialysis is CB buffer;

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

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

[0026] The second activation further comprises a second termination, and the second terminated reagent is sodium borohydride, the concentration of the sodium borohydride is 0.1% to 0.3%, and the concentration ratio of the sodium borohydride to the enzyme-labeled antibody 1 is 1:50.

[0027] The kit provided by the application comprises a coating solution, a washing solution, an antigen diluent, an enzyme-labeled antibody diluent, a blocking solution, a substrate buffer solution, and a substrate solution.

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

[0029] The washing solution comprises water, sodium chloride, disodium hydrogen phosphate, potassium hydrogen phosphate, and / or Twenn-20.

[0030] The antigen diluent comprises water, sodium chloride, potassium chloride, disodium hydrogen phosphate, and / or potassium hydrogen phosphate.

[0031] The enzyme-labeled antibody diluent comprises newborn calf serum and the washing solution.

[0032] The blocking solution comprises BSA and the antigen diluent.

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

[0034] Further, the kit provided by the application comprises a coating solution, a washing solution, an antigen diluent, an enzyme-labeled antibody diluent, a blocking solution, a substrate buffer solution, and a substrate solution.

[0035] The coating solution is a CB buffer solution.

[0036] The washing 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 potassium hydrogen phosphate, 3g / L to 4g / L dodecahydrate disodium hydrogen phosphate, and / or 0.4% to 0.6% Twenn-20.

[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 potassium hydrogen phosphate, and 3g / L to 4g / L dodecahydrate disodium hydrogen phosphate.

[0038] The enzyme-labeled antibody diluent comprises 5% to 10% newborn calf serum and the washing solution.

[0039] The blocking solution comprises 0.5% to 1.5% BSA and the antigen diluent.

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

[0041] Further, the kit comprises,

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

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

[0044] The enzyme-labeled antibody diluent comprises 10% new born calf serum by volume fraction and the cleaning solution;

[0045] The blocking solution comprises 1% BSA by mass fraction and the antigen diluent;

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

[0047] The kit further comprises TMB developing solution, positive control and / or negative control.

[0048] The antibodies in the kit are screened, 38 different HCBP6 antibodies are labeled by enzyme markers, and then the 38 antibodies are coated on enzyme-labeled plates respectively, and the antibody combination of the 38th antibody (antibody 1) labeled by enzyme and the 9th antibody (antibody 2) used for coating on the enzyme-labeled plate is screened by two-by-two combination pairing, and the antibody combination is used for detection of HCBP6, and compared with other antibody combinations, the sensitivity is the highest and the accuracy is the best.

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

[0050] The kit is used for HCBP6 detection and / or preparation of a fatty liver diagnostic product.

[0051] The kit is used for HCBP6 detection and / or preparation of a fatty liver diagnostic product.

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

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

[0054] Step 1, after adding antibody 2 to an enzyme-labeled plate and coating and blocking, an enzyme-labeled plate coated with antibody 2 is obtained;

[0055] Step 2, after adding samples and enzyme-labeled antibody 1 to the enzyme-labeled plate coated with antibody 2 and sequentially performing first incubation and second incubation, color development is performed in the presence of TMB color development solution, and the absorbance value at 450 nm is measured.

[0056] In specific embodiments of the present application, the coating and blocking comprise the following steps:

[0057] CB buffer is used to dilute antibody 2 (9# antibody) to a final concentration of 2 μg / mL, 0.1 mL / well is added to a 96-well enzyme-labeled plate, and after 4℃ reaction overnight, 1×PBST is used for washing twice, 0.2 mL / well of blocking solution is added, and 37℃ blocking is performed for 1 hour;

[0058] Specifically, the step 2 is that after the diluted reference or serum sample is added to 0.1 mL / well in the reaction plate and incubated at 37℃ for 1 hour, the enzyme-labeled plate is washed with 1×PBST for 6 times, and after being patted dry, the diluted horseradish peroxide-labeled antibody 0.1 mL / well is added, incubated at 37℃ for 1 hour, and then the enzyme-labeled plate is washed with 1×PBST for 6 times, and the solution obtained by mixing the substrate buffer and TMB color development solution at a ratio of 1:1 is added, 0.1 mL / well is colored at 37℃ for 10 min, and then the stop solution is added for termination. The absorbance value at 450 nm is measured.

[0059] In the present application, the detection method includes detection methods for diagnostic purposes and non-diagnostic purposes, and further, the detection method of the present application is a non-diagnostic purpose detection method, which is not directly aimed at obtaining health status. The non-diagnostic purpose detection method includes detection of environmental samples or detection for research purposes, wherein the environmental samples include but are not limited to food, drinking water, domestic water, domestic wastewater or object surface swabs. The diagnostic purpose detection method includes detection of human or animal bodies or ex vivo samples thereof.

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

[0061] Figure 1 is a linear fitting curve diagram;

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

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

[0064] Figure 4 is the effect diagram of three coating liquids;

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

[0066] The present application provides a kit for detecting hepatitis C virus core protein binding protein 6 and its application. Those skilled in the art can refer to the content of this paper and appropriately improve the process parameters to achieve it. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application of this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0067] The basic principle of the present application is: using two different antibodies, one is fixed on the detection plate, and the other is labeled with an enzyme, which is used to detect the target molecule in the sample. The sample to be tested is added to the detection plate hole covered by the fixed antibody, after the target molecule is combined with the fixed antibody, the sample is washed, and the enzyme-labeled antibody is added, and the enzyme is combined with the target molecule again to form a "sandwich" complex. Through the action of the substrate, the enzyme can convert the substrate into a color product. Only the enzyme-labeled antibody combined with the target molecule can be clamped, so the number of target molecules in the sample can be determined, and qualitative or quantitative detection of the target molecule can be realized.

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

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

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

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

[0072] The reagent consumables used in the present application are all ordinary commercially available products and can be purchased in the market.

[0073] The present application is further described below in conjunction with examples:

[0074] Example 1: Determination of hepatitis C virus core protein binding protein 6 in human serum

[0075] I. Preparation method

[0076] 1. Preparation of reagents

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

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

[0080] Enzyme-labeled antibody diluent: contains 10% volume fraction of newborn calf serum in the washing solution.

[0081] Blocking solution: contains 1% BSA in the antigen diluent.

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

[0083] TMB developing 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. Weigh 0.20 g ± 0.01 g of tetramethyl benzidine in 1 mL of DMSO, and mix the two solutions under ultrasonic conditions to dilute to 500 mL.

[0084] Termination solution: add 1 mL of concentrated dropwise to 17 mL of ultrapure water.

[0085] 2. Preparation of enzyme-labeled antibody

[0086] Dilute antibody 1 (38# antibody) to 2.0 mg / mL, and dialyze overnight with CB buffer. Prepare a 20 mg / mL HRP solution and a 20 mg / mL sodium periodate solution, mix the two solutions in a 1:1 ratio, and react in the dark at 4°C for 20 min, then add 15%-20% volume fraction of ethylene glycol to terminate the reaction for 30 min. Add the activated HRP (horseradish peroxidase) to the dialyzed monoclonal antibody in a certain proportion (mass ratio HRP / antibody = 1:2), react in the dark at 4°C for 4 hours, then add sodium borohydride (concentration of sodium borohydride is 5 mg / ml, 8 μl of 5 mg / mL is added to 1 mL of reaction system) to terminate the reaction for 1 hour, and dialyze overnight at 4°C.

[0087] 3. Detection steps

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

[0089] Enzyme immunoassay step: After the diluted reference or serum sample was added to 0.1 mL / well in the reaction plate and incubated at 37°C for 1 hour, the enzyme immunoassay plate was washed with 1 x PBST for 6 times, and then dried, the diluted horseradish peroxidase-labeled antibody was added to 0.1 mL / well, and incubated at 37°C for 1 hour. After washing the enzyme immunoassay plate with 1 x PBST for 6 times, the substrate buffer and TMB developing solution were mixed at a ratio of 1:1, and then 0.1 mL / well was added and developed at 37°C for 10 min. Finally, the stop solution (1M sulfuric acid) was added to terminate the reaction. The absorbance value at 450 nm was measured.

[0090] II. Experimental data results and analysis

[0091] 1. Detection limit

[0092] The blank sample detection was completed 20 times according to the enzyme immunoassay operation steps at A450 nm in the enzyme label instrument, and the repeated test results are shown in Table 1:

[0093] Table 1. Blank sample absorbance results

[0094] Number of 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 Number of 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 the minimum detection concentration CL were calculated according to the following formula.

[0096]

[0097] CL = KSb1 / S

[0098] Wherein, K: confidence factor, take 3;

[0099] Blank average value

[0100] Sb1: standard deviation of blank value

[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] Mean 0.1691 Standard Deviation 0.012 Limit of Detection 0.2051 Lowest Concentration Detected 0.2278

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

[0105] 2. Linear range

[0106] The antigen was diluted to a final concentration of 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, 32 ng / mL, 64 ng / mL, 128 ng / mL, 512 ng / mL, and 1024 ng / mL, respectively, using the above antigen diluent.

[0107] Table 3. Linear test results

[0108]

[0109] The concentration was used as the abscissa, and the absorbance value was used as the ordinate. Four-parameter Logistic curve fitting was performed using ELISA Calc, and the curve is shown in Figure 1

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

[0111] where A = 1.22093, B = -1.19440, C = 95.80041, D = 0.21917, and r2= 0.99553.

[0112] In the reference interval, the linear correlation coefficient r is greater than or equal to 0.990.

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

[0114] 3. Reproducibility

[0115] The same batch of reagents was selected for low, medium, and high concentration urine samples, and repeated testing was performed 10 times. The test results are shown in Table 4:

[0116] Table 4. Reproducibility test results

[0117]

[0118] For low, medium, and high concentration reference products, the coefficient of variation (CV) of the measured values was less than or equal to 30% for 10 repeated determinations, so the accuracy and tightness experiment was successfully verified, and met the expected acceptance criteria.

[0119] 4. Clinical sample detection

[0120] A total of 22 serum samples were collected for the clinical diagnosis of fatty liver, including 3 cases of fatty liver, 6 cases of non-fatty liver, and 13 cases of different degrees of liver disease, such as hepatitis B, hepatitis C, liver cancer, and cirrhosis. The levels of HCBP6 in these samples were detected, as shown in​Figure 2 The results are shown in the table 1.

[0121] For the detection of clinical samples, it can be concluded that HCBP6 has obvious color difference in serum samples with and without fatty liver, the fatty liver sample does not develop color, indicating that the content of HCBP6 is low; the normal sample develops color obviously, indicating that the content of HCBP6 is high. The level of HCBP6 in different degrees of liver disease and the reference interval of HCBP6 need to be further verified with a large number of samples.

[0122] The application 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 application can be used for the development of other platforms such as chemiluminescence detection kit.

[0123] Example 2 Optimization comparison of antibody and reagent

[0124] 1. Screening of 38 antibody pairs

[0125] The 38 antibodies obtained by research and development were coated with CB buffer at 2 μg / mL, and 38 enzyme-labeled antibodies were prepared, and enzyme immunoassay cross pairing was carried out as follows Figure 3 , Figure 3 Only some of the placed parts have better detection results (9# coating 38# labeling), and the best pairing is 9# coating 38# labeling, followed by 11# coating 1# labeling, and subsequent experiments use 9# and 38# pairing for experimental research.

[0126] 2. Optimization of coating solution

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

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

[0129]

[0130] With the concentration as the abscissa and the average value of the absorbance minus the blank value as the ordinate, the effect curve is as follows Figure 4 The above three kinds of coating solutions are used to detect different concentrations of HCBP6 recombinant protein, and it can be obviously seen from Figure 4 that the color development using CB as the coating solution is obviously higher than that of PB and Tris-Hcl.

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

[0132] Optimization condition 1: HRP solution and sodium periodate solution with a concentration of 5 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:1, and reacted for 20 min at 4°C in the dark;

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

[0134] Optimization condition 3: HRP solution and sodium periodate solution with a concentration of 20 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:1, and reacted for 20 min at 4°C in the dark;

[0135] Optimization condition 4: HRP solution and sodium periodate solution with a concentration of 30 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:1, and reacted for 20 min at 4°C in the dark;

[0136] Optimization condition 5: HRP solution and sodium periodate solution with a concentration of 20 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:2, and reacted for 20 min at 4°C in the dark;

[0137] Optimization condition 6: HRP solution and sodium periodate solution with a concentration of 20 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:3, and reacted for 20 min at 4°C in the dark;

[0138] Optimization condition 7: HRP solution and sodium periodate solution with a concentration of 20 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:1, and reacted for 10 min at 4°C in the dark;

[0139] Optimization condition 8: HRP solution and sodium periodate solution with a concentration of 20 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:1, and reacted for 30 min at 4°C in the dark;

[0140] Optimization condition 9: HRP solution and sodium periodate solution with a concentration of 20 mg / mL were prepared, and the two solutions were mixed at a ratio of 1:1, and reacted for 40 min at 4°C in the dark;

[0141] The enzyme-labeled antibodies prepared according to the above conditions were used to detect 4 repeated blank wells and 4 repeated recombinant HCBP6 protein with a concentration of 40 ng / mL, and the absorbance values obtained are shown in Table 6:

[0142] Taking the labeling optimization condition as the abscissa and the average absorbance value as the ordinate, the effect curve is as shown in Figure 5 Combined with the color development of 40 ng / mL and the blank value background, Figure 5The histogram directly reflects that the condition 3, 5 and 8 are better, but considering the cost of raw materials and time, the condition 3 is selected as the optimal scheme of the enzyme-labeled antibody labeling in the present study.

[0143] Table 6. Comparison of absorbance at 450 nm under enzyme-labeled antibody labeling condition optimization

[0144]

[0145]

[0146] The above is only the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

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

4.

2. The kit of claim 1, wherein The heavy chain constant region of the antibody 1 is a mouse IgG1 subtype, and the light chain constant region is a mouse kappa 1 type; The heavy chain constant region of the antibody 2 is a mouse IgG1 subtype, and the light chain constant region is a mouse kappa 1 type; The enzyme label of the enzyme-labeled antibody 1 comprises any one of horseradish peroxidase or alkaline phosphatase.

3. The kit of claim 1, wherein The enzyme label of the enzyme-labeled antibody 1 is horseradish peroxidase; The preparation method of the enzyme-labeled antibody 1 comprises the following steps: Step 1: obtaining an activated enzyme label by first reaction of enzyme label solution and sodium periodate solution; Step 2: obtaining the enzyme-labeled antibody 1 by second reaction of the activated enzyme label and the antibody 1; The concentration ratio of the enzyme label in the enzyme label to sodium periodate in the sodium periodate solution is 1:1; The concentration ratio of the activated enzyme label to the antibody 1 is 1:2; The first reaction condition is 4°C, light shielding, 15min~25min; The second reaction condition is 4°C, light shielding, 3~5h.

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

5. The kit of claim 4, wherein The coating solution is CB buffer; The washing solution comprises water, 6g / L~10g / L sodium chloride, 0.1g / L~0.3g / L potassium chloride, 0.1g / L~0.3g / L dipotassium hydrogen phosphate, 3g / L~4g / L dodecahydrate disodium hydrogen phosphate and / or 0.4%~0.6% volume fraction Twenn-20; The antigen diluent comprises water, 6g / L~10g / L sodium chloride, 0.1g / L~0.3g / L potassium chloride, 0.1g / L~0.3g / L dipotassium hydrogen phosphate and 3g / L~4g / L dodecahydrate disodium hydrogen phosphate; The enzyme-labeled antibody diluent comprises 5%~10% volume fraction of newborn calf serum and the washing solution; The blocking solution comprises 0.5%-1.5% BSA and antigen diluent by mass fraction; The substrate buffer solution comprises 12 g / L-14 g / L sodium acetate, 1 g / L-2 g / L citric acid and hydrogen peroxide.

6. The kit of claim 5, wherein, The washing solution comprises water, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.24 g / L potassium phosphate dibasic, 3.6 g / L sodium phosphate dibasic dodecahydrate and / or 0.5% Twenn-20 by volume fraction; The antigen diluent comprises water, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 0.24 g / L potassium phosphate dibasic and 3.6 g / L sodium phosphate dibasic dodecahydrate; The enzyme-labeled antibody diluent comprises 10% new born calf serum by volume fraction and the washing solution; The blocking solution comprises 1% BSA and antigen diluent by mass fraction; The substrate buffer solution comprises 13.6 g / L sodium acetate, 1.6 g / L citric acid and hydrogen peroxide.

7. The kit of claim 6, wherein The kit further comprises TMB developing solution, positive control and / or negative control.

8. Use of the kit of any one of claims 1-7 in the preparation of a product for detecting HCBP6 and / or diagnosing fatty liver.

9. A method for detecting HCBP6 for non-diagnostic purposes, characterized in that, The kit of any one of claims 1-7 is used to detect a sample.

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

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