Heterophile antibody blocking agent and myoglobin detection kit

Abstract

The present invention provides a heterophilic antibody blocker comprising a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2. The present invention also provides a use of the above-mentioned heterophilic antibody blocker in the preparation of a myoglobin detection kit and a corresponding kit. The heterophilic antibody blocker of the present invention is a self-developed product designed specifically for myoglobin detection and can effectively remove interfering substances in serum; the use of this heterophilic antibody blocker can greatly improve the performance of the reagent, reduce the false positive rate, and provide a reliable guarantee for the test results.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a heterophilic antibody blocker and a myoglobin detection kit. Background Art

[0002] Myoglobin (Mb) is a protein in muscle that stores and transports oxygen. Composed of a single polypeptide chain and a prosthetic group, heme, it has a relative molecular mass of approximately 16,700 kD and contains 153 amino acid residues. Its physiological function is to help myocytes transport oxygen to the mitochondria. When myocardial or skeletal muscle membrane permeability increases, myoglobin easily escapes from the cytoplasm into the bloodstream and is excreted through the kidneys. Therefore, myoglobin is a sensitive indicator of the extent of skeletal and myocardial muscle damage.

[0003] Currently, myoglobin is commonly measured using methods such as latex-enhanced turbidimetry, ELISA, and colloidal gold. However, during the test process, interference from heterophilic antibodies can result in false positives.

[0004] Heterophile antibodies (HA), also known as heterophile antibodies (HA), are endogenous autoantibodies secreted by the human immune system in response to known or unknown antigens. They bind nonspecifically to animal immunoglobulins. HA can be caused by close contact with animals, consumption of contaminated food, treatment with monoclonal animal antibody preparations, infection, and blood transfusions. The interference caused by heterophile antibodies is ultimately due to errors caused by nonspecific competition between the antigen and the antibody. To eliminate heterophile interference, heterophile blockers are commonly used on the market.

[0005] At present, blockers are widely used in in vitro diagnostic reagents, including Roche's MARK33 series, Scantibodies' HBR series, Meridian's TRUBLOCK series, Millipore's CHEMIBLOCK series and Feipeng's HIER series. However, the blocking effects of blockers of different types and manufacturers may vary greatly, and most of the blockers on the market are broad-spectrum and not very targeted.

[0006] Based on this, if a heterophilic antibody blocker specifically for myoglobin detection can be developed, it will greatly improve the performance of the reagent, reduce the false positive rate, and provide a reliable guarantee for the test results. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a heterophilic antibody blocker specifically for myoglobin and a myoglobin detection kit. The use of this heterophilic antibody blocker in combination with a self-developed myoglobin kit can greatly improve the performance of the reagent, reduce the false positive rate, and provide a reliable guarantee for the test results.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A heterophilic antibody blocker 4-MR-poly comprises a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2.

[0010] An application of the heterophilic antibody blocker in preparing a myoglobin detection kit.

[0011] A myoglobin detection kit is based on latex-enhanced immunoturbidimetry and the above-mentioned heterophilic antibody blocker.

[0012] As one of the preferred embodiments of the present invention, it specifically includes R1 reagent and R2 reagent;

[0013] The R1 reagent includes: 10-30 mmol / L Tris buffer, 90-110 g / L NaCl, 15-25 g / L polyethylene glycol-6000, 3-8 mL / L Tween-20, 3-8 mL / L Triton X-100, 3-8 g / L BSA, 0.5-1.5 mL / L PC300, and 50-70 mg / L heterophilic antibody blocking agent;

[0014] The R2 reagent comprises: 10-30 mmol / L Tris buffer, 0.5-1.5 mL / L Tween-20, 1.5-2.5 g / L BSA, 8-12 g / L NaCl, 0.5-1.5 mL / L PC300, and 30-50 mg / L latex myoglobin antibody.

[0015] As one of the preferred embodiments of the present invention, the preparation method of the R1 reagent is as follows: Tris is added to water and the pH is initially adjusted to 7.50-8.50; NaCl, polyethylene glycol-6000, Tween-20, Triton X-100, BSA, and PC300 are added and the pH is adjusted to 7.50±0.02; after filtering the solution with a cellulose acetate membrane, a heterophilic antibody blocker is added.

[0016] As one of the preferred embodiments of the present invention, a cellulose acetate membrane with a diameter of 0.45 μm is used to filter the solution.

[0017] As one of the preferred embodiments of the present invention, the preparation method of the R2 reagent is as follows: Tris is added to water and the pH is initially adjusted to 7.50-8.00; Tween-20, BSA, and PC300 are added and the pH is adjusted to 7.50±0.02; the solution is filtered using a cellulose acetate membrane and then a latex myoglobin antibody is added.

[0018] As one of the preferred embodiments of the present invention, a cellulose acetate membrane with a diameter of 0.45 μm is used to filter the solution.

[0019] The advantages of the present invention over the prior art are:

[0020] (1) The heterophilic antibody blocker 4-MR-poly of the present invention is a self-developed product designed specifically for myoglobin detection and can effectively remove interfering substances in serum. The use of this heterophilic antibody blocker in combination with the self-developed myoglobin test kit can greatly improve the performance of the reagent, reduce the false positive rate, and provide a reliable guarantee for the test results.

[0021] (2) The myoglobin detection kit of the present invention adopts latex immunoturbidimetry, and the self-developed 4-MR-poly blocking antibody is prepared by genetic engineering. The method is simple, easy to operate, and widely used. Compared with the blocking agents of other manufacturers, it has high blocking efficiency, strong targeting, and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the calibration curve of the reagents in the control group and experimental group 1 in the experimental example (in the figure, the horizontal axis represents the concentration in ng / mL, and the vertical axis represents the absorbance Abs);

[0023] Figure 2 3 and 4. It is the accelerated stability curve of the reagents of the control group and experimental group 1 in the experimental example (in the figure, the horizontal axis represents the concentration in ng / mL, and the vertical axis represents the absorbance Abs). DETAILED DESCRIPTION

[0024] The following embodiments of the present invention are described in detail. These embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the scope of protection of the present invention is not limited to the following embodiments. At the same time, the reagents and experimental methods used in the following embodiments are conventional reagents or methods in the art unless otherwise specified, and will not be repeated here.

[0025] Example 1

[0026] The self-developed 4-MR-poly blocker antibody (heterophilic antibody blocker) of this embodiment comprises a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 2. The 4-MR-poly blocker antibody of this embodiment is capable of binding to interfering substances such as heterophilic antibodies in serum.

[0027] Example 2

[0028] This embodiment is a preparation of a 4-MR-poly blocker antibody in the above embodiment:

[0029] (1) Expression of 4-MR-poly blocker antibody in CHO-K1 suspension expression system

[0030] a. Cell Thawing: Thaw a vial of CHO cells (approximately 1 x 10E7 cells) frozen in liquid nitrogen at 37°C in a water bath. Centrifuge at 300g for 5 minutes. Wipe the vial with alcohol wipes and place in a laminar flow hood. Aspirate the supernatant, resuspend in 20 mL of pre-warmed complete CHO medium, and culture in a 125 mL shake flask at 37°C, 5% CO2, and 120-130 rpm.

[0031] b. Plasmid Extraction: Inoculate the DH5a strain containing the pCDNA4.2 vector containing the 4-MR-poly blocker antibody (gene synthesized by a biotechnology company; the sequences of the 4-MR-poly blocker antibody are shown in SEQ ID NO. 1 and SEQ ID NO. 2) in advance and culture overnight at 37°C. Extract the plasmid using a commercial plasmid extraction kit.

[0032] c. Cell Transfection: When cell viability is greater than 95%, there are no obvious cell clumping, and the cell density is between 2 and 3 x 10E6, harvest 4 x 10E7 CHO cells and centrifuge at 300g for 5 minutes. Remove the supernatant and resuspend the CHO cells in 400µL of Celetrix commercial electroporation buffer. Add 25µg of plasmid to deplete 2 x 10E7 CHO cells. Electroporate using a Celetrix cell electroporator at 1250V. Allow the cells to recover for 24 hours.

[0033] d. Cell transfer and fed-batch culture: After cell recovery, transfer the cells to fermentation medium at a density of 0.5 x 106 cells. Culture until Day 5, then initiate fed-batch culture. Cool down the culture until Day 7.

[0034] e. Supernatant Collection: Culture until Day 15 or when cell viability drops below 60%. Centrifuge the culture supernatant to remove the cell pellet and retain the supernatant.

[0035] (2) Affinity purification of "4-MR-poly blocker antibody"

[0036] a. Affinity column filling: Calculate the required commercial nickel filler and load the filler onto the column. Wash the loaded filler with PBS equilibration buffer.

[0037] b. Loading and washing: Load the cell supernatant collected by centrifugation at a low flow rate. After loading, wash with equilibration buffer for 10 column volumes and pre-elution buffer A containing 20 mM imidazole for 10 column volumes.

[0038] c. Elution and dialysis: After washing the filler with the pre-elution solution, elute it with 250mM imidazole-containing Tris elution solution. The eluted protein is dialyzed overnight with 20mM PBS buffer. After dialysis, the antibody is aliquoted and stored.

[0039] Example 3

[0040] The myoglobin detection kit of this embodiment based on latex-enhanced immunoturbidimetry includes R1 reagent and R2 reagent.

[0041] R1 reagent includes: 10 mmol / L Tris buffer, 90 g / L NaCl, 15 g / L polyethylene glycol-6000, 3 mL / L Tween-20, 3 mL / L Triton X-100, 3 g / L BSA, 0.5 mL / L PC300, and 50 mg / L heterophilic antibody blocker (4-MR-poly blocker antibody developed by the present invention).

[0042] R2 reagents include: 10 mmol / L Tris buffer, 0.5 mL / L Tween-20, 1.5 g / L BSA, 8 g / L NaCl, 0.5 mL / L PC300, and 30 mg / L latex myoglobin antibody.

[0043] Preparation method:

[0044] (1) Prepare R1 reagent:

[0045] Tris was added to water and the pH was initially adjusted to 7.50. NaCl, polyethylene glycol-6000, Tween-20, Triton X-100, BSA, and PC300 were added and the pH was adjusted to 7.50±0.02. The solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm, and a heterophilic antibody blocking agent was added.

[0046] (2) Preparation of latex myoglobin antibody:

[0047] A. Washing and Activation of Latex Particles

[0048] ① Add the latex particles to 10mM MES buffer and centrifuge, discard the supernatant, and repeat the washing process three times;

[0049] ② Resuspend the pellet in 10 mM MES buffer and add EDC and NHS for activation for 30 min;

[0050] ③ After activation, the latex particles were washed three times with 10 mM MES buffer and then resuspended;

[0051] B. Antibody Coupling and Blocking

[0052] ① Take the activated and washed latex solution, add myoglobin antibody (Chengdu Huirui Xinyuan Biotechnology Co., Ltd., product number 301081, batch number JE0270), mix well, and couple in a shaker at 37°C at 220 rpm for 2 h;

[0053] ② Wash the microspheres with 10 mM MES buffer, repeat 3 times;

[0054] ③Add blocking agent to block for 2 hours;

[0055] ④ Wash the above microsphere particles with MES buffer, repeat 3 times, and obtain "latex myoglobin antibody".

[0056] (3) Prepare R2 reagent:

[0057] Tris was added to water and the pH was initially adjusted to 7.50; Tween-20, BSA, and PC300 were added and the pH was adjusted to 7.50±0.02; the solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm, and latex myoglobin antibody was added.

[0058] Example 4

[0059] The myoglobin detection kit of this embodiment based on latex-enhanced immunoturbidimetry includes R1 reagent and R2 reagent.

[0060] R1 reagent includes: 20 mmol / L Tris buffer, 100 g / L NaCl, 20 g / L polyethylene glycol-6000, 5 mL / L Tween-20, 5 mL / L Triton X-100, 5 g / L BSA, 1 mL / L PC300, and 60 mg / L heterophilic antibody blocker (the present invention's self-developed 4-MR-poly blocker antibody).

[0061] R2 reagents include: 20 mmol / L Tris buffer, 1 mL / L Tween-20, 2 g / L BSA, 10 g / L NaCl, 1 mL / L PC300, and 40 mg / L latex myoglobin antibody.

[0062] Preparation method:

[0063] (1) Prepare R1 reagent:

[0064] Tris was added to water and the pH was initially adjusted to 8.0. NaCl, polyethylene glycol-6000, Tween-20, Triton X-100, BSA, and PC300 were added and the pH was adjusted to 7.50±0.02. The solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm, and a heterophilic antibody blocking agent was added.

[0065] (2) Preparation of latex myoglobin antibody:

[0066] A. Washing and Activation of Latex Particles

[0067] ① Add the latex particles to 20mM MES buffer and centrifuge, discard the supernatant, and repeat the washing process three times;

[0068] ② Resuspend the pellet in 20 mM MES buffer and add EDC and NHS for activation for 30 min;

[0069] ③ The activated latex particles were washed three times with 20 mM MES buffer and then resuspended;

[0070] B. Antibody Coupling and Blocking

[0071] ① Take the activated and washed latex solution, add myoglobin antibody (Chengdu Huirui Xinyuan Biotechnology Co., Ltd., product number 301081, batch number JE0270), mix well, and couple in a shaker at 37°C at 220 rpm for 2 h;

[0072] ② Wash the microspheres with MES buffer, repeat 3 times;

[0073] ③Add blocking agent to block for 2 hours;

[0074] ④ Wash the above microsphere particles with MES buffer, repeat 3 times, and obtain "latex myoglobin antibody".

[0075] (3) Prepare R2 reagent:

[0076] Tris was added to water and the pH was initially adjusted to 7.8; Tween-20, BSA, and PC300 were added and the pH was adjusted to 7.50±0.02; the solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm, and latex myoglobin antibody was added.

[0077] Example 5

[0078] The myoglobin detection kit of this embodiment based on latex-enhanced immunoturbidimetry includes R1 reagent and R2 reagent.

[0079] R1 reagent includes: 30mmol / L Tris buffer, 110g / L NaCl, 25g / L polyethylene glycol-6000, 8mL / L Tween-20, 8mL / L Triton X-100, 8g / L BSA, 1.5mL / L PC300, and 70mg / L heterophilic antibody blocker (4-MR-poly blocker antibody developed by the present invention).

[0080] R2 reagents include: 30 mmol / L Tris buffer, 1.5 mL / L Tween-20, 2.5 g / L BSA, 12 g / L NaCl, 1.5 mL / L PC300, and 50 mg / L latex myoglobin antibody.

[0081] Preparation method:

[0082] (1) Prepare R1 reagent:

[0083] Tris was added to water and the pH was initially adjusted to 8.50. NaCl, polyethylene glycol-6000, Tween-20, Triton X-100, BSA, and PC300 were added and the pH was adjusted to 7.50±0.02. The solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm and a heterophilic antibody blocking agent was added.

[0084] (2) Preparation of latex myoglobin antibody:

[0085] A. Washing and Activation of Latex Particles

[0086] ① Add the latex particles to 30mM MES buffer and centrifuge, discard the supernatant, and repeat the washing process three times;

[0087] ② Resuspend the pellet in 30 mM MES buffer and add EDC and NHS for activation for 30 min;

[0088] ③ After activation, the latex particles were washed three times with 30 mM MES buffer and then resuspended;

[0089] B. Antibody Coupling and Blocking

[0090] ① Take the activated and washed latex solution, add myoglobin antibody (Chengdu Huirui Xinyuan Biotechnology Co., Ltd., product number 301081, batch number JE0270), mix well, and couple in a shaker at 37°C at 220 rpm for 2 h;

[0091] ② Wash the microspheres with MES buffer, repeat 3 times;

[0092] ③Add blocking agent to block for 2 hours;

[0093] ④ Wash the above microsphere particles with MES buffer, repeat 3 times, and obtain "latex myoglobin antibody".

[0094] (3) Prepare R2 reagent:

[0095] Tris was added to water and the pH was initially adjusted to 8.00; Tween-20, BSA, and PC300 were added and the pH was adjusted to 7.50±0.02; the solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm, and latex myoglobin antibody was added.

[0096] Comparative Example 1

[0097] This comparative example uses conventional animal immunization methods to prepare heterophilic antibody blocking agents:

[0098] Antigens are used to stimulate the body, producing an immunological response. Antibodies are a group of immunoglobulins synthesized and secreted by the body's plasma cells that have the ability to specifically bind to the antigen. The general process for preparing rabbit polyclonal antibodies is as follows: complete antigen preparation → rabbit immunization → titer testing and final release → antibody affinity purification → antibody concentration and storage. The specific experimental steps are as follows:

[0099] (1) Rabbit preparation

[0100] Two healthy 6-week-old New Zealand white rabbits (about 2.5 kg) were selected and allowed to adapt to the new living environment and stabilize for a few days before the first blood draw.

[0101] a. Pre-blood collection (as a negative reference).

[0102] b. Carefully place the rabbit into a fixed rack and calm it down.

[0103] c. Carefully shave the rabbit's ears to allow the blood vessels to be clearly visible.

[0104] d. Use a small cotton ball dipped in alcohol to smear the blood vessels to dilate them.

[0105] e. Draw approximately 10 mL of blood (approximately 5 mL of serum) from the ear vein using a syringe.

[0106] f. Carefully remove the needle, apply pressure to the wound to prevent bleeding, and then disinfect the wound with an alcohol cotton ball.

[0107] g. Inactivate the collected blood at 37°C for 30 minutes and finally place it at 4°C overnight to allow it to coagulate and release serum.

[0108] h. Centrifuge the coagulated blood at 12000 rpm for 10 min.

[0109] i. Collect the supernatant, which is the serum.

[0110] (2) Rabbit Immunity

[0111] a. 1 mL of special antigen is injected into two rabbits. The special antigen buffer solution must not contain any chemical reagents that are harmful to rabbits. 400 μg of antigen is suitable for each rabbit for the initial immunization, but it can be reduced appropriately for better results. 100 μg of antigen is sufficient for each subsequent immunization.

[0112] b. Thoroughly mix 1 mL of Freund's complete adjuvant with 1 mL of the prepared specific antigen until the mixture turns milky white.

[0113] c. Carefully remove the rabbits from the cage and immunize each rabbit at four locations (the back and thighs are both acceptable). Use 250 μL of the injection at each location. Insert the needle at a 45-degree angle 1 to 2 cm subcutaneously. Leave the injection for a few seconds to prevent the specific antigen from leaking out.

[0114] d. The immunization cycle is 20 days. Blood samples are collected 7 to 10 days after immunization (including blood samples for mid-term testing and final blood collection). A total of 5 to 6 immunizations are conducted.

[0115] (3) Potency testing

[0116] a. Reference: The negative reference is the pre-blood serum, which is diluted with the initial concentration of 1 antibody (blocking diluent); the positive reference is the previous positive serum.

[0117] b. Add 100 μL of 1 μg / mL specific antigen to each well of a 96-well plate and incubate at 4°C overnight or at 37°C for 2 hours.

[0118] c. Pour off the antigen solution, add 200 μL of blocking solution to each well, and incubate at 4°C overnight or at 37°C for 2 hours.

[0119] d. Pour off the blocking solution and tap on absorbent paper to remove as much residual liquid as possible. Wash the plate three times with detergent, patting as much residual liquid as possible each time. If the plate needs to be stored for a period of time, dry it at 37°C and seal it in a sealable bag and store it at -20°C.

[0120] e. Take a coated 96-well plate and add 100uL of negative reference solution to the first well, 100uL of positive reference solution to the second well, and 1:500 dilution of the test antiserum to the third well. Dilute each subsequent well in the same dilution ratio and incubate at 37°C for 1 hour.

[0121] f. Pour off the liquid, wash the plate three times with washing solution, pat dry, add 100 μL of HRP-labeled mouse anti-rabbit IgG (1:2000 dilution) to each well, and incubate at 37°C for 45 minutes.

[0122] g. Pour off the liquid, wash the plate three times with washing solution, and pat dry. Add 100 μL of TMB substrate (Sigma one-component TMB) to each well and incubate at 37°C for 5-20 minutes (the color development time will depend on the color depth).

[0123] h. Add 50uL of stop solution (2N H2SO4) to each well and read the absorbance at a wavelength of 450nm on a microplate reader.

[0124] i. After the antibody titer reaches 10,000 or above, the rabbit is bled and the antibody is purified.

[0125] (4) Antibody affinity---Preparation of affinity column

[0126] a. Weigh 1 mg of CNBr-Sepherose 4B agarose gel and add it to 2 mmol / L hydrochloric acid. Allow to fully swell at 4°C overnight to obtain 3 mL of swollen gel.

[0127] b. Transfer the gel into a purification column and wash the medium three times with approximately 20 mL of 2 mmol / L hydrochloric acid.

[0128] c. Wash the medium once with coupling buffer. Because the activated group is easily hydrolyzed at the pH value of the coupling buffer, this step is best completed within a few minutes.

[0129] d. Add 5-10 mg of antigen dissolved in 5 mL of coupling buffer to the gel.

[0130] e. Gently mix and shake at room temperature for 2-4 hours, or at 4°C overnight. If you need to determine the binding efficiency, take a small amount of the solution after this step. Wash the medium once with 20 mL of coupling buffer.

[0131] f. Add 15 mL of 1% BSA solution and incubate at room temperature for 2 h or at 4°C overnight.

[0132] g. Wash the medium with phosphate buffer at least three times, with at least 15 mL each time.

[0133] h. Antigen solid phase is completed and can be used for purification.

[0134] i. If not used immediately or after use, seal with 20% ethanol.

[0135] (5) Purification and preservation of antiserum

[0136] a. Dilute the antiserum with an equal volume of PBS, centrifuge at 5000-10000 rpm for 15 minutes, and collect the supernatant.

[0137] b. Wash the antigen affinity column with 10 volumes of PBS to equilibrate the column.

[0138] c. Add 10 mL of diluted antiserum to the equilibrated column.

[0139] d. Mix gently with shaking at room temperature for 2 to 4 hours, or at 4°C overnight.

[0140] e. Wash the antigen column with 10 volumes of PBS to remove the foreign proteins bound to the column.

[0141] f. Wash the column with 2 volumes of antibody elution buffer to obtain specific antibodies.

[0142] g. Equilibrate the column with 10 volumes of PBS.

[0143] h. Seal the column with 20% alcohol and store at 4°C.

[0144] i. After obtaining the eluted antibody, concentrate it with sucrose or polyethylene glycol and dialyze it against PBS to remove salts. Measure the antibody OD value at a wavelength of 280 nm using a UV-visible spectrophotometer. Divide the obtained OD value by 1.35 to obtain the measured antibody concentration, which is also the concentration of the blocking agent. Add 40-50% glycerol and then add Proclin 300. Store at -20°C for long-term storage.

[0145] Comparative Example 2

[0146] This comparative example utilizes hybridoma cells to secrete specific monoclonal antibodies to prepare a special antibody blocking agent:

[0147] (1) Hybridoma cells were obtained by hybridoma technology and cultured to 5×10 5 After collecting the cells, resuspend them in serum-free medium, inoculate them into a 5L fermenter, and add serum-free medium to 800mL;

[0148] (2) Entering the mammalian cell fermentation mode; after 24 hours, the count was 1.5×10 6 cells / mL, cell viability 90%, add 1000mL serum-free medium; count 2.0×10 after 48h 6 cells / mL, cell viability 90%, add 1000 mL serum-free medium; after 72 h, count 3.5×10 6 / mL, cell viability 90%, supplemented with 2000mL serum-free medium and 200mL 3g / L soybean hydrolyzate; after 96h, the count was 4.5×10 6 / mL, cell viability 70%, stop feeding;

[0149] (3) When the cell viability drops to 40%, the culture is collected and centrifuged to remove the cells to obtain the fermentation broth;

[0150] (4) The fermentation broth was filtered through a filter membrane with a pore size of 0.22 μm to obtain a sample; the AKTA PURE was turned on and UV was flushed to the baseline, zeroed, the Protein A purification column was connected to the column valve, equilibrated with PBS buffer until the conductivity parameters and UV parameters were at the baseline, and the sample was injected into the sample loop; after the injection was completed, the PBS buffer was used to equilibrate until the conductivity parameters and UV parameters were at the baseline, the eluate was connected to collect the purified product, and immediately neutralized with 1 M Tris buffer to obtain the purified active antibody;

[0151] (5) The purified active antibody was concentrated by replacing the liquid in a 15 mL, 30K ultrafiltration tube, and finally replaced with PBS buffer. It was sterile filtered using a filter membrane with a pore size of 0.22 μm in an ultra-clean bench. The sterile filtered filtrate was collected and sampled for SDS PAGE electrophoresis to verify its purity. The antibody concentration was detected using an ultra-micro spectrophotometer to obtain an active blocking agent containing 5 mg / mL active antibody, 2 mM KH2 PO4, 137 mM NaCl, 13 mM Na2HPO4, and 2.7 mM KCl, and stored at -20°C.

[0152] Experimental example

[0153] This experimental example is used to verify the application effect of the self-developed blocker 4-MR-poly of the present invention.

[0154] 1. Experimental methods:

[0155] Set up one control group and six experimental groups:

[0156] Control group: R1 reagent kit without any blocker added (taking the kit of Example 4 of the present invention as an example, the blocker was removed);

[0157] Experimental Group 1: R1 reagent was supplemented with a kit containing the "self-developed 4-MR-poly blocker antibody of the present invention" (using the kit of Example 4 of the present invention as an example);

[0158] Experimental Group 2: R1 reagent was supplemented with the kit containing the "animal immune production blocker of Comparative Example 1" (other reagent components except the blocker were the same as those of Experimental Group 1);

[0159] Experimental Group 3: R1 reagent was supplemented with the kit containing the hybridoma cell production blocker from Comparative Example 2 (other reagent components except the blocker were the same as those in Experimental Group 1);

[0160] Experimental group 4: purchased Japanese Seiken reagents (myoglobin assay kit Mb-CN: biochemical immunoassay reagents | Denka Co., Ltd.; specification: 120 ml; standard traceability: in-house)";

[0161] Experimental group 5: R1 reagent supplemented with "Haiyuan Blocker R and M1 (Hunan Haiyuan Medical Technology Co., Ltd.)" (other reagent components except the blocker are the same as those of experimental group 1);

[0162] Experimental Group 6: R1 reagent was supplemented with Jingda Heterophilic Antibody Blocker (P-heterophilic Antibody Blocker, Nanjing Jingda Biotechnology Co., Ltd., Catalog No.: HBR-5) (other reagent components except the blocker were the same as those in Experimental Group 1).

[0163] The control group and six experimental group reagents were used to conduct Landau quality control tests for abnormal serum and myocardial type. At the same time, the calibration results and accelerated stability results of the control group and experimental group 1 reagents were compared.

[0164] 2. Experimental results:

[0165] The abnormal serum and myocardial Randox quality control test results of the control group and each experimental group reagent are shown in Tables 1 and 2. The calibration results of the control group and experimental group 1 reagent are shown in Table 3, and the calibration curve is shown in Figure 1 The accelerated stability results are shown in Figure 2 shown.

[0166] Table 1 Abnormal serum test results (unit: ng / mL)

[0167]

[0168]

[0169] Table 2 Myocardial Landau quality control test results (unit: ng / mL)

[0170]

[0171]

[0172] Table 3 Calibration data (unit: S1-S5 (0-800) is ng / mL, others are absorbance Abs)

[0173]

[0174] The above results show that:

[0175] (1) The blocking effects of heterophilic antibody blockers produced by immunization of common animals (rabbits) and purchased Jingda heterophilic antibody blockers are both poor, and there is a risk of false positives;

[0176] (2) The blocking effects of monoclonal antibody blockers produced by hybridoma cells and the sea source blockers M1 and R are both good, but the process of monoclonal antibody produced by hybridoma cells is complicated and the cost is high. The sea source blocker requires the simultaneous use of M1 and R, which is also expensive.

[0177] (3) The test results of the "kit plus self-developed 4-MR-poly blocking antibody" of the present invention and the "Japan Seiken reagent" were similar, showing that they could effectively block interfering substances in serum. In addition, the addition of the self-developed 4-MR-poly blocking antibody to R1 did not affect the calibration results of the reagent, the Landau test results, or the accelerated stability.

[0178] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A heterophilic antibody blocking agent, characterized in that: It comprises a light chain and a heavy chain; the amino acid sequence of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

2.

2. Use of the heterophilic antibody blocker according to claim 1 in the preparation of a myoglobin detection kit.

3. A myoglobin detection kit, characterized in that, The method is based on latex-enhanced immunoturbidimetry and comprises the heterophilic antibody blocking agent as claimed in claim 1.

4. The myoglobin detection kit according to claim 3, characterized in that Specifically including R1 reagent and R2 reagent; The R1 reagent includes: 10-30 mmol / L Tris buffer, 90-110 g / L NaCl, 15-25 g / L polyethylene glycol-6000, 3-8 mL / L Tween-20, 3-8 mL / L Triton X-100, 3-8 g / L BSA, 0.5-1.5 mL / L PC300, and 50-70 mg / L heterophilic antibody blocking agent; The R2 reagent includes: 10-30 mmol / L Tris buffer, 0.5-1.5 mL / L Tween-20, 1.5-2.5 g / L BSA, 8-12 g / L NaCl, 0.5-1.5 mL / L PC300, and 30-50 mg / L latex myoglobin antibody.

5. The myoglobin detection kit according to claim 4, characterized in that The preparation method of the R1 reagent is as follows: Tris is added to water and the pH is initially adjusted to 7.50-8.50; NaCl, polyethylene glycol-6000, Tween-20, Triton X-100, BSA, and PC300 are added and the pH is adjusted to 7.50±0.02; the solution is filtered through a cellulose acetate membrane and a heterophilic antibody blocking agent is added.

6. The myoglobin detection kit according to claim 5, characterized in that The solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm.

7. The myoglobin detection kit according to claim 4, characterized in that The preparation method of the R2 reagent is as follows: adding Tris to water and initially adjusting the pH to 7.50-8.00; adding Tween-20, BSA, NaCl, and PC300 and adjusting the pH to 7.50±0.02; filtering the solution with a cellulose acetate membrane, and then adding latex myoglobin antibody.

8. The myoglobin detection kit according to claim 7, characterized in that The solution was filtered through a cellulose acetate membrane with a diameter of 0.45 μm.

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