Monoclonal antibody of CK-MB, preparation method and application

Abstract

The present invention discloses a monoclonal antibody, a preparation method and an application of CK-MB, and relates to the technical field of monoclonal antibodies. The proposed monoclonal antibody has an EC50 of ng / ml level for binding to an antigen, and a KD of 10 ‑11 As mentioned above, for IgG antibodies, it is generally believed in the art that when the KD value of binding to the antigen reaches 10 ‑9 M is considered to have high affinity, and the KD values ​​of the two monoclonal antibodies of the present invention for binding to the antigen both reached 10 ‑11 M is 100 times the above-defined value, so that the monoclonal antibody proposed in the present application has extremely strong antigen binding activity and affinity; the present invention is simple, effective, and easy to use.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibodies, and specifically relates to a preparation method and application of a monoclonal antibody of CK-MB. Background Art

[0002] The patent with publication number CN108333357A discloses a magnetic bead time-resolved fluorescent immunoquantitative detection kit for CK-MB, including immunomagnetic beads coated with CK-MB monoclonal antibodies, CK-MB calibration solution, europium-labeled CK-MB monoclonal antibody solution, cleaning solution and enhancement solution. The immunomagnetic beads coated with CK-MB monoclonal antibodies are superparamagnetic beads with a diameter of 1-3 μm modified with functional groups and covalently coupled with CK-MB monoclonal antibodies. The sensitivity of the present invention is high, the sensitivity of CK-MB is 1 ng / ml, and the serum (plasma) sample does not need to be diluted; the detection time is short, and the report is issued in 30 minutes; the sample requirement is small, and only 50 μl is required for one loading; it is equipped with a fully automatic time-resolved immunoanalyzer, which is simple to operate, has no human error and saves manpower. This kit rationally utilizes the space of the reagent strip, making the reagent strip structure more compact, easier to transport, convenient to use, and simple to operate and stable.

[0003] However, for CK-MB monoclonal antibodies, how to make them have extremely strong antigen binding activity and affinity is a problem; based on this, a solution is provided. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art; to this end, the present invention provides a monoclonal antibody to CK-MB;

[0005] Monoclonal antibodies to CK-MB, including:

[0006] Light chain amino acid sequence: SEQ ID NO: 7;

[0007] Heavy chain amino acid sequence: SEQ ID NO: 6;

[0008] Among them, the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 4, RAS and SEQ ID NO: 5, respectively; the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The monoclonal antibodies proposed in the present invention have an EC50 of ng / ml level for binding to antigens and a KD of 10 -11 As mentioned above, for IgG antibodies, it is generally believed in the art that when the KD value of binding to the antigen reaches 10 -9 M is considered to have high affinity, and the KD values ​​of the two monoclonal antibodies of the present invention for binding to the antigen both reached 10 -11 M is 100 times the above-defined value, so that the monoclonal antibody proposed in the present application has extremely strong antigen binding activity and affinity; the present invention is simple, effective, and easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A It is a schematic diagram of the SDS-PAGE detection results of the present invention;

[0012] Figure 1B It is a schematic diagram of the SDS-PAGE detection results of the present invention;

[0013] Figure 2A Schematic diagram of the binding activity curve between the antibody of the present invention and the antigen;

[0014] Figure 2B Schematic diagram of the binding activity curve between the antibody of the present invention and the antigen;

[0015] Figure 3 This is a schematic diagram of the structure of the colloidal gold immunochromatographic test strip of the present invention;

[0016] Figure 4 It is a schematic diagram of the coincidence rate curve between the test results of the test strip of the present invention and the test results of Roche. DETAILED DESCRIPTION

[0017] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] See also Figure 1A , Figure 1B , Figure 2A , Figure 2B , Figure 3 , Figure 4 ;

[0019] The present application provides a monoclonal antibody to CK-MB and a preparation method and application thereof;

[0020] The preparation of monoclonal antibodies against CK-MB is as follows:

[0021] (1) Expression and purification of CK-MB / CKMM / CKBB proteins:

[0022] The amino acid sequences of CreatinekinaseM-type (P06732) and CreatinekinaseB-type (P12277) were found on the UniProt website, and the corresponding gene sequences were deduced and synthesized using software. The obtained DNA sequences were cloned into the PCDNA3.4 vector, and then transformed and plated, clones were picked, and plasmids were extracted; the recombinant plasmids were transfected into N293 cells, and the transfection conditions were: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2μg plasmid / 1ml N293 cells; 5-7 days after transfection, the cell supernatant was collected; the CKMM protein and CKBB protein were purified using a nickel column, and the elution buffer was replaced with PBS by dialysis, and the antibody concentration was determined by the Bradford method.

[0023] CK-MB

[0024] The gene sequences of CK-M protein and CK-B protein were coupled together with the nucleotide base of Loop, cloned into the multiple cloning site BamH I / Xho I of the expression vector pET28a, and the expression plasmid pET28a-CKMB was obtained; the expression plasmid pET28a-CKMB was transformed into Escherichia coli BL21 (DE3), and the expression was induced by IPTG; after the culture was completed, the culture solution was stirred and shaken with Tris-hydrochloric acid buffer to resuspend the bacterial precipitate, ultrasonically broken the cell wall, centrifuged to obtain the supernatant, and sequentially passed through a nickel column, a strong anion exchange column Q column and a Superdex G200 molecular sieve chromatography column to obtain a purified CK-MB protein. The expression of CK-MB protein refers to patent CN107698683A1.

[0025] (2) Animal immunization:

[0026] The expressed CK-MB protein, with a specific concentration of 0.5 mg / ml, was used as an immunogen. 200 ug of the antigen, i.e. 400 ul, was fully emulsified with 400 ul of Freund's complete adjuvant and then injected subcutaneously to immunize rabbits. The rabbit species was: New Zealand white rabbits, female, 6-8 weeks old. The booster immunization was performed at intervals of two weeks for a total of 3 times. Blood was collected from the rabbit vein 5-8 days after the last booster immunization, and the serum titer was detected by ELISA. The specific experimental information is shown in Table 1 below.

[0027] Table 1: Experimental information

[0028]

[0029] The serum titer data detected by ELISA are shown in Table 2 ;

[0030] Table 2: ELIAS method for testing serum titer data

[0031] Dilution factor / K R0007 Negative control 0.0606 Negative control 0.0577 1 1.7550 2 1.3826 4 1.0542 8 0.7110 16 0.4707 32 0.2925 64 0.1879 128 0.1267 256 0.0980 512 0.0795

[0032] The rabbit serum titer was 64K, and peripheral blood mononuclear cell (PBMC) suspension was prepared.

[0033] (3) PBMC preparation:

[0034] Add Ficoll, a lymphocyte separation solution, to the centrifuge tube, take anticoagulated peripheral blood (whole blood) and sterile PBS and mix thoroughly at a ratio of 1:1-1:5, use a pipette to slowly superimpose on the stratified liquid surface along the tube wall, centrifuge horizontally at 400g×30 minutes, remove part of the upper liquid after centrifugation, and about 1ml is left. Use a pipette to insert into the cloud layer to absorb the mononuclear cells. Place in another centrifuge tube, add more than 5 times the volume of PBS, centrifuge at 300g×10 minutes, and wash the cells twice. After the last centrifugation, discard the supernatant, add red blood cell lysis solution, incubate at room temperature for 2 minutes, lyse the red blood cells, add 10ml PBS, centrifuge at 300g×10 minutes, and wash the cells twice. Discard the supernatant, add RPMI1640 containing 10% FBS, resuspend the cells, and count;

[0035] (4) Obtaining single B cells:

[0036] The prepared spleen single cell suspension is used to label the antigen and perform cell screening using a single cell screening platform to obtain antigen-specific B cells.

[0037] Through the above steps, a total of 37 monoclonal antibodies against CK-MB protein were obtained, and they were all IgG antibodies after identification, and their names are as shown in Table 3. In the following examples, the light and heavy chain gene sequences of the monoclonal antibodies were detected and expressed, and the clones of the cell supernatant after expression were placed in a 96-well plate according to the layout of Table 3 for binding activity detection;

[0038] Table 3: 37 antibody clone numbers

[0039] 1 2 3 4 5 A 1M7A7 1M3C3 1M3G7 1M4A1 1M4D4 B 1M7C9 1M3D3 1M3D8 1M4B1 1M4F4 C 1M7C10 1M3G3 1M3G8 1M4A2 1M4B5 D 1M3E1 1M3C4 1M3E9 1M4C2 1M4F5 E 1M3C2 1M3D4 1M3G9 1M4E2 1M4A6 F 1M3E2 1M3E5 1M3D11 1M4F2 Negative control G 1M3G2 1M3C6 1M3E12 1M4G2 Negative control H 1M3A3 1M3E7 1M3G12 1M4C4

[0040] 2. Specific detection of monoclonal antibodies against CK-MB protein;

[0041] The supernatant of the cell clones in Example 1 was tested for antigen specificity using ELISA:

[0042] ① Antigen coating: dilute CK-MB / CK-MM / CKBB proteins to 2µg / ml respectively, then add 100μl / well to an empty ELISA plate, place the ELISA plate in a 4℃ refrigerator, and incubate overnight;

[0043] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0044] ③ Incubate with primary antibody: add the culture supernatant of 96-well plate to the wells of ELISA plate, 100 μl per well, incubate at 37°C for 1 hour; wash the plate 5 times with a plate washer and dry;

[0045] ④ Incubate with secondary antibody: dilute HRP-labeled goat anti-rabbit secondary antibody at 1:10000, then add to the ELISA plate, 100 μl per well, incubate at 37°C for 30 min; wash the plate 5 times with a plate washer and dry;

[0046] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37℃ / 15min;

[0047] ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, the detection results are shown in Table 4-Table 6.

[0048] Table 4: CK-MB plate specificity test results

[0049] CKMB 1 2 3 4 5 A 2.6959 0.9491 1.2852 0.1698 0.1667 B 1.0982 0.8581 2.1397 2.5481 0.4724 C 0.0831 0.1373 0.2215 0.4832 0.3783 D 0.1240 1.7838 1.8155 0.0978 0.4253 E 0.3977 0.7336 1.1408 0.3477 0.0598 F 0.6091 2.0104 1.9866 1.5410 0.0559 G 0.2744 0.4594 1.6568 2.0098 0.0540 H 1.9767 0.1264 0.8094 0.2113

[0050] Table 5: CK-MM plate specificity test results

[0051] CKMM 1 2 3 4 5 A 3.2253 0.1272 0.0913 1.8276 0.1477 B 0.0690 0.0699 0.0719 2.2278 0.0938 C 0.1114 0.0576 0.0777 0.3617 0.0585 D 0.1299 0.0624 0.0705 0.0833 0.1561 E 0.0967 0.0611 0.0721 0.2407 0.0723 F 1.9607 0.2511 0.0720 0.0800 0.0516 G 0.4098 0.0661 0.0661 0.2682 0.0524 H 0.0540 0.1148 0.1134 0.1343

[0052] Table 6: CK-BB plate specificity test results

[0053] CKBB 1 2 3 4 5 A 1.5214 0.2188 0.2068 0.0885 0.0943 B 0.2765 0.1391 0.0720 2.1170 0.1600 C 0.1138 0.0723 0.1115 0.3835 0.0763 D 0.1301 0.3356 0.2892 0.0895 0.1316 E 0.1300 0.0850 0.1429 0.2383 0.0576 F 0.1077 0.3442 0.3494 0.2187 0.0527 G 0.2986 1.2453 0.2627 0.5459 0.0523 H 0.0823 0.1276 0.1639 0.1047

[0054] ⑦Data analysis: By comparing the signal strengths, two clones with stronger reaction signals with CK-MB and no binding with CK-MM and CK-BB (i.e., CK-MB mAb1 clone number: 1M3A3; CK-MB mAb2 clone number: 1M3D8) were finally selected for subsequent amplification, expression and purification.

[0055] 3. Obtaining the amino acid sequences of light and heavy chains of monoclonal antibodies against CK-MB protein

[0056] (1) cDNA preparation:

[0057] RNA was extracted from the cells corresponding to the two clones determined in Example 2, and reverse transcription was performed using the RNA as a template to obtain cDNA;

[0058] (2) Acquisition of light and heavy chain genes:

[0059] Amplification was performed using specific primers, and the amplified DNA sequences were cloned into the PCDNA3.4 vector, and then transformed and plated, clones were picked, and plasmids were extracted. The plasmids were sent for sequencing to obtain the gene sequences of the two monoclonal antibodies, and the heavy and light chain amino acid sequences of the two monoclonal antibodies were deduced. The heavy chain amino acid sequences of CK-MB mAb1 and CK-MB mAb2 are shown in SEQ ID NO: 8 and SEQ ID NO: 17, respectively, and the light chain amino acid sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 18, respectively;

[0060] CK-MB mAb1:

[0061] The full length of the heavy chain is specifically: SEQ ID NO: 8;

[0062] The full length of the light chain is specifically: SEQ ID NO: 9;

[0063] CK-MB mAb2:

[0064] The full length of the heavy chain is specifically: SEQ ID NO: 17;

[0065] The full length of the light chain is specifically: SEQ ID NO: 18;

[0066] The heavy chain variable regions (FV) and light chain variable regions (FV) of the two monoclonal antibodies were then analyzed based on the sequencing results, and finally the amino acid sequences of their heavy chain variable regions and light chain variable regions were obtained, as shown below, respectively, where the underlined regions are their CDR regions.

[0067] CK-MB mAb1:

[0068] The heavy chain FV is specifically: SEQ ID NO: 6;

[0069] The light chain FV is specifically: SEQ ID NO: 7;

[0070] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 4, RAS and SEQ ID NO: 5, respectively;

[0071] CK-MB mAb2:

[0072] The heavy chain FV is specifically: SEQ ID NO: 15;

[0073] The light chain FV is specifically: SEQ ID NO: 16;

[0074] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 13, RAS and SEQ ID NO: 14, respectively;

[0075] IV. Expression and purification of monoclonal antibodies against CK-MB protein

[0076] (1) Cultivate N293 suspension cells until the density reaches (1-3)×10 6 When the cell count is 10 cells / ml and the survival rate is >80-90%, cell transfection is performed;

[0077] (2) The light and heavy chain genes of the two monoclonal antibodies CK-MB mAb1 and CK-MB mAb2 obtained in Example 3 were respectively constructed into the pcDNA3.4 plasmid; then, the recombinant plasmids containing the antibody light and heavy chain genes were transfected into N293 cells under the following transfection conditions: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2 μg plasmid / 1 ml N293 cells;

[0078] (3) 5-7 days after transfection, collect the cell supernatant and perform affinity purification using Protein A resin to obtain high-purity monoclonal antibodies. Replace the elution buffer with PBS by dialysis, and measure the antibody concentration using Nanodrop.

[0079] Next, the purity of the antibody was tested by SDS-PAGE as follows:

[0080] SDS-PAGE detection:

[0081] Take 2μg of the antibody to be tested, add an appropriate amount of SDS-PAGE protein loading buffer to make the total volume 20μl; prepare reducing SDS-PAGE samples and non-reducing SDS-PAGE samples at the same time, and then load them; first, electrophoresis at 80V for 30min, and then electrophoresis at 120V until the bands are clearly separated; remove the PAGE gel after electrophoresis, stain with Coomassie Brilliant Blue, remove the staining solution after 15min, rinse with clean water, and then add decolorizing solution for decolorization until clear bands are seen. The results are as follows Figure 1A , Figure 1BThe results showed that under reducing conditions, the two monoclonal antibodies CK-MB mAb1 and CK-MBmAb2 had two electrophoretic bands, and the corresponding molecular weights of the two electrophoretic bands were consistent with the sizes of their respective light and heavy chains; under non-reducing conditions, the monoclonal antibodies CK-MB mAb1 and CK-MB mAb2 had a single band, and the corresponding molecular weights of the electrophoretic bands were consistent with the sizes of their respective complete antibodies; these results indicated that the expression of this monoclonal antibody was accurate.

[0082] In addition, all electrophoresis bands had clear edges and no foreign bands, indicating that the monoclonal antibodies obtained by the above expression and purification steps had high purity.

[0083] 5. Antigen Binding Activity and Affinity Testing of Monoclonal Antibodies Against CK-MB Protein

[0084] In this example, the monoclonal antibodies CK-MB mAb1 and CK-MBmAb2 expressed and purified in Example 4 were tested for antigen activity and affinity, respectively.

[0085] ① Antigen coating: dilute the antigen (i.e., the aforementioned CK-MB protein) to an appropriate concentration, and then add it to an empty ELISA plate at a volume of 100 μl / well; place the ELISA plate in a 4°C refrigerator and incubate overnight;

[0086] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0087] ③ Incubate the primary antibody: dilute the antibody to be tested to a suitable concentration gradient with 1×PBS; add the diluted antibody to be tested to the ELISA plate, 100 μl per well, reaction time: 37℃ / 1h; wash the plate 5 times with a plate washer and dry;

[0088] ④ Incubate the secondary antibody: dilute the enzyme-labeled secondary antibody in an appropriate ratio, and then add it to the ELISA plate, 100 μl per well, reaction time: 37°C / 30 min; wash the plate 5 times with a plate washer and dry;

[0089] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37℃ / 15min;

[0090] ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, export the detection results into Excel table.

[0091] ⑦Data analysis: Use software to analyze and plot data, fit EC50, and derive its KD.

[0092] The test data are shown in Table 7.

[0093] Table 7: OD450 values ​​of the tested antibodies at various concentrations

[0094] Concentration (ng / ml) CK-MBmAb1 CK-MBmAb2 4000 2.2972 2.4206 1333.333 2.2758 2.3954 444.444 2.2071 2.3105 148.148 2.0874 2.1859 49.383 1.7653 1.9835 16.461 1.3186 1.5722 5.487 0.9445 0.9223 1.829 0.6006 0.6242 0.610 0.4806 0.4318 0.203 0.3867 0.357 0.068 0.3379 0.282 0 0.0691 0.0644

[0095] According to Table 7, the binding activity curve of antibody and antigen was prepared. The results are as follows Figure 2A , Figure 2B As shown, the data in Table 7 were fitted with a four-parameter curve using the "ELISAcalc" software to obtain the EC50 values ​​of the two antibodies and the KD values ​​derived therefrom. The data are shown in Table 8.

[0096] Table 8: EC50 and KD values ​​of an antibody

[0097] Antibody ID EC50 (ng / ml) KD CK-MBmAb1 13.51 <![CDATA[9*10 -11 M]]> CK-MBmAb2 10.4 <![CDATA[6.9*10 -11 M]]>

[0098] like Figure 2A , Figure 2B Shows: EC50 of monoclonal antibody CK-MB mAb binding to antigen reaches ng / ml level, KD reaches 10 -11 As mentioned above, for IgG antibodies, it is generally believed in the art that when the KD value of binding to the antigen reaches 10 -9 M is considered to have high affinity, and the KD values ​​of the two monoclonal antibodies of the present invention for binding to the antigen both reached 10 -11 M is 100 times the above-defined value, from which it can be concluded that the monoclonal antibody of the present invention has extremely strong antigen binding activity and affinity.

[0099] 6. Preparation of colloidal gold immunochromatographic test strips based on anti-CK-MB monoclonal antibodies;

[0100] In this step, the CK-MB mAb1 and CK-MB mAb2 expressed and purified in step 4 are paired to prepare a colloidal gold immunochromatographic test strip for detecting CK-MB based on the principle of double antibody sandwich method. The schematic diagram of the structure of the test strip is shown in FIG. Figure 3 As shown; the preparation method of the test strip is as follows:

[0101] (1) Preparation of Au-anti-CK-MB monoclonal antibody complex

[0102] ① Take 3 ml of colloidal gold particles (colloidal gold particle size: 20 nm, maximum absorption peak wavelength of 529 nm, absorbance of 2.0 A), add 18 μl of 0.1 M K2CO3, mix well, add 10 μg of detection antibody (CK-MB mAb1), and react at room temperature for 1 hour;

[0103] ② Add 100 μl 5% BSA blocking solution to the system in step ① and block at room temperature for 30 minutes;

[0104] ③ After blocking, centrifuge at 11000 rpm at 10°C for 15 min, discard the supernatant, and re-dissolve the precipitate with 50 μl of gold-labeled diluent (i.e., concentrated 60 times, 60×) to obtain the Au-anti-CK-MB monoclonal antibody complex; the gold-labeled diluent contains 100 mM Tris buffer at pH 7.5-8.5, 5% sucrose, 1% trehalose, and 5% BSA.

[0105] (2) Deposit

[0106] ① Draw a closed line: Take 5% BSA solution, set the water output of the instrument to 3μl / cm, and place the drawn bottom plate in a 37℃ oven to dry for 2 hours;

[0107] ② Dilute the Au-anti-CK-MB monoclonal antibody complex to a final concentration of 50×, set the water output to 2 μl / cm, draw a line at the closed line, and then place the bottom plate in a 37°C oven to dry for 2 hours. Then, place the marking pad in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for storage;

[0108] (3) Coating quality control line C, detection line T

[0109] ① Preparation of coating solution for quality control line C and test line T: Take goat anti-rabbit antibody and dilute it with 10mM PBS buffer at pH 7.4 to make an antibody solution of 1.0mg / ml; Take capture antibody (CK-MB mAb2) and dilute it with 10mM PBS buffer at pH 7.4 to make an antibody solution of 1.0mg / ml;

[0110] ② Coating: Coat the control line C and test line T coating solutions on the control line and test line of the nitrocellulose membrane, respectively. The interval between the C and T lines is 8 mm, and the coating amount is 1 μl / cm2. Place the coated PVC substrate in a 37°C oven for 16 hours, and then place the PVC substrate in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for future use.

[0111] (4) Pretreatment of sample pad

[0112] Clean the drying net, soak the sample pad with the sample pad pretreatment solution, and then drain it by rotating until no water drops. Then, dry it in a 37°C oven for 4 hours, and then seal it in an aluminum foil bag with desiccant added in advance at a humidity not higher than 36% for storage.

[0113] The sample pad pretreatment solution contains 10 mM Tris buffer at pH 8.5, 0.1% PVP10, 0.1% PEG20000, 0.1% Tween20 and 0.5% sodium caseinate;

[0114] (5) Assembly and cutting of membrane materials

[0115] At a humidity not higher than 36%, take out the absorbent pad and the treated sample pad, cut them into a size of 10mm×300mm, take out the coated PVC base plate, and assemble them according to the following process: first stick the sample pad on the PVC base plate so that the sample pad part presses the nitrocellulose membrane for 2mm, and finally stick the absorbent pad so that the absorbent pad presses the nitrocellulose membrane for 2mm; cut the assembled PVC base plate into 4mm, which is the colloidal gold immunochromatographic test strip for detecting CK-MB.

[0116] VII: Performance test of colloidal gold immunochromatographic test strips based on anti-CK-MB monoclonal antibodies

[0117] In this step, the colloidal gold immunochromatographic test strip prepared in step 6 was used to detect CK-MB and interferences such as CK-MM and CK-BB in clinical serum samples, and the clinical samples were assigned samples for Roche detection reagents.

[0118] (1) Testing clinical serum samples

[0119] Specifically, for the colloidal gold immunochromatographic test strip prepared in step six, 75 μl of clinical serum sample was directly aspirated and added, and tested with a colloidal gold immunoassay analyzer after 15 minutes, the detection signal value was read, and the data was processed. The signal value detected by the test strip was compared with the concentration assigned by the Roche reagent to produce a clinical correlation curve.

[0120] The specific results are as follows:

[0121] Table 9 shows the test results of the colloidal gold immunochromatographic test strips, in which the first column represents the concentration of clinical samples detected by Roche reagent (unit: ng / ml), and the second column represents the signal value of the colloidal gold immunochromatographic test strips of the present invention for the corresponding clinical samples;

[0122] Table 9

[0123] Concentration (ng / ml) T / C 0.8 0.48 1.2 0.67 2.8 0.87 4.6 1.07 6.2 1.58 9.5 2.01 12.7 2.14 18.3 2.54 22.3 3.11 27.6 3.31 36.5 4.37 40.7 4.26 44.8 4.71 52.6 5.13 59.3 5.61 66.3 5.21 67.2 5.96 81.6 6.82 84.6 6.57 89.6 7.29 92.1 8.61 97.8 8.04 111.5 9.36 127.6 9.31 135.8 9.98 175.4 13.28 180.3 13.64 192.5 15.27

[0124] According to the test results in Table 9, the clinical relevance of colloidal gold immunochromatographic test strips for detecting CK-MB in serum samples was prepared, that is, the coincidence rate curve between the test results of colloidal gold immunochromatographic test strips and the test results of Roche was shown in the following figure. Figure 4 As shown; the results show that the clinical correlation R2 of the colloidal gold immunochromatographic test strip of the present invention for detecting CK-MB in serum samples is greater than 0.98.

[0125] The above results show that the CK-MB colloidal gold immunochromatographic test strip prepared based on the antibody of the present invention has a high coincidence rate with the Roche test result, with R2 being above 0.98, and therefore has a good clinical relevance.

[0126] (2) Testing for CK-MM and CK-BB interferents;

[0127] Specifically, CK-MB antigen was diluted with PBS buffer to 6 concentration gradients of 200, 50, 20, 5, 1, and 0 ng / ml, 75 μl was added to the colloidal gold immunochromatographic test strip, and the colloidal gold immunoassay was used for detection after 15 minutes, and the detection value was read, and the data was processed to obtain the standard curve for CK-MB detection; then CK-MM and CK-BB antigens were diluted with PBS buffer to two concentrations of 10000 and 5000 ng / ml, respectively, and 75 μl was added to the colloidal gold immunochromatographic test strip, and the colloidal gold immunoassay was used for detection after 15 minutes, and the detection value was read, and the detected T / C value was brought into the curve to back-calculate the concentration value of CK-MB, and then the corresponding cross-reaction rate was obtained, and the results are shown in Table 10. The results show that the cross-reactivity of the colloidal gold immunochromatographic test strip prepared based on the CK-MB monoclonal antibody developed by the present invention for CK-MM and CK-BB is less than about 0.01%, indicating no cross-reaction.

[0128] Table 10

[0129]

[0130] The above embodiments are only used to illustrate the technical method of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical method of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical method of the present invention.

Claims

1. A monoclonal antibody against CK-MB, characterized in that: include: Light chain variable region amino acid sequence: SEQ ID NO: 7; Heavy chain variable region amino acid sequence: SEQ ID NO: 6; Among them, the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 4, RAS and SEQ ID NO: 5, respectively; the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. A colloidal gold immunochromatographic test strip of monoclonal antibodies against CK-MB, characterized in that: The invention comprises the monoclonal antibody against CK-MB as claimed in claim 1.

3. A method for preparing a colloidal gold immunochromatographic test strip of an anti-CK-MB monoclonal antibody as claimed in claim 2, characterized in that: The method specifically comprises the following steps: (1) Preparation of Au-anti-CK-MB monoclonal antibody complex; ① Take 3 ml of colloidal gold particles, add 18 μl of 0.1 M K2CO3, mix well, add 10 μg of the monoclonal antibody of CK-MB as described in claim 1, and react at room temperature for 1 hour; ② Add 100 μl 5% BSA blocking solution to the system in step ① and block for 30 min at room temperature; ③ After the blocking is completed, centrifuge at 11000 rpm at 10°C for 15 min, discard the supernatant, and re-dissolve the precipitate with 50 μl of gold-labeled diluent, i.e., concentrate 60 times to obtain the Au-anti-CK-MB monoclonal antibody complex; the gold-labeled diluent contains 100 mM Tris buffer at pH 7.5-8.5, 5% sucrose, 1% trehalose, and 5% BSA; (2) gold; ① Draw a closed line: Take 5% BSA solution, set the instrument water output to 3μl / cm, and place the drawn bottom plate in a 37℃ oven to dry for 2 hours; ② Dilute the Au-anti-CK-MB monoclonal antibody complex to a final concentration of 50×, set the water output to 2 μl / cm, draw a line at the closed line, and then place the bottom plate in a 37°C oven to dry for 2 hours, and then place the marking pad in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for storage; (3) Coating quality control line C, detection line T; ① Preparation of coating solution for quality control line C and test line T: dilute goat anti-rabbit antibody with 10mM PBS buffer at pH 7.4 to 1.0mg / ml antibody solution; dilute CK-MB monoclonal antibody with 10mM PBS buffer at pH 7.4 to 1.0mg / ml antibody solution; ② Coating: Coat the control line C and the detection line T coating solution to the control line and the detection line of the nitrocellulose membrane respectively, with the interval between the C and T lines being 8mm, and the coating amount being 1μl / cm2; Place the coated PVC substrate in a 37°C oven for 16 hours, and then place the PVC substrate in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for future use; (4) Pretreatment of sample pad; Clean the drying net, soak the sample pad with the sample pad pretreatment solution, and then drain it until no water drops. Then, dry it in a 37°C oven for 4 hours, and then seal it in an aluminum foil bag with desiccant added in advance at a humidity not higher than 36% for storage; The sample pad pretreatment solution contains 10 mM Tris buffer at pH 8.5, 0.1% PVP10, 0.1% PEG20000, 0.1% Tween20 and 0.5% sodium caseinate; (5) Assembly and cutting of membrane materials; At a humidity not higher than 36%, take out the absorbent pad and the treated sample pad, cut them into a size of 10 mm × 300 mm, take out the coated PVC base plate, and assemble them according to the following process: first stick the sample pad on the PVC base plate so that the sample pad part presses the nitrocellulose membrane for 2 mm, and finally stick the absorbent pad so that the absorbent pad presses the nitrocellulose membrane for 2 mm; cut the assembled PVC base plate into 4 mm, which is the colloidal gold immunochromatographic test strip for detecting CK-MB.

4. Colloidal gold immunochromatographic test paper of the monoclonal antibody against CK-MB according to claim 3 The method for preparing a strip is characterized in that Colloidal gold particle size: 20nm, maximum absorption peak wavelength is 529nm, The absorbance is 2.0.

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