ELISA kit for detecting CA125 and its application

Abstract

The present application provides an ELISA kit for detecting CA125 and its application, which includes monoclonal antibody 125E7, whose heavy chain CDR1 sequence is SEQ ID NO.3; CDR2 sequence is SEQ ID NO.4; CDR3 sequence is SEQ ID NO.5; light chain CDR1 sequence is SEQ ID NO.7; CDR2 sequence is SEQ ID NO.8; CDR3 sequence is SEQ ID NO.9 and monoclonal antibody 125G1; whose heavy chain CDR1 sequence is SEQ ID NO.11; CDR2 sequence is SEQ ID NO.12; CDR3 sequence is SEQ ID NO.13; light chain CDR1 sequence is SEQ ID NO.15; CDR2 sequence is SEQ ID NO.16; CDR3 sequence is SEQ ID NO.17.

Description

Field of the Invention

[0001] The present application belongs to the field of antibodies in the field of proteins. Specifically, the present application provides an ELISA kit for detecting CA125 and its application. Background Art

[0002] Currently, CA125 / MUC16 has been widely used as a marker in clinical practice and physical examinations to diagnose or assist in the diagnosis of various cancers and inflammatory diseases. The most commonly used method for serum CA125 detection is the ELISA kit, which consists of a solid phase carrier coated with an antigen or antibody, an enzyme-labeled antibody, a substrate, a stop solution, a control, and other components.

[0003] CA125 is also expressed at a slightly higher level in some normal people, and often changes in some gynecological diseases and in the elderly female population. Therefore, CA125, as a disease marker, especially when used alone as a disease marker, is not very specific, and the sensitivity and specificity of its detection kit need to be continuously improved. Summary of the invention

[0004] The performance of an ELISA test kit depends largely on the performance of the antibody. Therefore, in view of the above situation, the applicant of the present application has developed a new CA125 detection antibody and provided a test kit based on the antibody.

[0005] In one aspect, the present application provides an ELISA kit for detecting CA125, the kit comprising CA125 monoclonal antibody 125E7 and CA125 monoclonal antibody 125G1;

[0006] The heavy chain CDR1 sequence of the CA125 monoclonal antibody 125E7 is SEQ ID NO.3; the heavy chain CDR2 sequence is SEQ ID NO.4; the heavy chain CDR3 sequence is SEQ ID NO.5; the light chain CDR1 sequence is SEQ ID NO.7; the light chain CDR2 sequence is SEQ ID NO.8; and the light chain CDR3 sequence is SEQ ID NO.9;

[0007] The heavy chain CDR1 sequence of the CA125 monoclonal antibody 125G1 is SEQ ID NO.11; the heavy chain CDR2 sequence is SEQ ID NO.12; the heavy chain CDR3 sequence is SEQ ID NO.13; the light chain CDR1 sequence is SEQ ID NO.15; the light chain CDR2 sequence is SEQ ID NO.16; and the light chain CDR3 sequence is SEQ ID NO.17.

[0008] Furthermore, the CA125 monoclonal antibody 125E7 in the kit is coated on a solid phase carrier.

[0009] Furthermore, the CA125 monoclonal antibody 125G1 is connected with an enzyme label.

[0010] Furthermore, the enzyme marker is horseradish peroxidase.

[0011] Furthermore, the kit further comprises one or more components selected from the group consisting of a substrate solution, a negative control, a positive control, a diluent, a washing solution, and a stop solution.

[0012] Furthermore, the heavy chain variable region sequence of the CA125 monoclonal antibody 125E7 is SEQ ID NO.2; the light chain variable region sequence is SEQ ID NO.6

[0013] Furthermore, the heavy chain variable region sequence of the CA125 monoclonal antibody 125G1 is SEQ ID NO.10; and the light chain variable region sequence is SEQ ID NO.14.

[0014] On the other hand, the present application provides the use of the above-mentioned kit in preparing a reagent for diagnosing a tumor.

[0015] Furthermore, the reagent is used to detect the CA125 protein level in serum.

[0016] Based on the known ELISA detection principle (such as the double antibody sandwich method), those skilled in the art can routinely select enzymes (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, etc.) and select suitable substrates (such as TMB, hydrogen peroxide, etc.). Those skilled in the art can routinely adjust the parameters such as the dosage and concentration of each component in the kit to obtain the desired detection effect.

[0017] The kit of the present application uses the monoclonal antibody screened and developed by the applicant as the coating and labeling antibody, and has good sensitivity and specificity. The actual test results show that its effect is better than that of imported kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the detection principle of the kit of this application.

[0019] Figure 2 This is a diagram of the expression and purification results of CA125 protein; WL: whole lysate; out: ultrafiltration liquid; FT: flow-through; W1-W3: Wash1-wash3; E1-E3: Elution (MACS buffer + 100mM imidazole), 3 times; +: known CA125-HIS protein.

[0020] Figure 3The results of ELISA screening of fusion cells: immune mouse spleen cells and myeloma cells (SP2 / 0) were fused and plated (a total of 6 plates), 125G1 and 125E7 were both on this plate (indicated by arrows). According to the results of ELISA and Western blot, the wells with good signals were selected for subcloning.

[0021] Figure 4 This is the result of ELISA screening of 125E7 subclones; the two rows of data for each subclone are 450nm and 595nm respectively.

[0022] Figure 5 This is a diagram of the Western blot validation results of 125E7 subclone; where -: colon cancer cell (SW480) lysate (125 negative); +: SW480 lysate + 125 immune protein.

[0023] Figure 6 This is the result of ELISA screening of 125G1 subclones; the two rows of data for each subclone are 450nm and 595nm respectively.

[0024] Figure 7 This is a diagram of the Western blot verification results of the 125G1 subclone; where -: colon cancer cell (SW480) lysate (125 negative); +: SW480 lysate + 125 immune protein.

[0025] Figure 8 This is a sensitivity comparison chart of the antibody composition kit of this application and the international leading brand detection kit. DETAILED DESCRIPTION

[0026] Example 1 Expression and purification of antigens

[0027] The antigen shown in SEQ ID NO.1 was cloned into the prokaryotic expression vector pET28, and after sequencing to verify that the sequence was completely correct, a small-scale expression test (Western Blot method, anti-HIS antibody, Thermo) was performed to confirm that the expression vector expressed the target protein. Subsequently, the expression bacteria were cultured with 1 liter of LB medium until the bacterial optical density OD600 was about 0.6-08, 1mMIPTG was added to induce the expression of the target protein overnight (12-16 hours), and the bacteria were collected by centrifugation at 5000rpm, and the bacterial precipitate was washed once with 200mlPBS, and 100ml of MACS buffer (300mM NaCl, 50mM NaH2PO4) was added to resuspend the bacterial block, and the bacterial body was lysed by ultrasonication, and the lysate was obtained by high-speed centrifugation (12000RPM, 30min).

[0028] The CA125 protein in the supernatant has a 6XHIS tag, and Ni-NTS beads are used to purify the CA125 protein as an immunogen. The protein purification method is implemented according to the experimental steps of Ni-NTA beads (Qiagen): 1ml beads slurry is added to 50ml lysis supernatant, rotated on a rotary mixer for 2 hours to allow the target protein to bind to the beads, and washed with 40ml MACS buffer + 5mM imidazole (5 times), added to the chromatography column, and then 10ml of 10mL Elution Buffer (MACS Buffer + 100mM imidazole) eluted the target protein 3 times ( Figure 2 ), the purified CA125 protein will be used as an immunogen for immunizing mice and as an antigen for screening hybridomas (ELISA and Western blot).

[0029] SEQ ID NO.1 (HIS tag)

[0030] ATGGCTGCTGGCCCTCTCCTGGTACCATTCACCCTCAACTTCACCATCACCAACCTGCAGTATGGGGAG

[0031] GACATGGGTCACCCTGGCTCCAGGAAGTTCAACACCACAGAGAGGGTCCTGCAGGGTCTGCTTGGTCCC

[0032] ATATTCAAGAACACCAGTGTTGGCCCTCTGTACTCTGGCTGCAGACTGACCTCTCTCAGGTCTGAGAAG

[0033] GATGGAGCAGCCACTGGAGTGGATGCCATCTGCATCCATCATCTTGACCCCAAAAGCCCTGGACTCAAC

[0034] AGAGAGCGGCTGTACTGGGAGCTGAGCCAACTGACCAATGGCATCAAAGAGCTGGGCCCCTACACCCTG

[0035] GACAGGAACAGTCTCTATGTCAATGGTTTCACCCATCGGACCTCTGTGCCCACCAGCAGCACTCCTGGG

[0036] ACCTCCACAGTGGACCTTGGAACCTCAGGGACTCCATTCTCCCTCCCAAGCCCCGCAACTGCTGGCTCC

[0037] TGGTGCTGTTCACCCTCAACTTCACCATCACCAACCTGAAGTATGAGGAGGACATGCATCGCCCTGGCT

[0038] CCAGGAAGTTCAACACCACTGAGAGGGTCCTGCAGACTCTGCTTGGTCCTATGTTCAAGAACACCAGTG

[0039] TTGGCCTTCTGTACTCTGGCTGCAGACTGACCTTGCTCAGGTCCGAGAAGGATGGAGCAGCCACTGGAG

[0040] TGGATGCCATCTGCACCCACCGTCTTGACCCCAAAAGCCCTGGAGTGGACAGGGAGCAGCTATACTGGG

[0041] AGCTGAGCCAGCTGACCAATGGCATCAAAGAGCTGGGCCCCTACACCCTGGACAGGAACAGTCTCTATG

[0042] TCAATGGTTTCACCCATTGGATCCCTGTGCCCACCAGCAGCACTCCTGGGACCTCCACAGTGGACCTTG

[0043] GGTCAGGGACTCCATCCTCCCTCCCCAGCCCCACAACTGCTGGCCCTCTCCTGGTGCCGTTCACCCTCA

[0044] ACTTCACCATCACCAACCTGAAGTACGAGGAGGACATGCATTGCCCTGGCTCCAGGAAGTTCAACACCA

[0045] CAGAGAGAGTCCTGCAGAGTCTGCTTGGTCCCATGTTCAAGAACACCAGTGTTGGCCCTCTGTACTCTG

[0046] GCTGCAGACTGACCTTGCTCAGGTCCGAGAAGGATGGAGCAGCCACTGGAGTGGATGCCATCTGCACCC

[0047] ACCGTCTTGACCCCAAAAGCCCTGGAGTGGACAGGGAGCAGCTATACTGGGAGCTGAGCCAGCTGACCA

[0048] ATGGCATCAAAGAGCTGGGTCCCTACACCCTGGACAGAAACAGTCTCTATGTCAATGGTTTCACCCATC

[0049] AGACCTCTGCGCCCAACACCAGCACTCCTGGGACCTCCACAGTGGACCTTGGGACCTCAGGGACTCCAT

[0050] CCTCCCTCCCCAGCCCTACATCTGCTGGCCCTCTCCTGGTGCCATTCACCCTCAACTTCACCATCACCA

[0051] ACCTGCAGTACGAGGAGGACATGCATCACCCAGGCTCCAGGAAGTTCAACACCACGGAGCGGGTCCTGC

[0052] AGGGTCTGCTTGGTCCCATGTTCAAGAACACCAGTGTCGGCCTTCTGTACTCTGGCTGCAGACTGACCT

[0053] TGCTCAGGCCTGAGAAGAATGGGGCAGCCACTGGAATGGATGCCATCTGCAGCCACCGTCTTGACCCCA

[0054] AAAGCCCTGGACTCAACAGAGAGCAGCTGTACTGGGAGCTGAGCCAGCTGACCCATGGCATCAAAGAGC

[0055] TGGGCCCCTACACCCTGGACAGGAACAGTCTCTATGTCAATGGTTTCACCCATCGGAGCTCTGTGGCCC

[0056] CCACCAGCACTCCTGGGACCTCCACAGTGGACCTTGGGACCTCAGGGACTCCATCCTCCCTCCCCAGCC

[0057] CCACAACAGCTGTTCCTCTCCTGGTGCCGTTCACCCTCAACTTTACCATCACCAATCTGCAGTATGGGG

[0058] AGGACATGCGTCACCCTGGCTCCAGGAAGTTCAACACCACAGAGAGGGTCCTGCAGGGTCTGCTTGGTC

[0059] CCTTGTTCAAGAACTCCAGTGTCGGCCCTCTGTACTCTGGCTGCAGACTGATCTCTCTCAGGTCTGAGA

[0060] AGGATGGGGCAGCCACTGGAGTGGATGCCATCTGACCCACCACCTTAACCCTCAAAGCCCTGGACTGG

[0061] ACAGGGAGCAGCTGTACTGGCAGCTGAGCCAGATGACCAATGGCATCAAAGAGCTGGGCCCCTACACCC

[0062] TGGACCGGAACAGTCTCTACGTCAATGGTTTCACCCATCGGAGCTCTGGGCTCACCACCAGCACTCCTT

[0063] GGACTTCCACAGTTGACCTTGGAACCTCAGGGACTCCATCCCCCGTCCCCAGCCCCACA CATCATCAC CATCACCAT TAA.

[0064] Example 2 Mouse immunization

[0065] 15ug / mouse immunized 4 Balb / c mice aged 8-12 weeks, the first immunization was emulsified with Freund's complete adjuvant (antigen: adjuvant volume ratio was 1:1), and then immunized again every 1 month (emulsified with Freund's incomplete adjuvant, antigen: adjuvant ratio was 1:1). After completing 3 immunizations, the antibody titer of the mice was tested (ELISA method, the coated antigen was immune protein), and the antibody titer was >1:100,000. The results are shown in Table 1:

[0066] Table 1 Mouse serum titer results

[0067]

[0068] The titers of 3 mice were higher than 3 million dilutions; HIS: control anti-His monoclonal antibody.

[0069] Example 3 Screening and identification of monoclonal antibodies

[0070] Mouse spleen cells were fused with SP2 / 0 myeloma cell line (PEG method), and the fused cells were plated on 6 96-well plates and cultured with DMEM+20% FBS+HAT medium. After about 8-10 days, about 30-50 clones were observed to grow in each well. At this time, 100ul of culture supernatant from each well was taken for indirect ELISA screening (coating antigen, detecting whether there is antibody in the culture supernatant, the result is as follows Figure 3 The positive wells are then checked by Western Blot to see if the antibody recognizes the target antigen and whether the antibody has excellent specificity and sensitivity. After that, the wells with good specificity and sensitivity are selected for subcloning until a single clone of 1 clone / well is obtained.

[0071] The results of subclone screening of 125E7 are as follows Figure 4 and Figure 5 As shown: A total of 5 wells of supernatant (100ul) were verified, and the specificity (the results of SW480 cell lysates with / without CA125 added were significantly different) and sensitivity were relatively ideal. Subclone H9 was a monoclonal clone and was selected for antibody sequencing, antibody production and subsequent research

[0072] The subclone screening results of 125G1 are as follows Figure 6 and Figure 7 As shown: A total of 5 wells of supernatant (100ul) were verified, with ideal specificity and sensitivity. Subclone E6 was a monoclonal clone and was selected for antibody sequencing, antibody production and subsequent research.

[0073] 125E7 and 125G1 hybridoma cells were cultured to 1X10 7 Cells were collected and centrifuged to remove the culture supernatant. 1 ml of Trizol reagent (Thermo) was added and sent to General Biotechnology (Anhui) Co., Ltd. (Chuzhou, Anhui) on dry ice for V region gene amplification and sequencing. The CDR sequence was analyzed using the website: https: / / www.imgt.org / IMGT_vquest / input:

[0074] The sequencing results of 125E7 (heavy chain: Ig2a, light chain: κ) are as follows:

[0075] Heavy chain variable region, SEQ ID NO.2 (underlined CDR region): EVKLVESGGGLANPGGSLKLSCAAS G FTF SRYT MSWLRQNPEKRLEWVAT ISVGGIYAYYPDSVKG RFTISRDNAQNTLYLQMSSLKSEDTAMYYC ARDGTGDYVSFDY WGQGTTLTVSS

[0076] CDR1, SEQ ID NO.3: GFTFSRYT

[0077] CDR2, SEQ ID NO.4: ISVGGIYAYYPDSVKG

[0078] CDR3, SEQ ID NO.5: ARDGTGDYVSFDY

[0079] Light chain variable region, SEQ ID NO.6 (underlined CDR region): DIVMTQSPLTLSVTIGQPASISCKSS QSL LDSDG LSWLLQRPGQSPKRLIY LVSKLES GVPDRFTGSGSGTDFTLRISRVEAEDLGVYYC WQ GTHFPWT FGGGTKLEIK

[0080] CDR1, SEQ ID NO.7: QSLLDSDGKTY

[0081] CDR2, SEQ ID NO.8: LVSKLES

[0082] CDR3, SEQ ID NO.9: WQGTHFPWT

[0083] The sequencing results of 125G1 (heavy chain: IgG1, light chain: κ) are as follows:

[0084] Heavy chain variable region, SEQ ID NO.10 (underlined CDR region): QVQLKESGAELVRPGSSVKISCKAS GY FSAYW MNWVKQRPGQGLEWIG QIYPGDGDTNYNGKFKG KGTLTADKSSSTAYMQLSSLTSEDSAVYFC ARGHYGSLDN WGQGTTLTVSS

[0085] CDR1, SEQ ID NO.11: GYAFSAYW

[0086] CDR2, SEQ ID NO.12: QIYPGDGDTNYNGKFKG

[0087] CDR3, SEQ ID NO.13: ARGHYGSLDN

[0088] Light chain variable region, SEQ ID NO.14 (underlined CDR region): QAVVTQESALTTSPGETVTLTCRSS TGA VTTNNF ANWVQEKPDHLFTGLIG GTNNRAP GVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWY SNH FGGGTKLTVL

[0089] CDR1, SEQ ID NO.15: TGAVTTNNF

[0090] CDR2, SEQ ID NO. 16: GTNNRAPCDR3, SEQ ID NO. 17: ALWYSNHLV.

[0091] Monoclonal Antibody Affinity Determination:

[0092] Biomembrane optical interference technology (ForteBio Octet) was used to determine the binding constant and dissociation constant of the antigen-antibody interaction, and the antibody affinity was determined. The affinity K of the monoclonal antibody of the present invention was calculated based on the binding and dissociation curves of the BXMD12 antibody provided by the present invention in combination with CA125-His protein at different dilution concentration gradients (500nM-31nM). D value:

[0093] 125E7: 6.9×10 -10 (R 2 =0.988);

[0094] 125G1: 5.5×10 -10 (R2=0.965).

[0095] Actual detection effect of the test kit of Example 4

[0096] The antibodies 125E7 and 125G1 obtained in Example 3 were used to prepare a double antibody sandwich kit. 125E7 was used as the coating antibody and 125G1 was used as the labeled antibody (HRP labeled) to form a CA125 tumor marker ELISA detection kit (the basic principle is as follows Figure 1As shown): 300ng of 125E7 was added to each well of a 96-well ELISA plate in 100ul carbonate coating buffer (15mM Na2CO3, 35mM NaHCO3, PH9.5), incubated overnight, poured out and patted dry the next day, added 300ul blocking solution (PBST+3% BSA) for more than 2 hours, poured out the blocking solution, added the sample to be tested and incubated for 2 hours, washed 3 times (PBST), added 100ul HRP-labeled 125G1 labeled antibody, incubated for 1 hour, washed 5 times with PBST, patted dry on absorbent paper each time, added 100ul TMB substrate, and color developed in the dark for 20 minutes. At this time, the positive signal is blue, and 200ul 1N HCL is added to terminate. At this time, the optical density (OD) can be read at a wavelength of 450nm, and the non-specific absorbance of the ELISA plate can be read at a wavelength of 595nm.

[0097] The detection sensitivity is higher than that of the international leading brand kits that have obtained the Chinese approval number (see Figure 8 ). The left side is the kit of this application, and the right side is the kit of an international brand (with a batch number in my country). Part A: After ELISA is added with TMB substrate and incubated for 20 minutes, the color is developed. The signal of the kit of this application is significantly stronger than that of the kit that has obtained a batch number in China; Part B: After adding 100ul of 1N HCL and stopping, the 450OD value is read. The kit of this application detects 3.25ul of the sample.

[0098] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An ELISA kit for detecting CA125, characterized in that: The kit includes monoclonal antibody 125E7 and monoclonal antibody 125G1; The heavy chain CDR1 sequence of the monoclonal antibody 125E7 is SEQ ID NO.3; the heavy chain CDR2 sequence is SEQ ID NO.4; the heavy chain CDR3 sequence is SEQ ID NO.5; the light chain CDR1 sequence is SEQ ID NO.7; the light chain CDR2 sequence is SEQ ID NO.8; and the light chain CDR3 sequence is SEQ ID NO.9; The heavy chain CDR1 sequence of the monoclonal antibody 125G1 is SEQ ID NO.11; the heavy chain CDR2 sequence is SEQ ID NO.12; the heavy chain CDR3 sequence is SEQ ID NO.13; the light chain CDR1 sequence is SEQ ID NO.15; the light chain CDR2 sequence is SEQ ID NO.16; and the light chain CDR3 sequence is SEQ ID NO.

17. 2 . The kit according to claim 1 , wherein the monoclonal antibody 125E7 in the kit is coated on a solid phase carrier. 3 . The kit according to claim 1 , wherein the monoclonal antibody 125G1 in the kit is linked to an enzyme label. The kit according to claim 3 , wherein the enzyme label is horseradish peroxidase.

5. The kit according to claim 1, further comprising one or more components selected from the group consisting of a substrate solution, a negative control, a positive control, a diluent, a washing solution, and a stop solution. 6 . The kit according to claim 1 , wherein the heavy chain variable region sequence of the monoclonal antibody 125E7 is SEQ ID NO. 2; and the light chain variable region sequence is SEQ ID NO.

6.

7. The kit according to claim 1, wherein the heavy chain variable region sequence of the monoclonal antibody 125G1 is SEQ ID NO.10; and the light chain variable region sequence is SEQ ID NO.

14.

8. Use of the kit according to any one of claims 1 to 7 in preparing a reagent for diagnosing a tumor.

9. The use according to claim 8, wherein the reagent is used to detect the CA125 protein level in serum.

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