CN115819598A

CN115819598A - Antibody for detecting carbohydrate antigen CA242 and application thereof

Info

Publication number: CN115819598A
Application number: CN202211497275.1A
Authority: CN
Inventors: 李伟; 苏晶; 张敏; 王玉婷
Original assignee: Nanjing Pertai Biotechnology Co ltd
Current assignee: Nanjing Pertai Biotechnology Co ltd
Priority date: 2022-11-28
Filing date: 2022-11-28
Publication date: 2023-03-21

Abstract

The invention discloses an antibody for detecting carbohydrate antigen CA242 and application thereof, wherein the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2; the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO.3, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 4. The variable region sequences of CDR1, CDR2 and CDR3 of the heavy chain are shown in SEQ ID No. 5-7, and the variable region sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 8-10. The application of the antibody in preparing a reagent or a kit for detecting carbohydrate antigen CA 242. The antibody of the invention has the characteristics of high affinity, good specificity and high titer.

Description

Antibody for detecting carbohydrate antigen CA242 and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to an antibody for detecting carbohydrate antigen CA242 and application thereof.

Background

Carbohydrate antigen 242 (CA 242) is found in human rectal cancer cells, is immunized by a human colorectal cancer cell line Cold205, and is a tumor associated antigen recognized by a monoclonal antibody C242 obtained by a hybridoma technology, has a sialylated carbohydrate structure, is a mucin, can recognize determinants of CA50 and CAl9-9, and is a marker with high specificity. It is present in normal pancreatic and colorectal mucosa, and has low expression level and very low content in normal state. In the body of a malignant tumor patient, the expression of the antigen decision cluster has higher specificity. The CA242 in the serum has higher level than squamous carcinoma in non-squamous tissue malignant tumor, and can be used for auxiliary diagnosis and treatment monitoring of patients with pancreatic cancer and colorectal cancer.

The purpose of carbohydrate antigen (CA 242) detection mainly has several important roles: normally, the carbohydrate antigen 242 has a normal value of less than 20U/mL, and if the carbohydrate antigen 242 is higher than the normal value, the carbohydrate antigen 242 can be used for diagnosing whether a tumor exists in a patient or not, and also can be used for differential diagnosis between tumor diseases or judging the severity and prognosis of the diseases. 1. And (3) diagnosing tumors: carbohydrate antigen 242 is a mucin-associated antigen, and can be used to indicate whether disease conditions of pancreatic cancer, rectal cancer, colon cancer, lung cancer and the like exist in patients, and the index can also be used for differential diagnosis, for example, the serum carbohydrate antigen 242 level of a lung squamous cell carcinoma patient is often lower than that of a lung adenocarcinoma patient and a lung large cell lung cancer patient. 2. Judging the severity: generally, the degree of increase of carbohydrate antigen 242 in serum is positively correlated with the stage of the tumor, i.e., the higher the index, the later the stage. In addition, when cancer is metastasized at a distance, the index is significantly elevated, and the more severe and extensive the disease may be. 3. And (3) judging prognosis: the carbohydrate antigen 242 has different sensibility for different patients, the index has low diagnosis value for the non-small cell lung cancer, but has certain significance for the curative effect after the non-small cell lung cancer treatment and the prognosis of the life cycle of the patients. In addition, carbohydrate antigen 242 has a higher sensitivity to diseases which are already obviously increased before the operation, and after the surgical excision treatment, if the indexes are found to be increased, the possibility of relapse and metastasis is suggested. CA242 has higher specificity in early diagnosis of pancreatic cancer and differential diagnosis of pancreatic cancer and benign pancreatic disease than CA 19-9.

Disclosure of Invention

The invention provides an antibody for detecting carbohydrate antigen CA242, which has the characteristics of high affinity, good specificity and high titer, the coating titer is 2 mug/ml, and the titer of a labeled antibody is 1/4W.

The technical scheme of the invention is as follows:

an antibody for detecting carbohydrate antigen CA242, wherein the amino acid sequence of a heavy chain of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain of the antibody is shown as SEQ ID NO. 2.

The amino acid sequence of the variable region of the heavy chain of the antibody is shown as SEQ ID NO.3, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 4.

The CDR1, CDR2 and CDR3 variable region sequences of the antibody heavy chain are shown in SEQ ID NO. 5-7, and the CDR1, CDR2 and CDR3 variable region sequences of the antibody light chain are shown in SEQ ID NO. 8-10.

A nucleic acid molecule encoding the carbohydrate antigen CA242 detection antibody.

The nucleic acid molecules for coding the variable regions CDR1, CDR2 and CDR3 of the heavy chain have the nucleotide sequences shown in SEQ ID N0.11-13, and the nucleic acid molecules for coding the variable regions CDR1, CDR2 and CDR3 of the light chain have the nucleotide sequences shown in SEQ ID N0.14-16.

Biological material containing said nucleic acid molecule, said biological material being an expression cassette, a vector or a host cell.

The expression cassette is a recombinant nucleic acid molecule obtained by linking the upstream or downstream of the nucleic acid molecule to regulatory elements for transcription and translation.

The vector is a nucleic acid carrier into which a nucleic acid molecule can be inserted, and includes, but is not limited to, plasmids, artificial chromosomes, phages, animal viruses, and the like, and may be an expression vector, or a cloning vector or a non-replicating vector.

The host cell may be a microbial cell (e.g., escherichia coli, yeast cell, etc.) or an animal cell (e.g., insect cell, CHO cell, BHK cell, HEK293 cell, etc. for expression and production of an antibody), wherein the animal cell is a cell that cannot be propagated into an animal individual.

An antibody conjugate, which is obtained by coupling the carbohydrate antigen CA242 detection antibody with a carrier or a drug, or is obtained by coupling the antibody with a chemical or biological marker.

A detection reagent comprising said antibody or said antibody conjugate.

A pharmaceutical composition comprising said antibody or said antibody conjugate.

The antibody or the nucleic acid molecule or the biological material is used for preparing a reagent or a kit for detecting carbohydrate antigen CA 242.

Has the advantages that: the antibody of the invention has high affinity, good specificity, wide detection range and high sensitivity, the self-detection range is 1-150U/mL, and the normal value is as follows: the titer is high, the coating titer is 2 mu g/mL, and the labeled antibody titer is 1/4W.

Drawings

FIG. 1: CA242 clinical relevance test chart.

Detailed Description

EXAMPLE 1 obtaining of antibodies

Antigen immunization process

Antigen preparation process: taking carbohydrate antigen CA242, wherein CA242 is a surface marker of colo-205 human colon cancer cells, can directly take colo-205 cells as immunogen, the cell surface carries CA242, the antigen is diluted to 1mg/mL, and 50 muL of antigen solution and 50 muL of cytokine adjuvant interleukin-2 are taken for primary immunization; the subsequent immunization took 50. Mu.L of antigen solution, 50. Mu.L of cytokine adjuvant. Two 2mL syringes are used for respectively sucking the antigen solution and the adjuvant, air in the syringes is discharged as much as possible and linked by a tee joint, the antigen is pushed into the adjuvant, and the connected syringes are repeatedly pushed for about 20-40 min until the emulsified antigen is not diffused in water.

The mouse immunization process comprises the following steps: healthy experimental mice are selected, the mice with no obvious trauma on the body surface, active food and 6 to 8 weeks old are suitable. The method comprises immunizing once every 21 days by intraperitoneal injection, taking blood after cutting off tail 7 days after each immunization to detect titer, stopping immunization when titer meets requirement, and fusing.

Cell fusion process

Preparing materials:

preparing HAT culture medium, and subpackaging DMEM and PEG; surgical instruments, 50mL plastic centrifuge tubes, 50mL glass centrifuge tubes were sterilized.

SP2/0 cells were revived one week earlier, cultured and allowed to grow well.

Feeder cells were harvested and plated one day in advance with HAT medium and 5 feeder cell plates were plated for one mouse.

Fusing:

processing SP2/0: blowing, transferring to a50 mL plastic centrifuge tube, centrifuging for 5min at 1000r, discarding supernatant, adding DMEM, blowing, centrifuging for 5min at 1000r, adding DMEM to about 10mL, blowing, and counting for later use.

Extracting splenocytes:

mice were bled from their eye sockets, sacrificed by cervical dislocation, and blood samples were reserved as positive controls.

The mouse spleen was isolated, the exterior of the spleen was washed with DMEM first, the interior of the spleen was washed with DMEM again, and the washings were collected and transferred to a 10mL glass centrifuge tube and counted.

Centrifuging at 1000r for 5min, discarding the supernatant, adding DMEM to blow off and transferring to a50 mL glass centrifuge tube, simultaneously transferring the SP2/0 treated previously to the 50mL glass centrifuge tube, centrifuging at 1000r for 5min, discarding the supernatant, and grinding evenly on the back of the hand. Warm water bath at 37 ℃, 1mL of PEG is added within 1min, shaking is carried out while adding, and standing is carried out for 90s. Adding 1mL of DMEM within 1min, adding 1mL of DMEM within 30s, gradually accelerating, adding DMEM to 40-50mL, standing for 10min at 37 ℃, centrifuging for 6min at 800r, discarding the supernatant, and adding HAT for plating.

Half the medium change after 3 days, and full medium change after one week, and HAT medium change.

Subclone selection process

And (4) observing the cell state, carrying out whole plate detection after a cell mass which can be observed by naked eyes exists in 96 holes, selecting holes with higher positive, and carrying out subcloning, wherein one positive hole is arranged on each plate.

And after cell clusters which can be observed by naked eyes exist in the 6 holes, performing subcloning and picking, selecting 12 holes of single cell clusters from each plate, performing the next subcloning on the cells with high positive and good cell states for at least three times, and finally selecting monoclonal cell cluster strains with high positive and good cell states.

And transferring the picked cell mass to 24-hole culture, and transferring to a small square bottle for culture after the 24-hole cells grow well. After the cells in the small square bottle grow well, freezing and storing and preparing ascites.

Process for the preparation of ascites

Mice were injected one week earlier with paraffin, 0.5mL per mouse.

Blowing off after the cells in the small square bottle grow well, transferring to a 10mL glass centrifuge tube, centrifuging for 5min at 1000r, discarding the supernatant, adding 1mLDMEM, counting, injecting 0.8 × 10 per mouse ⁶ Each mouse was diluted by injecting 0.5mL of each cell, and then the mouse was intraperitoneally injected with the cells.

Observing the state of the mouse, and starting to extract and collect ascites when the abdomen of the mouse obviously rises.

Antibody purification process

Preparation of reagents: equilibration/dialysis buffer: 10mM PB (pH 7.5) +100mM NaCl

100% saturated ammonium sulfate

Column wash: 20mM Tris (pH8.0) +1.5M NaCl

Preparing equipment: a Q column, a low-temperature high-speed centrifuge, a balance, a peristaltic pump and a nucleic acid protein detector.

Sample preparation for 33% ammonium sulfate precipitation: the frozen ascites is melted in a constant temperature water bath at 25 ℃ and filtered by filter paper to remove grease.

Measuring 120mL of ascites, adding 10mM PB (pH7.5) +100mM NaCl with the volume 1 time that of the ascites, and mixing uniformly; adding saturated ammonium sulfate solution with volume 1 time of ascites volume, stirring and precipitating at 20-25 deg.C for 10min, wherein the saturation of ammonium sulfate is 33%; centrifuging at 9000rpm and 5 ℃ for 10min, reserving precipitate, and discarding supernatant; suspending the precipitate with 10mM PB (pH7.5) +100mM NaCl in 2/3 times of ascites volume, and mixing;

dialysis and desalting: dialyzing twice with 10mM PB (pH 7.5) +100mM NaCl dialysate at a dialysis ratio of 1And (5) 25h. After dialysis, the antibody was recovered and filtered through filter paper to remove impurities for use. Column purification: filling a50 mL Q column, cleaning with 0.1M NaOH 2 times the column volume, and washing with ultrapure water 5 times the column volume for later use; balancing the column with a balance buffer solution with 5 times of column volume, and adjusting the display value of the protein ultraviolet detector to 0 as a base line after the column is balanced; loading the dialyzed sample at a speed of 5-6mL/min, collecting the flow-through liquid of the Q column in the whole process, A ₂₈₀ Starting collection when the detection value is more than 0.2, stopping collection until the detection value is reduced to 0.2, and collecting the target protein in different tubes; after the protein collection, the column was washed with 3 column volumes of 20mM Tris (pH8.0) +1.5M NaCl until no protein flowed out; washing the packing with 5 column volumes of ultrapure water, and then preparing a second pass through a Q column using 5 column volumes of 10mM PB (pH 7.5) +100mM NaCl equilibrium column;

sampling a Q column for the first time, and collecting target protein in the same steps;

after the protein collection, the column is cleaned by using 20mM Tris (pH8.0) +1.5M NaCl with the column volume of 3 times until no protein flows out, then the filler is washed by using ultrapure water with the column volume of 5 times, and the column is stored at the temperature of 5-8 ℃ after being not used for a long time;

antibody treatment: measuring the volume of the collected antibody, and immediately adjusting the NaCl concentration of the preservation solution to 300mM by using 5M NaCl;

diluting with 10mM PB (pH 7.5) +300mM NaCl buffer solution, adjusting antibody concentration to 3mg/mL, adding 100% Proclin300 to final concentration of 0.1%, filtering with 0.22 μm filter membrane for sterilization, and packaging as required.

Antibody sequencing process

Carrying out reverse transcription on a cell sample to obtain cDNA; amplifying and obtaining heavy chain and light chain variable regions; cloning to pMD18-T vector, sequencing; the sequencing results were aligned using IMGT/V-QUEST, after which the entire sequence was obtained. The specific sequence is shown in a sequence table.

Example 2 functional Activity identification of antibodies

The prepared CA242 antibody pairs are used for respectively detecting a calibration disk of a kit of Huakotai and a calibration disk prepared by dilution of self-produced antigen (the self-produced antigen is the CA242 antigen obtained by purifying tumor cell culture supernatant): with reference to the Huakotai calibration plate, the dilution of the self-produced antigen achieved a level of detected luminescence that was substantially consistent with the control, and the results are shown in Table 1.

TABLE 1

The method comprises the steps of selecting 53U/ml, 20U/ml and 5.3U/ml of middle-low three batches of fixed value blood samples in Caniger for multi-well detection, calculating the average value M and standard deviation SD of 21 multi-well measurement results, obtaining the coefficient of variation CV according to the formula CV = SD/M multiplied by 100%, wherein the Coefficient of Variation (CV) is not more than 15%, respectively obtaining the coefficient of variation CV, wherein the CV values are respectively 3.99%, 2.46% and 3.17%, the CV average value is 3.20%, the precision detection is better, and the detection results are shown in Table 2 by adopting a double-antibody sandwich method.

TABLE 2

The method verifies 200 clinical samples, 63 samples of the clinical samples are intercepted, the detection luminescence value of the low-value area sample is low, the back calculation concentration is between S0 and S1 (0 to 10U/mL), and the specific detection is excellent.

TABLE 3

Example 3 use of the antibodies of the invention for the detection of the carbohydrate antigen CA242

1, experimental process: double antibody sandwich method detection

1.1 preparation of coated plates

1.1.1 coating solution preparation: coating the CA242 antibody; coating is carried out according to the concentration of the active material, the coating concentration is 2 mug/ml, the correct volume (diluted according to the concentration of the coated antibody to 2ug/ml, for example, the concentration of the coated antibody is 5 mg/ml) of the antibody to be coated is added into the coating solution (CB buffer solution) to prepare 10ml of the coating solution, namely, 4ul of the coated antibody is added, the mixture is fully mixed, and the mixture is kept stand for 20-30min.

1.1.2 coating: adding the prepared coating liquid into a blank luminescent plate according to 100 mul/hole, sealing the plate sealing film, fully mixing in a plate mixer, and standing for 16-24 hr at 2-8 ℃.

1.1.3 plate washing: and (4) selecting a proper program for the plate washing machine, washing the plate 1 time by using the washing liquid, and drying by buckling. Meanwhile, the sealing liquid is taken out in advance to be recovered to the room temperature for later use.

1.1.4 blocking: sealing within 2 minutes after washing the plate, adding sealing liquid according to 150 mul/hole, sealing the plate sealing film, and standing for 16-24 hr at 2-8 ℃.

1.1.5 the sealing liquid of the coated plate is thrown out and dried, and the pre-coated plate with the sealing liquid is dried in an incubator (37 ℃ plus or minus 2 ℃) for 5-8 hours for use.

1.2 preparation of enzyme conjugates

Preparing an enzyme-labeled antibody by using a labeled antibody naked antibody labeled HRP; according to the preparation proportion of the enzyme-labeled antibody of 1 40000, the titer difference can be preliminarily judged according to the labeling yield difference, and if the yields are basically consistent, the titers are all as follows: 40000; according to the principle of stepwise dilution, adding enzyme-labeled antibody into enzyme diluent to dilute enzyme working solution with required titer, mixing well, standing for at least 1hr, and keeping.

1.3 taking out the plate coated in advance, the prepared enzyme working solution in advance and the benchmarking sample to recover the room temperature, and marking the experimental layout.

1.4 sample adding: add 25. Mu.L of constant blood sample and 100. Mu.L/well of enzyme working solution, which is enzyme-labeled sample diluted with enzyme diluent, separately, incubate at 37 ℃ for 60min. Control and blank wells were left.

1.5 plate washing: the plate was washed 5 times with PBST and dried.

1.6 luminescence reading: luminescent substrates A and B are semi-finished products of outsourcing kit manufacturers, the component A is 0.1% of luminol (Tris buffer solution), and the component B is 0.01% of hydrogen peroxide (LM buffer solution). Mixing at equal ratio (just prepared), and adding 50 uL/hole; within 1-5min, measuring the luminescence value with luminometer.

1.7 judging the result: from the data, the assay reagents were linearly related to the benchmarks.

Linear range of CanAg: 1-150U/mL; normal values: < 20U/mL

2 analysis of the results of the experiment

95 samples with clinical values of the Canagliptin are collected, and the CA242 pairing reagent detects the samples, so that the correlation between the samples and the samples with the Canagliptin values can reach more than 0.96.

Claims

1. The antibody for detecting the carbohydrate antigen CA242 is characterized in that the amino acid sequence of a heavy chain of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain of the antibody is shown as SEQ ID NO. 2.

2. The antibody according to claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the antibody is represented by SEQ ID No.3, and the amino acid sequence of the variable region of the light chain is represented by SEQ ID No. 4.

3. The antibody according to claim 1, wherein the variable region sequences of CDR1, CDR2 and CDR3 of the heavy chain of the antibody are represented by SEQ ID No. 5-7, and the variable region sequences of CDR1, CDR2 and CDR3 of the light chain of the antibody are represented by SEQ ID No. 8-10.

4. A nucleic acid molecule encoding the carbohydrate antigen CA242 detection antibody according to claim 1.

5. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encoding the CDR1, CDR2, and CDR3 variable regions of the heavy chain has a nucleotide sequence as set forth in SEQ ID Nos. 0.11-13, and the nucleic acid molecule encoding the variable region of the light chain has a nucleotide sequence as set forth in SEQ ID Nos. 0.14-16.

6. Biological material comprising a nucleic acid molecule according to claim 5, wherein the biological material is an expression cassette, a vector or a host cell.

7. An antibody conjugate obtained by coupling the antibody for detecting the saccharide antigen CA242 according to claim 1 to a carrier or a drug, or by coupling the antibody according to claim 1 to a chemical or biological label.

8. A detection reagent comprising the antibody of claim 1 or the antibody conjugate of claim 5.

9. A pharmaceutical composition comprising the antibody of claim 1 or the antibody conjugate of claim 5.

10. Use of the antibody of claim 1 or the nucleic acid molecule of claim 2 or the biological material of claim 4 in the preparation of a reagent or kit for the detection of carbohydrate antigen CA 242.