CN116462762B

CN116462762B - Anti-prostate specific antigen monoclonal antibody and application thereof

Info

Publication number: CN116462762B
Application number: CN202310703122.6A
Authority: CN
Inventors: 林铁; 邓德文; 王立凯; 张文杰; 谌红彬
Original assignee: TJBiotechnologies Tianjin Co ltd
Current assignee: TJBiotechnologies Tianjin Co ltd
Priority date: 2023-06-14
Filing date: 2023-06-14
Publication date: 2023-08-25
Anticipated expiration: 2043-06-14
Also published as: CN116462762A

Abstract

The invention relates to the field of antibodies, in particular to an anti-prostate specific antigen monoclonal antibody and application thereof. The test of the titer and the electrophoretogram of the monoclonal antibody as a raw material of the kit shows that the monoclonal antibody has excellent performance in the detection of the prostate specific antigen, and can be used as a target for further production and optimization.

Description

Anti-prostate specific antigen monoclonal antibody and application thereof

Technical Field

The invention relates to the field of antibodies, in particular to an anti-prostate specific antigen monoclonal antibody and application thereof.

Background

The prostate specific antigen (Prostate specific antigen, PSA) is a serine glycoprotein enzyme with trypsin and chymotrypsin activity. PSA breaks down the clotting proteases and fibronectin in semen, allowing the coagulated semen to liquefy again after ejaculation. In normal prostate tissue, PSA is produced mainly by differentiated columnar secretory cells in the glandular epithelium, which are distributed within the prostate acini. PSA is a glycoprotein with a relative molecular weight of 28430 and consists of 237 amino acid residues. PSA exists in the human body in 4 molecular forms: (1) free molecular form (fPSA), about 10% to 30%; (2) Forming a complex form (PSAACT) with a 1-antichymotrypsin, accounting for about 70% -90%; (3) Form a complex with a1 protease inhibitor (PSA API); (4) Form a complex with α2-macroglobulin (PSA α2m). The first 3 forms all retain the epitope of PSA and can be assayed for content. Wherein PSA α1m does not display an epitope and cannot be detected. In addition, there are other PSA complexes or complexed forms. PSA in serum exists in both free and bound states, with a major portion of the PSA molecules being present in bound and a minor portion in free state. Whereas the free PSA is only 50-70% enzymatically active. tPSA is generally considered to be the sum of fPSA and PSAACT.

There are 3 common possibilities of elevated serum PSA in prostate disease. Firstly, the barrier permeability of the surrounding environment of the normal prostate catheter system changes, PSA secretion into blood increases, such as prostatitis; secondly, the volume of the prostate is increased, which causes the secretion amount of PSA to be increased, such as Benign Prostatic Hyperplasia (BPH); thirdly, the barrier of the prostate is destroyed, such as by prostate cancer or other mechanical infiltration factors. About 7% -20% of the serum PSA of patients with acute prostatitis and prostatic hyperplasia are slightly increased, and 70% -80% of the serum PSA of patients with prostatic cancer are obviously increased, which shows that the PSA is closely related to the occurrence and development of the prostatic diseases.

The pathological changes of prostatitis are mainly manifested by infiltration of a large number of inflammatory cells in the stroma and acini, including lymphocytes, plasma cells, large mononuclear cells, etc., and occasionally invade glandular epithelium. These inflammatory reactions disrupt the integrity of the prostatic ducts and the original physiological barriers, allowing PSA within the prostatic ducts and acini to leak into the blood circulation, causing elevated serum PSA. (1) acute and chronic prostatitis: in acute prostatitis, macrophages appear in the prostate epithelium, stroma and lumen of the gland; in chronic prostatitis, lymphocytes and plasma cells infiltrate into the interstitium and surround the gland. That is, acute prostatitis is more affected by the acinus or ductal epithelium of the prostate than chronic prostatitis, and the extent of the affected is also greater. Acute prostatitis results in a greater elevation of serum PSA levels than chronic prostatitis. (2) bacterial and non-bacterial prostatitis: most scholars believe that the extent of serum PSA elevation caused by bacterial and non-bacterial prostatitis is different. Since prostatitis can raise serum PSA, almost half or more of men without any clinical symptoms who have significantly elevated serum PSA levels have evidence of inflammation of the prostate tissue, and such subclinical infections can also cause elevated serum PSA levels. It is thought that PSA secretion is positively correlated with prostate volume, and that prostate epithelial cells that produce PSA are increased significantly when the prostate volume is large. It was considered that every 1g increase in BPH tissue increased serum PSA by 0.3 ng/ml; the PSA level increased by approximately 32% for each 1.0 mL increase in prostate volume.

The majority of prostate cancer patients in bed can be histopathologically diagnosed by a systemic biopsy of the prostate. However, initially suspected prostate cancer is typically examined by a prostate digital examination or serum prostate specific antigen and then a determination is made as to whether a prostate biopsy is to be performed. Digital rectal examination in combination with PSA testing is the currently accepted method of forescreening for the best early detection of prostate cancer.

At present, the domestic chemiluminescent technology mainly comprises a horseradish peroxidase (HRP) luminescent system, an Alkaline Phosphatase (AP) luminescent system and an Acridine Ester (AE) direct luminescent system according to different labels. With the full automation of the domestic chemiluminescence technology, the tubular magnetic particle method has become the most dominant method, and the direct light emission of acridinium ester has a plurality of advantages over enzymatic light emission, and also becomes the most dominant light emitting system.

The acridinium ester-magnetic particle system has a number of advantages: 1) The cost can be effectively saved: about ten kits (100T) can be made by releasing 1mg of antibody, about 20-40 kits can be made by enzymatic plate luminescence, and about 100 kits can be made by chemiluminescence based on an acridinium ester-magnetic particle system; 2) Short incubation time and simple operation: the magnetic particle system has high reaction speed and can report after 15-30 min. And the chemiluminescent method has a rapid development trend in China thanks to the development of the full-automatic chemiluminescence analyzer.

However, at present, it is difficult to find anti-PSA monoclonal antibodies with good specificity and high affinity in China, and mainly depends on imported products, so that development of an anti-PSA monoclonal antibody with high affinity and high specificity for developing a PSA detection kit is urgently needed.

Disclosure of Invention

In order to overcome the problems, the invention provides an anti-prostate specific antigen monoclonal antibody 4TJ28 with good specificity and high sensitivity, which is characterized by comprising the following components:

the monoclonal antibody comprises three antigen complementarity determining regions on the heavy chain variable region: CDR1, CDR2 and CDR3 and three antigen complementarity determining regions CDR4, CDR5, CDR6 on the light chain variable region; 6. the individual complementarity determining regions CDR1, CDR2, CDR3, CDR4, CDR5 and CDR6 are shown as SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, respectively; the amino acid sequence of the heavy chain variable region is shown as SEQ.ID.NO. 1; the amino acid sequence of the light chain variable region is shown as SEQ.ID.NO. 2.

The invention also provides a method for coating magnetic particles by monoclonal antibodies, which comprises the following steps:

1) 1ml of 10mg/ml of magnetic particles having a particle size of 1.5 μm and 1mg of monoclonal antibody 4TJ28 according to claim 1 are added to 5ml of MES buffer;

2) 1ml of EDC solution with the concentration of 10mg/ml is added while swirling;

3) After 3h at room temperature, magnetic separation and 1ml of 0.2M glycine solution were added;

4) After 1h reaction at room temperature, magnetic separation and 1ml of 2% BSA were added and reacted for 1h at room temperature;

5) Magnetic separation is carried out, 5ml TBS-T buffer solution is used for cleaning for three times, after the supernatant is removed by magnetic separation again, 1ml PBS buffer solution is added, and the magnetic particles coated with the monoclonal antibody 4TJ28 are prepared.

In addition, the invention also discloses a chemiluminescent kit capable of specifically detecting the prostate specific antigen.

The beneficial effects are that:

the invention provides an anti-prostate specific antigen monoclonal antibody 4TJ28, and a detection kit prepared by using the antibody has good accuracy, and the detection limit can reach below 1 ng/ml.

Drawings

FIG. 1 is a SDS-PAGE electrophoresis of the purified anti-prostate specific antigen monoclonal antibody 4TJ 28.

FIG. 2 is a standard graph of the kit for detecting PSA standards.

Detailed Description

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

1. Preparation of anti-prostate specific antigen monoclonal antibodies.

The PSA antigens used in this example were obtained commercially. PSA antigen (50 ug/only) was dissolved with complete adjuvant (50 ug/only) in PBS (0.5 ml/only). Shaking in a 2ml EP tube until the emulsion is completed, and packaging in a syringe to perform intraperitoneal injection on the mice.

The first boost is carried out two weeks after the primary immunization, the boost injection dosage is the same as or halved from the primary immunization, the adjuvant used in the boost step is incomplete Freund's adjuvant, other processes are completely consistent with the primary immunization, the mice are numbered 10 days after the first boost, blood collection is carried out to detect serum titers, and the immune effect is evaluated: on day 10 after each boost, blood was collected from the mouse canthus using 09-11mm inner diameter glass capillaries. After the murine blood was allowed to stand at room temperature for 1-3 hours, it was centrifuged at 3000 rpm for 15 minutes. Serum was collected and dissolved in Blocking Buffer at dilution ratios of 1:100, 1:300, 1:900, 1:2.7k, 1:8k, 1:24k and 1:72 k. And (3) selecting a Blocking Buffer as a blank control, and performing ELISA detection.

The second reinforcement was performed two weeks after the first reinforcement, and the procedure for reinforcement was identical to that of the first reinforcement. Mice were terminally boosted three weeks after the last boost, with the same antigen dose as the primary immunization, and cell fusion was performed 3-5 days after final boost.

Mice were sacrificed and spleens were removed and placed in a cell screen, the spleens were sheared with scissors and rinsed with 5mL mem and the spleens were ground with a 5mL syringe push head until completely crushed. The pusher and filter were rinsed with DMEM, and the splenocyte solution was transferred to a 50mL centrifuge tube, supplemented with DMEM to 45mL. Taking out the T-75 cell culture flask of the CO2 incubator in advance, and tapping the flask to separate the cells from the flask wall under 7-10. Transfer into 50ml centrifuge tubes and replenish the original flask with DMEM medium containing 10% fbs,1 x ht, shake spleen cells and sp2/0 cells into centrifuge tubes twice, centrifuge for 5min at 1000 rpm, discard supernatant, re-suspend spleen cells and sp2/0 cells, respectively, and add 45ml ldmem, respectively. 40. Mu.L of the counted cells were taken. The cells were inverted twice and shaken well, centrifuged again, and the cells counted.

Spleen cells and sp2/0 cells were resuspended, respectively, added to 30ml of DMEM into a spleen cell centrifuge tube, mixed well in 10ml of DMEM into a sp2/0 centrifuge tube, and sp2/0 cells were mixed with spleen cells according to the ratio of sp2/0 cells to spleen cells 1:5, depending on the number of sp2/0 cells. The supernatant was discarded after centrifugation again, resuspended and allowed to stand for one minute. Cell fusion was performed by taking PEG1500 pre-heated for 15 min with a 2ml pipette, and then slowly dropping the pipette port into the cell fluid in an amount of 1ml/1.0 x 108 splenocytes, and stirring the cell fluid uniformly while gently sliding the pipette. The cells were allowed to stand for 30 seconds, 1ml of DMEM was added dropwise to the centrifuge tube at a constant rate over an exact period of 1 minute, and the tube was gently shaken while adding. In the same manner as above, 2ml of DMEM was added dropwise over the second 1 minute, 5ml of DMEM was added dropwise over the third 1 minute, and 10ml of DMEM was added dropwise over the fourth 1 minute. DMEM was supplemented to 50ml and centrifuged at 1000 rpm for 5min.

The fused cells were resuspended and dissolved in DMEM medium containing 30% fbs,1×b.s, 2×h.a.t at a concentration of 1.5-2×106/ml of spleen cells. 100. Mu.l/well was dispensed into feeder cell plates. And placing the split-packed cell culture plates into a CO2 incubator for culture. The culture plates were supplemented with 50 μl of medium, which was DMED medium containing 20% fbs,1 x ps, 10% hcf, on the fourth day after cell fusion. ELISA screening positive clone wells 7-14 days after cell fusion when clones grew to a size that could be observed visually.

Positive hybridoma cells were transferred out of the vial or one of the cryopreservation tubes was resuscitated and transferred to the medium vial. The cells were incubated with DMEM medium containing 10% fbs,1 x p.s for 3-5 days, and when the cells were expanded enough for injection, centrifugation was performed at 1000 rpm for 5 minutes, and the supernatant was discarded. Washed twice with DMEM, centrifuged at 1000 rpm for 5min, and the supernatant was discarded. Cells were resuspended in DMEM at a final cell concentration of 2-4x106cells/ml and injected into the mouse abdominal cavity at a dose of 1-2 x106 cells/0.5ml/mouse within 1 hour after resuspension. The injected mice need to be immunized with liquid paraffin 14-30 days ago, or with incomplete Freund's adjuvant 7-32 days ago. Mice were observed daily for their abdomen. Obvious swelling of the abdomen of the small phoenix occurs 7-10 days after injecting the cells, the ascites is continuously collected every day by using a 1.2# syringe needle until the mice die or stop producing the ascites, the collected ascites is centrifuged every day, and the supernatant is taken after centrifugation at 4000 rpm for 15 minutes and stored in a refrigerator at 4 ℃.

The anti-PSA mab 4TJ28 was detected using ELISA antibody titer detection kit and the results are shown in table 1.

TABLE 1 results of anti-PSA monoclonal antibody 4TJ28 potency detection

When the titer detection result P/N is more than 2, the result in the table 1 shows that the titer of the 4TJ28 monoclonal antibody reaches more than 1:81000, which indicates that the monoclonal antibody has better activity and can be used for subsequent experiments, and the SDS-PAGE electrophoresis of the purified antibody is shown in the attached figure 1 of the specification.

2. Preparation of monoclonal antibody 4TJ28 coated magnetic particles.

10mL of EP was rinsed three times with MES buffer (pH=5.0), 5mL of MES buffer (pH=5.0) was added to the tube, 1mL of magnetic particles (JSR Life Science, MS160/Carboxyl,1%,10mg/mL,1.5 μm) was added to the EP tube, vortexing was performed to form a uniform magnetic particle suspension, 1mL of EDC solution (10 mg/mL) was added while vortexing, the reaction was performed for 45min with shaking, 1mg of monoclonal antibody 4TJ28 was added, the reaction was performed for 4h with shaking at room temperature, 1mL of 0.2M glycine solution was added after magnetic separation was performed, 2% BSA was added after magnetic separation was performed, and incubation was performed at room temperature for 1h at 4 ℃. After the next day of magnetic separation, 1ml of TBS-T buffer was added to wash the beads three times, and after the last magnetic separation, 1ml of PBS buffer (containing 0.5% mouse serum) was added and stored at 4℃for further use.

3. The detection effect of the prostate specific antigen detection kit.

The specificity and sensitivity of the magnetic particle chemiluminescence method to monoclonal antibodies are utilized. The magnetic microparticles coated with 4TJ28 were diluted 40 times as the magnetic microparticle solution component of the kit, a labeled antibody prepared by using a purchased anti-PSA antibody to link acridinium ester was used as the acridinium ester labeling component of the kit, PSA antigen was prepared as a calibrator for detection with 20% fetal bovine serum at concentrations of 0ng/ml, 3ng/ml, 9ng/ml, 22ng/ml, 52ng/ml, 125ng/ml, PSA concentrations as abscissa, and luminescence values corresponding to each concentration of PSA as ordinate, and a standard curve was drawn by a four-parameter method. The results of the luminescence values are shown in Table 2, and the standard curves are shown in FIG. 2 of the specification.

And (5) determining a standard curve.

The experimental method comprises the steps of repeatedly detecting 6 concentration points of a working calibrator for 3 times, then calculating an average value, and drawing a standard curve by taking the concentration value as an abscissa and the average value of the luminous value as an ordinate.

TABLE 2 Standard Curve luminescence value results f

As shown in fig. 2, the standard curve r= 0.9972 after four parameter fitting.

And (5) verifying accuracy.

Experimental method, a PSA sample A having a concentration of about 50ng/mL (the concentration deviation thereof is allowed to be + -20%) is added to a serum B having a concentration in the range of 1ng/mL to 2.5ng/mL, the volume ratio between the added PSA and the serum B is 1:9, according to the formula(R-recovery, V-volume of sample A added, V ₀ Adding the volume of the sample B, C-the detection concentration of the mixed solution of the sample A and the sample B, C ₀ Concentration of sample B, C _S -concentration of sample a) the recovery R is calculated, which should be in the range of 85% -115%.

TABLE 3 recovery rate experimental results

The results in Table 3 show that the accuracy of the kit meets the standard.

The lowest detection limit is confirmed.

The experimental method comprises the steps of detecting by using a zero-concentration calibrator as a sample, repeatedly measuring for 20 times to obtain an RLU value (relative luminescence value) of 20 measurement results, calculating an average value (M) and a Standard Deviation (SD) of the RLU value to obtain an RLU value corresponding to M+2SD, performing two-point regression fitting according to a concentration-luminescence value result between the zero-concentration calibrator and an adjacent calibrator to obtain a first-order equation, introducing the RLU value corresponding to M+2SD into the equation, and obtaining a corresponding concentration value, namely the lowest detection limit, wherein the result is not more than 0.5ng/mL.

TABLE 4 minimum limit of detection experiment results

The results in Table 4 show that the minimum detection limit of the kit is 0.026ng/ml, and meets the minimum detection limit standard.

Through the electrophoresis chart after the purification of the antibody and the performance analysis of the kit containing the monoclonal antibody 4TJ28 coated magnetic particle component, the monoclonal antibody provided by the invention has excellent performance in the detection of the prostate specific antigen, and can be used as a target for further production and optimization.

Claims

1. A monoclonal antibody 4TJ28 that specifically recognizes a prostate-specific antigen, comprising three complementarity determining regions of the heavy chain variable region: CDR1, CDR2 and CDR3 and three antigen complementarity determining regions CDR4, CDR5, CDR6 on the light chain variable region; the 6 complementarity determining regions CDR1, CDR2, CDR3, CDR4, CDR5 and CDR6 are shown as SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, respectively; the amino acid sequence of the heavy chain variable region is shown as SEQ.ID.NO. 1; the amino acid sequence of the light chain variable region is shown as SEQ.ID.NO. 2.

2. A method for coating magnetic particles with monoclonal antibodies, which is characterized by comprising the following steps: 1) 1ml of 10mg/ml of magnetic particles having a particle size of 1.5 μm and 1mg of monoclonal antibody 4TJ28 according to claim 1 are added to 5ml of MES buffer; 2) 1ml of EDC solution with the concentration of 10mg/ml is added while swirling; 3) After 3h at room temperature, magnetic separation and 1ml of 0.2M glycine solution were added; 4) After 1h reaction at room temperature, magnetic separation and 1ml of 2% BSA were added and reacted for 1h at room temperature; 5) Magnetic separation is carried out, 5ml TBS-T buffer solution is used for cleaning for three times, after the supernatant is removed by magnetic separation again, 1ml PBS buffer solution is added, and the magnetic particles coated with the monoclonal antibody 4TJ28 are prepared.

3. A detection kit capable of specifically detecting a prostate specific antigen in human serum, which is characterized by comprising the monoclonal antibody 4TJ 28-coated magnetic particles prepared by the method for coating magnetic particles with a monoclonal antibody according to claim 2.