CN118754983A - A testosterone sandwich rabbit monoclonal antibody and its application - Google Patents

Abstract

The present application belongs to the field of immunoassay technology, and discloses a testosterone sandwich rabbit monoclonal antibody and its application. The testosterone sandwich monoclonal antibody mAb2 provided by the present application, comprising a light chain variable region and a heavy chain variable region, the LCDR1 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and LCDR3 comprises the sequence shown in SEQ ID NO:3; the HCDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises the sequence shown in SEQ ID NO:6. The monoclonal antibody of the present application can bind to the testosterone complex with high specificity, is suitable for double antibody sandwich immunoassay, is applied to the immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence, and the double antibody sandwich chemiluminescence platform detects testosterone standard antigen, and the detection sensitivity is less than 0.5ng/ml, and the magnetic chemiluminescence method detects clinical samples, within the range of 0.4-16ng/mL sample, and has good correlation with clinical comparison.

Description

A testosterone sandwich rabbit monoclonal antibody and its application

Technical Field

The present application relates to the technical field of immunoassay, and in particular to a testosterone sandwich rabbit monoclonal antibody and its application.

Background Art

Testosterone is the most important androgen in the human body. It is mainly synthesized by interstitial cells in the testicles, and the adrenal glands can secrete androgens. In addition, female ovaries can also secrete a small amount of androgens. Most of the testosterone in the blood can bind to plasma proteins, and only a small amount exists in free form. Only free testosterone is biologically active. The main physiological effects of testosterone are to promote the development and growth of reproductive organs, stimulate sexual desire, and at the same time, promote and maintain the development of male secondary sexual characteristics, maintain the function of the prostate and seminal vesicles and spermatogenesis. In addition, it can also promote protein synthesis, bone growth, and red blood cell production. This indicator can be used to evaluate the secretion function of testicular androgen and help rule out diseases such as abnormal male sexual development, testicular tumors, and infertility.

Testosterone content reflects the normal range of male hormones in the human body and is often used clinically to diagnose certain endocrine diseases, such as male sexual dysfunction, female sexual abnormalities, precocious puberty, sexual immaturity, etc. The clinical significance of testosterone level determination is very important for women. When the serum testosterone level of women decreases, it usually has no obvious clinical significance. However, when the testosterone level increases, it may be related to gynecological diseases, adrenal diseases, pregnancy, and exogenous factors.

At present, the "gold standard" for testosterone detection is mass spectrometry, but mass spectrometry detection requires complex and time-consuming sample pre-treatment processes and high clinical automation requirements, making it difficult to apply on a large scale. Therefore, immunoassays are usually used in clinical testing. Immunoassay technology is simple to operate and fast to detect, and is a practical alternative to small molecule mass spectrometry detection. Because small molecules lack antigenic epitopes, competitive methods are currently commonly used in clinical immunoassays. Competitive methods have low sensitivity, poor specificity, are prone to false positives, and have complex processes.

In order to solve the technical difficulties in the clinical application of the competition method in small molecules, it is urgently necessary to develop highly specific and sensitive monoclonal antibodies for the testosterone sandwich method. It is crucial to break through the limitations of the traditional competition method and realize the double antibody sandwich method for testosterone detection.

Summary of the invention

In order to overcome the above technical problems, the present application provides a testosterone sandwich rabbit monoclonal antibody with good specificity and high affinity, and its application in an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, to provide clinical management for prostate diseases.

In the first aspect, the present application provides a testosterone sandwich rabbit monoclonal antibody mAb2, wherein the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the LCDR1 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the LCDR3 comprises the sequence shown in SEQ ID NO:3; the HCDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the HCDR3 comprises the sequence shown in SEQ ID NO:6.

Further, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, the full length of VL is 110 amino acids, the number of amino acids in the four domains of FR are 26, 16, 36 and 10 respectively, the number of amino acids in the three domains of LCDR are 8, 3 and 11 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-34aa, 51aa-53aa and 90aa-100aa respectively, and their amino acid sequences are: QSVYSRNW (SEQ ID NO.1), KAS (SEQ ID NO.2), AGGYSGNIYYA (SEQID NO.3).

Further, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, the full length of VH is 120 amino acids, the number of amino acids in the four domains of FR are 25, 16, 38 and 11 respectively, the number of amino acids in the three domains of HCDR are 8, 7 and 15 respectively, HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 50aa-56aa and 95aa-109aa respectively, and their amino acid sequences are GFSLSSYA (SEQ ID NO.4), ISSSGST (SEQ ID NO.5), ARGVSYDDYGDPFNL (SEQ IDNO.6), respectively.

By adopting the above technical scheme, the testosterone sandwich rabbit monoclonal antibody mAb2 of the present application can bind to testosterone with high specificity and high affinity, and its affinity constant Ka can reach 6.2×10 ⁸ L/mol.

On the other hand, the present application also provides an isolated polynucleotide, which encodes the rabbit monoclonal antibody mAb2.

On the other hand, the present application also provides a nucleic acid construct, wherein the nucleic acid construct contains the polynucleotide, preferably, the polynucleotide can be operably linked to at least one expression control element

On the other hand, the present application also provides a recombinant expression vector, which contains the polynucleotide or the nucleic acid construct.

On the other hand, the present application also provides a host cell, which contains the polynucleotide, or the nucleic acid construct, or the recombinant expression vector.

On the other hand, the present application also provides the use of the rabbit monoclonal antibody mAb2, the polynucleotide, the nucleic acid construct, the recombinant expression vector and/or the host cell in the preparation of a detection reagent or kit for detecting testosterone.

On the other hand, the present application also provides a detection reagent or a detection kit for detecting testosterone, wherein the detection reagent or the detection kit contains the rabbit monoclonal antibody mAb2.

Furthermore, the detection kit is an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the rabbit monoclonal antibody mAb2 is the detection antibody.

On the other hand, the present application also provides a method for preparing the rabbit monoclonal antibody mAb2, which comprises expressing the rabbit monoclonal antibody mAb2 using the host cell under conditions suitable for the expression of the rabbit monoclonal antibody mAb2, and recovering the expressed monoclonal antibody from the culture of the host cell.

Compared with the prior art, the testosterone sandwich rabbit monoclonal antibody of the present application can bind to testosterone with high specificity and high affinity, and its affinity constant Ka can reach 6.2×10 ⁸ L/mol. The testosterone sandwich rabbit monoclonal antibody of the present application can also be prepared into various immunoassay kits for detecting testosterone, especially, it can be used in immunoassay kits prepared by double antibody sandwich ELISA or chemiluminescence method. The double antibody sandwich chemiluminescence platform detects testosterone standard antigen with a detection sensitivity of less than 0.5ng/ml. The magnetic chemiluminescence method detects clinical samples with a sample range of 0.4-16ng/mL, and has a good correlation with the R clinical comparison.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an electrophoresis diagram of the full-length amplified products of mAb2 heavy chain and light chain, where M is a DNA molecular weight marker.

FIG2 shows the detection of cross-protein and target antigen samples by the mAb2 sandwich ELISA method, and the ordinate is the OD value of the detection.

Figure 3 shows the mAb2 magnetic microparticle chemiluminescence method for testing testosterone standard antigen, the horizontal axis is the testosterone standard antigen concentration (ng/ml), and the vertical axis is the luminescence value of the test. The R ² of the standard curve is 0.9964, the linear detection range is 0.4-16n/mL, and the sample concentration calculation formula is: y=162200x-51092.

Figure 4 shows the mAb2 magnetic microparticle chemiluminescence method for clinical samples, with the horizontal axis representing the testosterone concentration of clinical samples (ng/ml) and the vertical axis representing the luminescence value of the test. The R ² of the standard curve is 0.9676, with a linear detection range of 0.4-16 n/mL, and the formula for calculating the sample concentration is: y=27870x+61136.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by manufacturers.

Example 1 Preparation of testosterone sandwich rabbit monoclonal antibody

1) Preparation of immunogen

The testosterone complex was prepared with an immunogen purity of more than 90%, meeting the purity requirement for preparing monoclonal antibodies.

2) Animal immunization

The testosterone complex prepared above was emulsified with complete Freund's adjuvant at a volume ratio of 1:1, and New Zealand white rabbits weighing about 2 kg were immunized by subcutaneous injection, with an immunization dose of 800 μg/rabbit. A second immunization was performed two weeks later, emulsified with incomplete Freund's adjuvant at a volume ratio of 1:1, with an immunization dose of 400 μg/rabbit. After two immunizations, tail blood was collected to determine the serum titer by gradient dilution using the ELISA method; based on the OD450 greater than 1.0 when the ELISA titer was 128,000, it was determined whether to collect PBMCs or continue immunization based on the results, and the rabbit with the highest antibody titer was selected for PBMCs collection.

3) PBMCs isolation, specific B cell sorting, and cloning recombination

The rabbit was fixed on the operating table in supine position, the hair at the heart area was trimmed, the skin was disinfected with alcohol, and the most obvious heartbeat was selected for puncture with a 50ml syringe. Blood flowed into the syringe after the needle pierced the heart. After obtaining the required amount of blood, the needle was quickly pulled out, and the whole blood in the syringe was transferred to a sterile 50ml tube, mixed with an equal amount of PBS, and then slowly added drop by drop to the lymphocyte separation solution. Centrifuged at room temperature at 400×g for 30 minutes. After centrifugation, the liquid surface was divided into four layers from top to bottom: yellow plasma layer, white film layer, i.e., mononuclear cell layer, separation solution layer and red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation solution to obtain rabbit PBMCs.

Antigen-specific B cells were further sorted from rabbit PBMCs for culture, and the supernatant of cultured B cells was screened for positive clones using an antigen-coated ELISA plate. The cells of the positive clones were collected and lysed, and RNA was extracted and reverse transcribed into cDNA. The full-length sequences of the naturally paired rabbit monoclonal antibody light chain and heavy chain were amplified from the cDNA of the corresponding positive clones. The rabbit monoclonal antibody expression vector was constructed by cloning and recombination methods, and the sequence was determined by sequencing. The results of the amplified full-length PCR product are shown in Figure 1.

4) Preparation and purification of monoclonal antibodies

In order to obtain multiple rabbit monoclonal antibodies that recognize testosterone small molecules, the heavy chain and light chain genes of rabbit monoclonal antibodies were loaded on the expression vector, and the plasmid was transfected into KEK293 cells. The culture supernatant was obtained after 120-144 hours of transfection to obtain recombinant rabbit monoclonal antibodies that recognize testosterone small molecules. The cell suspension was collected, the supernatant was centrifuged, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then the purified antibody was packaged and lyophilized. The purified antibody was named rabbit monoclonal antibody mAb2.

Example 2 Testosterone Sandwich Rabbit Monoclonal Antibody Identification

1) Specificity identification of rabbit monoclonal antibodies

Indirect ELSA method was used for detection. The ELISA plate was coated with cross protein and testosterone complex antigen at a concentration of 1 μg/ml, overnight at 4°C, blocked with PBST containing 1% BSA, added with 10 ^4- fold diluted purified rabbit monoclonal antibody, reacted at 37°C for 50 min, washed 3 times with PBST, added with HRP-goat anti-rabbit Ig secondary antibody, reacted at 37°C for 50 min, washed 5 times with PBST, added with TMB color development for 10 min, added with stop solution, and measured A450 with an ELISA reader.

The results in Figure 2 show that the cross protein reacts negatively with the rabbit monoclonal antibody mAb2, with OD450 less than 0.2; the testosterone complex reacts positively with the mAb2 antibody, with an OD value much higher than that of the cross protein, indicating that the testosterone sandwich method rabbit monoclonal antibody of the present application specifically recognizes the testosterone complex.

2) Determination of affinity constant of rabbit monoclonal antibody

The affinity constant (Ka) was determined by non-competitive ELISA.

Coating: Dilute the antigen with carbonate buffer to a concentration of 1, 0.5, 0.1, and 0.05 μg/mL, add 100 μL/well to a 96-well ELISA plate for coating, and incubate at 4°C for 24 h;

Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL/well, and incubate at 37°C for 2 h;

Adding monoclonal antibody: Wash the plate 4 times with PBST, dilute the rabbit monoclonal antibody starting at 100 μg/mL with carbonate buffer, add 100 μL to each well, and incubate at 37°C for 2 h;

Add enzyme-labeled secondary antibody: wash the plate 4 times with PBST, add 100 μL of 1:10000-fold diluted HRP enzyme-labeled goat anti-rabbit Ig secondary antibody to each well, and place at 37°C for 30 min;

Color development and termination: Wash the plate 4 times with PBST, add 100 μL of substrate color development solution to each well, and react at 37°C in the dark for 15 min; add 50 μL of 1.0 mol/L H ₂ SO ₄ stop solution to each well to terminate the reaction;

Detection: Measure the absorbance value at a wavelength of 450nm (A450nm).

An S-shaped curve was drawn with the logarithm of the antibody concentration as the abscissa and the OD value as the ordinate. After calculation, the affinity constant Ka of the testosterone sandwich monoclonal antibody mAb2 was 6.2×10 ⁸ L/mol.

3) Sandwich antibody pairing

In order to select the best combination of coating antibody and detection antibody, the testosterone complex antibody was coated on the ELISA plate at 4°C overnight. The next day, the ELISA plate was removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed 3 times with PBST; 100 μl of testosterone complex was added to each well at a concentration of 20 ng/ml, and incubated at 37°C for 1 hour; after the incubation, the ELISA plate was removed, washed 3 times with PBST, and HRP-labeled rabbit monoclonal antibody mAb2 was added as the detection antibody, respectively, and incubated at 37°C for 1 hour. Washed 5 times with PBST, TMB substrate was added, and color was developed at 37°C for 10 minutes. After removal, stop solution was added and OD450 reading was measured on the ELISA instrument. According to the OD value of the sample and the background value of the negative control, the most ideal antibody pair was selected. The paired screening results are shown in Table 1.

Table 1 Antibody pairing experiment screening results

It can be seen that the antibody mAb2 involved in the present application is best used for sandwich detection.

Example 3 Analysis of variable region genes and amino acid sequences of rabbit monoclonal antibodies

The recombinant plasmid of the antibody was used as a DNA template, and the sequencing primers for the light chain variable region and the heavy chain variable region were designed according to the vector sequences at the 5' end of the light chain and heavy chain on the template, and the sequencing was performed using a sequencer ABI 3730. The nucleotide sequences of the light chain and heavy chain variable regions of the rabbit monoclonal antibody were obtained by sequencing.

The nucleotide sequences of the light chain variable region and the heavy chain variable region were sequenced and analyzed using the IMGT/V-QUEST analysis software on the Internet at http://www.imgt.org. The amino acid sequence of the light chain variable region of the rabbit monoclonal antibody mAb2 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8. The total length of VL is 110 amino acids, the amino acid numbers of its four domains of FR are 26, 16, 36 and 10 respectively, the amino acid numbers of its three domains of LCDR are 8, 3 and 11 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-34aa, 51aa-53aa and 90aa-100aa respectively, and their amino acid sequences are: QSVYSRNW (SEQ ID NO.1), KAS (SEQ ID NO.2), AGGYSGNIYYA (SEQID NO.3); the total length of VH is 120 amino acids, the amino acid numbers of its four domains of FR are 25, 16, 38 and 11 respectively, the amino acid numbers of its three domains of HCDR are 8, 7 and 15 respectively, the regions of HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 50aa-56aa and 95aa-109aa respectively, and their amino acid sequences are: GFSLSSYA (SEQ ID NO.4). NO.4), ISSSGST (SEQ ID NO.5), ARGVSYDDYGDPFNL (SEQ ID NO.6).

Example 4 Testosterone sandwich rabbit monoclonal antibody for magnetic microparticle chemiluminescent immunoassay

1. Detection principles and methods

The magnetic particle chemiluminescence immunoassay technology based on the principle of double antibody sandwich method was used. Testosterone antibody was labeled with biotin, and the biotinylated antibody was fixed to SA magnetic beads, ALP was coupled to testosterone complex rabbit monoclonal antibody, and testosterone, ALP substrate and corresponding buffer components were placed on the Cosma automatic magnetic particle chemiluminometer, and the instrument program was set for detection. The result was judged to be positive based on the luminescence value signal-to-noise ratio greater than 2.0. The size of the luminescence value reflects the amount of bound enzyme-labeled antibody, which is proportional to the concentration of testosterone in the sample. A standard curve was drawn according to the luminescence value of the measured standard, and the testosterone concentration value in the sample to be tested can be obtained from the standard curve. The test results are shown in Figure 3.

2. Composition of the magnetic particle chemiluminescence detection kit for testosterone detection

1. SA magnetic beads combined with biotin-testosterone antibody: take 50μl magnetic beads into a 0.5ml centrifuge tube, place it on a magnetic rack, and remove the supernatant after 1 minute; wash the magnetic beads 3 times with 0.5ml antibody diluent; add a certain amount of biotin-labeled testosterone antibody and rotate and mix at room temperature for 60 minutes; after magnetic separation, resuspend in magnetic storage buffer with a working concentration of 0.5mg/ml.

2. mAb2 coupled with ALP: First, use 2-IT to reduce the antibody mAb2; second, form the ALP-SMCC intermediate, and finally couple ALP-SMCC with the reduced antibody. After the coupling is completed, use ALP storage buffer to dilute to the working concentration.

3. The washing buffer is conventional PBST at pH 7.4, containing 0.05% Proclin 300, and prepared into a 20-fold concentrated solution.

4. Chemiluminescent colorimetric solution was purchased from Avid Biotechnology.

5. The sample diluent contains 1% BSA, 0.05% Proclin 300 in PBST, which is sterilized by filtration.

6. Standard testosterone small molecule, using PBS containing 1% BSA, 5% sucrose, 10% glycerol, 0.05% Proclin300 as diluent, dilute the sample to 5 μg/ml, filter sterilize, and aseptically package.

3. Testing of clinical testosterone samples

Clinical samples with different testosterone concentrations were processed, testosterone antibody was used as coating antibody, mAb2 antibody was used as detection antibody, and the above detection method was used to detect clinical samples with different concentrations. The detection results are shown in Figure 4.

According to the result data, the rabbit monoclonal antibody described in the present application is used for magnetic microparticle chemiluminescent immunoassay reagent, and has good correlation with clinical comparison within the sample range of 0.4-16 ng/mL.

In summary, the testosterone sandwich rabbit monoclonal antibody mAb2 of the present application is applied to the immunoassay kit prepared by the double antibody sandwich ELISA or chemiluminescence method. The double antibody sandwich chemiluminescence platform detects the testosterone standard antigen with a detection sensitivity of less than 0.5 ng/ml. The magnetic chemiluminescence method detects clinical samples with a clinical composite rate of >0.95 within the sample range of 0.4-16 ng/mL, which is significantly higher than the traditional competitive detection method. The process is simple, breaking through the limitations of the traditional competitive method.

This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims (9)

1. A testosterone sandwich rabbit monoclonal antibody mAb2, characterized in that the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the LCDR1 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and LCDR3 comprises the sequence shown in SEQ ID NO:3; the HCDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises the sequence shown in SEQ ID NO:6. 2. The rabbit monoclonal antibody according to claim 1, characterized in that the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8. 3. An isolated polynucleotide, characterized in that the isolated polynucleotide encodes the rabbit monoclonal antibody mAb2 according to any one of claims 1-2. 4. A nucleic acid construct, characterized in that the nucleic acid construct contains the polynucleotide according to claim 3, preferably, the polynucleotide can be operably linked to at least one expression control element The recombinant expression vector is characterized in that the recombinant expression vector contains the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4. 5. A host cell, characterized in that the host cell contains the polynucleotide according to claim 3, or the nucleic acid construct according to claim 4, or the recombinant expression vector according to claim 5. 6. Use of the rabbit monoclonal antibody mAb2 according to any one of claims 1-2, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, the recombinant expression vector according to claim 5 and/or the host cell according to claim 6 in the preparation of a detection reagent or kit for detecting testosterone. 7. A detection reagent or a detection kit for detecting testosterone, characterized in that the detection reagent or the detection kit contains the rabbit monoclonal antibody mAb2 according to any one of claims 1-2. 8. The detection reagent or detection kit according to claim 8, characterized in that the detection kit is an immunodetection kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the rabbit monoclonal antibody mAb2 is the detection antibody. 9. A method for preparing the rabbit monoclonal antibody mAb2 according to claim 1 or 2, characterized in that the method comprises expressing the rabbit monoclonal antibody mAb2 using the host cell according to claim 6 under conditions suitable for the expression of the rabbit monoclonal antibody mAb2, and recovering the expressed monoclonal antibody from the culture of the host cell.