A testosterone sandwich rabbit monoclonal antibody mAb1 and its application

Abstract

The present application belongs to the field of immunoassay technology, and discloses a rabbit monoclonal antibody mAb1 and its application. The testosterone sandwich monoclonal antibody provided by the present application includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1-3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:3. The light chain variable region includes LCDR1-3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR2 is SAS, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:5. The rabbit monoclonal antibody of the present application can bind to the testosterone complex with high specificity, and is applied to the immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the double antibody sandwich chemiluminescence platform detects the 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 samples, and has good correlation with clinical comparison.

Description

Technical Field

[0001] The present application relates to the technical field of immunoassay, and in particular to a testosterone sandwich rabbit monoclonal antibody mAb1 and its application. Background Art

[0002] Testosterone (also known as testosterone, testosterone or testosterone) is a steroid hormone secreted by the testicles of men or the ovaries of women. The adrenal glands also secrete a small amount of testosterone. It is the main male hormone and anabolic steroid. Most of the testosterone in the blood can be bound to plasma proteins, and only a small amount exists in free form. Free testosterone is biologically active.

[0003] The main physiological effects of testosterone are to promote the development and growth of reproductive organs, stimulate sexual desire, 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 erythropoiesis. This indicator can be used to evaluate the testicular androgen secretion function and help to exclude diseases such as male sexual development abnormalities, 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 characteristics abnormalities, precocious puberty, sexual immaturity, etc. The clinical significance of testosterone level determination is also very important for women. When the serum testosterone level of women is reduced, there is usually no obvious clinical significance. However, when the testosterone level is elevated, it may be related to gynecological diseases, adrenal diseases, pregnancy, and exogenous factors.

[0004] The commonly used method for detecting testosterone 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. Since 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.

[0005] Therefore, there is an urgent need to develop highly specific and sensitive monoclonal antibodies for the testosterone sandwich method, break through the limitations of the traditional competitive method, realize the double antibody sandwich method for testosterone detection, and provide new ideas for clinical immunoassay of testosterone. Summary of the invention

[0006] In order to overcome the above technical problems, the present application provides a testosterone sandwich rabbit monoclonal antibody mAb1 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.

[0007] In the first aspect, the present application provides a testosterone sandwich rabbit monoclonal antibody mAb1, wherein the rabbit monoclonal antibody mAb1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:3.

[0008] Furthermore, the light chain variable region includes LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is SAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:5.

[0009] Further, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6, the full length of VH is 112 amino acids, the number of amino acids in the four domains of FR are 24, 17, 36 and 11 respectively, the number of amino acids in the three domains of HCDR are 8, 7 and 9 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-56aa and 93aa-101aa respectively, and their amino acid sequences are GFSLSSHA (SEQ ID NO.1), INTGGSA (SEQ ID NO.2), VRGDSNDWT (SEQ ID NO.3), respectively.

[0010] Further, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, the full length of VL is 106 amino acids, the number of amino acids in the four domains of FR are 26, 17, 36 and 11 respectively, the number of amino acids in the three domains of LCDR are 6, 3 and 7 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-95aa respectively, and their amino acid sequences are: ESIFSY (SEQ ID NO.4), SAS, LGSYSSI (SEQ ID NO.5).

[0011] By adopting the above technical scheme, the testosterone sandwich rabbit monoclonal antibody mAb1 of the present application can bind to testosterone with high specificity and high affinity, and its affinity constant Ka can reach 6.05×10 8 L / mol.

[0012] On the other hand, the present application also provides an isolated polynucleotide, which encodes the rabbit monoclonal antibody mAb1.

[0013] On the other hand, the present application also provides a nucleic acid construct, which contains the polynucleotide. Preferably, the polynucleotide can be operably linked to at least one expression regulatory element.

[0014] On the other hand, the present application also provides a recombinant expression vector, which contains the polynucleotide or the nucleic acid construct.

[0015] 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.

[0016] On the other hand, the present application also provides the use of the rabbit monoclonal antibody mAb1, the polynucleotide, the nucleic acid construct and / or the recombinant expression vector in the preparation of a detection reagent or kit for detecting testosterone.

[0017] 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 mAb1.

[0018] Furthermore, the detection kit is an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the rabbit monoclonal antibody mAb1 is the detection antibody.

[0019] On the other hand, the present application also provides a method for preparing the rabbit monoclonal antibody mAb1, the method comprising, under conditions suitable for the expression of the rabbit monoclonal antibody mAb1, using a host cell containing the polynucleotide, or the nucleic acid construct, or the recombinant expression vector to express the rabbit monoclonal antibody mAb1, and recovering the expressed monoclonal antibody from the culture of the host cell. And recovering the expressed monoclonal antibody from the culture of the host cell.

[0020] 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 6.05×10 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 clinical comparison of R. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is the electrophoresis diagram of the full-length amplified products of the heavy and light chains of the rabbit monoclonal antibody mAb1. M is the DNA molecular weight marker.

[0022] Figure 2 This is an indirect sandwich ELISA method for detecting cross-protein and target antigen samples. The ordinate is the OD value of the detection.

[0023] Figure 3 The magnetic particle chemiluminescence method is used to detect the testosterone standard antigen. The horizontal axis is the testosterone standard antigen concentration (ng / ml), and the vertical axis is the luminescence value detected. 2 =0.9917, linear detection range 0.4-16n / mL, the sample concentration calculation formula is: y=59177x-1082.9.

[0024] Figure 4 The magnetic particle chemiluminescence method is used to detect clinical samples. The horizontal axis is the testosterone concentration of clinical samples (ng / ml), and the vertical axis is the luminescence value of the detection. 2 =0.9641, linear detection range 0.4-16n / mL, the sample concentration calculation formula is: y=225180x+21742. DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1 Preparation of testosterone sandwich rabbit monoclonal antibody

[0027] 1) Preparation of immunogen

[0028] Different schemes were optimized to prepare testosterone complexes, and the purity of the immunogen was above 90%, meeting the purity requirement for preparing monoclonal antibodies.

[0029] 2) Animal immunization

[0030] 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 and serum titer was determined by gradient dilution by ELISA; OD450 greater than 1.0 when the ELISA titer was 128,000 was used as the standard, and whether to collect PBMCs or continue immunization was determined according to the results, and the rabbit with the highest antibody titer was selected for PBMCs collection.

[0031] 3) PBMCs isolation, specific B cell sorting, and cloning recombination

[0032] 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.

[0033] Antigen-specific B cells were further sorted from rabbit PBMCs for culture. 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 .

[0034] 4) Preparation and purification of monoclonal antibodies

[0035] 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 mAb1.

[0036] Example 2 Identification of testosterone sandwich rabbit monoclonal antibody mAb1

[0037] 1) Specificity identification of rabbit monoclonal antibodies

[0038] The indirect ELSA method was used for detection. The ELISA plate was coated with the cross protein and testosterone complex antigen at a concentration of 1 μg / ml, incubated overnight at 4°C, and blocked with PBST containing 1% BSA. 4The purified rabbit monoclonal antibody mAb1 was diluted 50 times, reacted at 37℃ for 50 min, washed 3 times with PBST, added with HRP-goat anti-rabbit Ig secondary antibody, reacted at 37℃ for 50 min, washed 5 times with PBST, added with TMB for color development for 10 min, added with stop solution, and measured A450 with an enzyme reader.

[0039] Figure 2 The results showed that the cross protein reacted negatively with the rabbit monoclonal antibody mAb1, and the OD450 was less than 0.25; the testosterone complex reacted positively with the mAb1 antibody, and the OD value was much higher than that of the cross protein, indicating that the testosterone sandwich method rabbit monoclonal antibody mAb1 of the present application specifically recognizes the testosterone complex.

[0040] 2) Determination of affinity constant of rabbit monoclonal antibody

[0041] The affinity constant (Ka) was determined by non-competitive ELISA.

[0042] 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;

[0043] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h;

[0044] Adding monoclonal antibody: Wash the plate 4 times with PBST, dilute the monoclonal antibody MAB1 in carbonate buffer starting at 100 μg / mL, add 100 μL to each well, and incubate at 37°C for 2 h;

[0045] 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;

[0046] 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 H2SO4 stop solution to each well to terminate the reaction;

[0047] Detection: Measure the absorbance value at a wavelength of 450nm (A450nm).

[0048] The S-shaped curve was drawn with the logarithm of antibody concentration as the horizontal axis and the OD value as the vertical axis. After calculation, the affinity constant Ka of the testosterone sandwich monoclonal antibody mAb1 was 6.05×10 8 L / mol.

[0049] 3) Antibody pairing

[0050] 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 incubation, the ELISA plate was removed, washed 3 times with PBST, and HRP-labeled rabbit monoclonal antibodies mAb1 and mAb0 were added as detection antibodies, 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 readings were measured on the ELISA reader. 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.

[0051] Table 1 Antibody pairing experiment screening results

[0052]

[0053] It can be seen that the antibody mAb1 involved in the present application is best used for sandwich detection.

[0054] Example 3 Analysis of variable region genes and amino acid sequences of rabbit monoclonal antibody mAb1 The recombinant plasmid of mAb1 antibody was used as a DNA template, and the sequencing primers of 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 the heavy chain on the template, and 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 mAb1 were obtained by sequencing.

[0055] 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 http: / / www.imgt.org on the Internet. The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody mAb1 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7. The total length of VH is 112 amino acids, and the amino acid numbers of the four domains of FR are 24, 17, 36 and 11, respectively, and the amino acid numbers of the three domains of HCDR are 8, 7 and 9, respectively. HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-56aa and 93aa-101aa, respectively, and their amino acid sequences are GFSLSSHA (SEQ ID NO.1), INTGGSA (SEQ ID NO.2), VRGDSNDWT (SEQ ID NO.3), respectively. The total length of VL is 106 amino acids, the number of amino acids in the four domains of FR are 26, 17, 36 and 11 respectively, the number of amino acids in the three domains of LCDR are 6, 3 and 7 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-95aa respectively, and their amino acid sequences are: ESIFSY (SEQ ID NO.4), SAS, LGSYSSI (SEQ ID NO.5).

[0056] Example 4 Testosterone sandwich method rabbit monoclonal antibody mAb1 used for magnetic microparticle chemiluminescent immunoassay

[0057] 1. Detection principles and methods

[0058] The magnetic particle chemiluminescence immunoassay technology based on the principle of double antibody sandwich method is used. Testosterone antibody is labeled with biotin, and the biotinylated antibody is fixed to SA magnetic beads. ALP is coupled to testosterone complex rabbit monoclonal antibody mAb1. At the same time, testosterone, ALP substrate and corresponding buffer components are placed on the Cosma automatic magnetic particle chemiluminometer, and the instrument program is set for detection. The result is judged as 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 is 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.

[0059] 2. Composition of the magnetic particle chemiluminescence detection kit for testosterone detection

[0060] 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.

[0061] 2. mAb1 coupled with ALP: First, use 2-IT to reduce the antibody mAb1; 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.

[0062] 3. The washing buffer is conventional PBST at pH 7.4, containing 0.05% Proclin 300, and prepared into a 20-fold concentrated solution.

[0063] 4. Chemiluminescent colorimetric solution was purchased from Avid Biotechnology.

[0064] 5. The sample diluent contains 1% BSA, 0.05% Proclin 300 in PBST, which is sterilized by filtration.

[0065] 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.

[0066] 3. Testing of clinical testosterone samples

[0067] Clinical samples with different testosterone concentrations were processed, and the testosterone antibody was used as the coating antibody and the mAb1 antibody was used as the detection antibody. The clinical samples with different concentrations were detected using the above detection method.

[0068] Figure 3 It is a magnetic particle chemiluminescence method for detecting testosterone standard antigen. Figure 4 It is a magnetic microparticle chemiluminescence method for detecting clinical samples. As can be seen from the figure, the double antibody sandwich chemiluminescence platform detects testosterone standard antigen with a detection sensitivity of less than 0.5ng / ml. At the same time, the testosterone sandwich rabbit monoclonal antibody of this application is applied to the immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the magnetic chemiluminescence method is used to detect clinical samples. In the sample range of 0.4-16ng / mL, its clinical compound rate is greater than 0.95, which is significantly higher than the traditional competitive detection method, breaking through the limitations of the traditional competitive method.

[0069] 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, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A testosterone sandwich rabbit monoclonal antibody mAb1, characterized in that: The rabbit monoclonal antibody mAb1 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes HCDR1-3, the amino acid sequence of the HCDR-1 is shown in SEQ ID NO:1, the amino acid sequence of the HCDR-2 is shown in SEQ ID NO:2, and the amino acid sequence of the HCDR-3 is shown in SEQ ID NO:3; the light chain variable region includes LCDR1-3, the amino acid sequence of the LCDR-1 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR-2 is SAS, and the amino acid sequence of the LCDR-3 is shown in SEQ ID NO:

5.

2. The rabbit monoclonal antibody mAb1 according to claim 1, characterized in that The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

7.

3. An isolated polynucleotide, characterized in that Encodes the rabbit monoclonal antibody mAb1 described in any one of claims 1-2.

4. A nucleic acid construct, characterized in that The nucleic acid construct contains the polynucleotide of claim 3, which is operably linked to at least one expression control element.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4.

6. Use of the rabbit monoclonal antibody mAb1 according to any one of claims 1-2, the polynucleotide according to claim 3, the nucleic acid construct according to claim 4 and / or the recombinant expression vector according to claim 5 in the preparation of a detection reagent or kit for detecting testosterone.

7. A detection reagent or detection kit for detecting testosterone, characterized in that: The detection reagent or detection kit contains the rabbit monoclonal antibody mAb1 according to any one of claims 1-2.

8. The detection reagent or detection kit according to claim 7, characterized in that: The detection kit is an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the rabbit monoclonal antibody mAb1 is the detection antibody.

9. A method for preparing the rabbit monoclonal antibody mAb1 according to any one of claims 1 to 2, characterized in that: The method comprises expressing the rabbit monoclonal antibody mAb1 under conditions suitable for the expression of the rabbit monoclonal antibody mAb1 using a host cell containing the polynucleotide of claim 3, or the nucleic acid construct of claim 4, or the recombinant expression vector of claim 5, and recovering the expressed monoclonal antibody from the culture of the host cell.

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