A T3 sandwich rabbit monoclonal antibody mAb3 and its application

Abstract

The present application belongs to the field of immunoassay technology, and discloses a T3 sandwich rabbit monoclonal antibody mAb3 and its application. The T3 sandwich monoclonal antibody mAb3 provided by the present application comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, wherein the LCDR1 comprises an amino acid sequence NIYNS, wherein the LCDR2 comprises an amino acid sequence YAS, wherein the LCDR3 comprises an amino acid sequence QSHYGFSSYGAA; wherein the heavy chain variable region comprises HCDR1-3, wherein the HCDR1 comprises an amino acid sequence GFDVSSYN, wherein the HCDR2 comprises an amino acid sequence IDTGANA, wherein the HCDR3 comprises an amino acid sequence ARVFYDNSFDP. The monoclonal antibody of the present application can bind to the T3 complex with high specificity, and is suitable for double antibody sandwich immunoassay, and is applied to an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, wherein the double antibody sandwich chemiluminescence platform detects T3 standard antigen, and the detection sensitivity is less than 0.3ng / ml, and the magnetic chemiluminescence method detects clinical samples, and within the range of 1-80 pmol / L samples, the correlation with clinical comparison is good.

Description

Technical Field

[0001] The present application relates to the technical field of immunoassay, and in particular to a T3 sandwich rabbit monoclonal antibody and its application. Background Art

[0002] T3, or triiodothyronine, is an important hormone secreted by the thyroid gland and plays a vital role in the human body. In the human body, about 99.7% of T3 specifically binds to thyroid hormone binding protein (TBG) and exists in a bound state; while the truly metabolically active free T3 (FT3) accounts for only about 0.3%. T3 is involved in almost every physiological process of the body, including growth and development, metabolism, regulation of body temperature and heart rate, etc. It is one of the key factors in maintaining normal human metabolism. T3 can accelerate cell metabolism of substances such as carbohydrates, fats and proteins, thereby accelerating the body's metabolism. It works together with thyroxine (T4) to regulate the body's energy consumption and material conversion. The production of T3 is activated by thyroid-stimulating hormone (TSH). When T3 is released into the blood, its increased concentration will inhibit the production of TSH in the anterior pituitary, forming a negative feedback regulation mechanism to stabilize the level of thyroid hormone in the blood.

[0003] T3 is an important clinical indicator for evaluating thyroid function. Combined detection of FT3, FT4 and high-sensitivity TSH is the first choice for thyroid function assessment. T3 and FT3 are of great clinical significance for the diagnosis and efficacy testing of thyroid diseases such as hyperthyroidism and hypothyroidism. FT3, in particular, is of great significance for differential diagnosis of whether thyroid function is normal, hyperthyroid or hypothyroid, and is a specific indicator for diagnosing T3-type hyperthyroidism.

[0004] At present, the "gold standard" for T3 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 practice. 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] In order to solve the technical difficulties in the clinical application of the competition method for small molecules, it is urgently necessary to develop highly specific and sensitive monoclonal antibodies for the T3 sandwich method. It is crucial to break through the limitations of the traditional competition method and realize the double antibody sandwich method for T3 detection. Summary of the invention

[0006] In order to overcome the above technical problems, the present application provides a T3 sandwich rabbit monoclonal antibody mAb3 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 thyroid diseases.

[0007] In the first aspect, the present application provides a T3 sandwich rabbit monoclonal antibody mAb3, wherein the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, wherein LCDR1 comprises an amino acid sequence NIYNS (SEQ ID NO.1), wherein LCDR2 comprises an amino acid sequence YAS (SEQ ID NO.2), and wherein LCDR3 comprises an amino acid sequence QSHYGFSSYGAA (SEQ ID NO.3); wherein the heavy chain variable region comprises HCDR1-3, wherein HCDR1 comprises an amino acid sequence GFDVSSYN (SEQ ID NO.4), wherein HCDR2 comprises an amino acid sequence IDTGANA (SEQ ID NO.5), and wherein HCDR3 comprises an amino acid sequence ARVFYDNSFDP (SEQ ID NO.6).

[0008] 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 27, 17, 36 and 10 respectively, the number of amino acids in the three domains of LCDR are 5, 3 and 12 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 28aa-32aa, 50aa-52aa and 89aa-100aa respectively, and their amino acid sequences are: NIYNS (SEQ ID NO.1), YAS (SEQ ID NO.2), QSHYGFSSYGAA (SEQID NO.3).

[0009] Further, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, the full length of VH is 114 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 11 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-56aa and 93aa-103aa respectively, and their amino acid sequences are GFDVSSYN (SEQ ID NO.4), IDTGANA (SEQ ID NO.5), ARVFYDNSFDP (SEQ IDNO.6), respectively.

[0010] By adopting the above technical scheme, the T3 sandwich rabbit monoclonal antibody mAb3 of the present application can bind to T3 with high specificity and high affinity, and its affinity constant Ka can reach 7×10 8 L / mol.

[0011] On the other hand, the present application also provides an isolated polynucleotide, which encodes the rabbit monoclonal antibody mAb3.

[0012] 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.

[0013] On the other hand, the present application also provides a recombinant expression vector, which contains the polynucleotide or the nucleic acid construct.

[0014] 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.

[0015] On the other hand, the present application also provides the use of the rabbit monoclonal antibody mAb3, 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 T3.

[0016] On the other hand, the present application also provides a detection reagent or a detection kit for detecting T3, wherein the detection reagent or the detection kit contains the rabbit monoclonal antibody mAb3.

[0017] Furthermore, the detection kit is an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the rabbit monoclonal antibody mAb3 is the detection antibody.

[0018] On the other hand, the present application also provides a method for preparing the rabbit monoclonal antibody mAb3, which comprises expressing the rabbit monoclonal antibody mAb3 using the host cell under conditions suitable for the expression of the rabbit monoclonal antibody mAb3, and recovering the expressed monoclonal antibody from the culture of the host cell.

[0019] Compared with the prior art, the T3 sandwich rabbit monoclonal antibody of the present application can bind to T3 with high specificity and high affinity, and its affinity constant Ka can reach 7×10 8L / mol. The T3 sandwich rabbit monoclonal antibody of the present application can also be prepared into various immunoassay kits for detecting T3, especially, it can be used in immunoassay kits prepared by double antibody sandwich ELISA or chemiluminescence method. The double antibody sandwich chemiluminescence platform detects T3 standard antigen with a detection sensitivity of less than 0.3ng / ml. The magnetic chemiluminescence method detects clinical samples with a good correlation with R clinical comparison within the sample range of 1-80pmol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 1 is the electrophoresis diagram of the full-length amplified products of mAb3 heavy chain and light chain, and M is the DNA molecular weight marker.

[0021] Figure 2 This is the mAb3 sandwich ELISA method for detecting cross-protein and target antigen samples, and the vertical axis is the OD value of the detection.

[0022] Figure 3 The mAb3 magnetic microparticle chemiluminescence method is used to detect the T3 standard antigen. The horizontal axis is the T3 standard antigen concentration (ng / ml), and the vertical axis is the luminescence value detected. 2 =0.9951, linear detection range 0.3-10 ng / mL, the sample concentration calculation formula is: y=22386x-6614.3.

[0023] Figure 4 The mAb3 magnetic microparticle chemiluminescence method is used to detect clinical samples. The horizontal axis is the free T3 concentration (pmol / L) of the clinical samples, and the vertical axis is the luminescence value of the detection. 2 =0.9616, linear detection range 1-80pmol / L, the sample concentration calculation formula is: y=33915x-128398. DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1 Preparation of rabbit monoclonal antibody using the T3 sandwich method

[0026] 1) Preparation of immunogen

[0027] The purity of the immunogen in the preparation of T3 complex was above 90%, which met the purity requirement for the preparation of monoclonal antibodies.

[0028] 2) Animal immunization

[0029] The T3 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, with incomplete Freund's adjuvant emulsified 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.

[0030] 3) PBMCs isolation, specific B cell sorting, and cloning recombination

[0031] 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.

[0032] 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 .

[0033] 4) Preparation and purification of monoclonal antibodies

[0034] In order to obtain multiple rabbit monoclonal antibodies that recognize T3 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 containing recombinant rabbit monoclonal antibodies that recognize T3 small molecules was obtained after 120-144 hours of transfection. 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 divided and lyophilized. The purified antibody was named rabbit monoclonal antibody mAb3.

[0035] Example 2 Identification of rabbit monoclonal antibodies using the T3 sandwich method

[0036] 1) Specificity identification of rabbit monoclonal antibodies

[0037] The indirect ELSA method was used for detection. The ELISA plate was coated with cross protein and T3 complex antigen at a concentration of 1 μg / ml, incubated overnight at 4°C, and blocked with PBST containing 1% BSA. 4 The purified rabbit monoclonal antibody 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.

[0038] Figure 2 The results showed that the cross protein reacted negatively with the rabbit monoclonal antibody mAb3, with OD450 less than 0.2; the T3 complex reacted positively with the mAb3 antibody, with an OD value much higher than that of the cross protein, indicating that the T3 sandwich rabbit monoclonal antibody of the present application specifically recognizes the T3 complex.

[0039] 2) Determination of affinity constant of rabbit monoclonal antibody

[0040] The affinity constant (Ka) was determined by non-competitive ELISA.

[0041] 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;

[0042] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h;

[0043] 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;

[0044] Add enzyme-labeled secondary antibody: wash the plate 4 times with PBST, add 100 μL of 1:10000 diluted HRP enzyme-labeled goat anti-rabbit Ig secondary antibody to each well, and place at 37°C for 30 min;

[0045] 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;

[0046] Detection: Measure the absorbance value at a wavelength of 450nm (A450nm).

[0047] 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 monoclonal antibody mAb3 in the T3 sandwich method was 7×10 8 L / mol.

[0048] 3) Sandwich antibody pairing

[0049] In order to select the best combination of coating antibody and detection antibody, the T3 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 T3 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 mAb3 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 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.

[0050] Table 1 Antibody pairing experiment screening results

[0051]

[0052] It can be seen that the antibody mAb3 involved in the present application is best used for sandwich detection.

[0053] Example 3 Analysis of variable region genes and amino acid sequences of rabbit monoclonal antibodies

[0054] 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.

[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 light chain variable region of the rabbit monoclonal antibody mAb3 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, and the amino acid numbers of the four domains of FR are 27, 17, 36 and 10, respectively, and the amino acid numbers of the three domains of LCDR are 5, 3 and 12, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 28aa-32aa, 50aa-52aa and 89aa-100aa, respectively, and their amino acid sequences are: NIYNS (SEQ ID NO.1), YAS (SEQ ID NO.2), QSHYGFSSYGAA (SEQID NO.3). The total length of VH is 114 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 11 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-56aa and 93aa-103aa respectively, and their amino acid sequences are GFDVSSYN (SEQ ID NO.4), IDTGANA (SEQ ID NO.5), ARVFYDNSFDP (SEQ ID NO.6), respectively.

[0056] Example 4: T3 Sandwich Rabbit Monoclonal Antibody 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. The biotinylated T3 antibody is fixed to the SA magnetic beads, and the ALP is coupled to the T3 complex rabbit monoclonal antibody. At the same time, the T3, 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 T3 in the specimen. A standard curve is drawn according to the luminescence value of the measured standard, such as Figure 3 , the T3 concentration value in the sample to be tested can be obtained from the standard curve.

[0059] 2. Composition of the magnetic microparticle chemiluminescence detection kit for detecting T3

[0060] 1. SA magnetic beads combined with biotin-T3 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 T3 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. mAb3 coupled with ALP: First, use 2-IT to reduce the antibody mAb3; 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 T3 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 T3 clinical samples

[0067] Clinical samples with different T3 concentrations were processed, T3 antibody was used as coating antibody, mAb3 antibody was used as detection antibody, and clinical samples with different concentrations were detected using the above detection method. The test results are shown in Figure 4 .

[0068] 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 1-80pmol / L.

[0069] In summary, the T3 sandwich rabbit monoclonal antibody mAb3 of the present application is applied to the immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method. The double antibody sandwich chemiluminescence platform detects the T3 standard antigen with a detection sensitivity lower than 0.3 ng / ml. The magnetic chemiluminescence method detects clinical samples with a clinical compound rate of >0.95 within the sample range of 1-80 pmol / L, which is significantly higher than the traditional competitive detection method. The process is simple, breaking through the limitations of the traditional competitive method.

[0070] 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 T3 sandwich rabbit monoclonal antibody mAb3, characterized in that: The rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, the amino acid sequence of LCDR1 is NIYNS, the amino acid sequence of LCDR2 is YAS, and the amino acid sequence of LCDR3 is QSHYGFSSYGAA; the heavy chain variable region comprises HCDR1-3, the amino acid sequence of HCDR1 is GFDVSSYN, the amino acid sequence of HCDR2 is IDTGANA, and the amino acid sequence of HCDR3 is ARVFYDNSFDP.

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 mAb3 according to 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. 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.

7. Use of the rabbit monoclonal antibody mAb3 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 T3.

8. A detection reagent or detection kit for detecting T3, characterized in that: The detection reagent or detection kit contains the rabbit monoclonal antibody mAb3 according to any one of claims 1-2.

9. The detection reagent or detection kit according to claim 8, characterized in that: The detection kit is an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and the rabbit monoclonal antibody mAb3 is the detection antibody.

10. A method for preparing the rabbit monoclonal antibody mAb3 according to claim 1 or 2, characterized in that: The method comprises expressing the rabbit monoclonal antibody mAb3 using the host cell of claim 6 under conditions suitable for the expression of the rabbit monoclonal antibody mAb3, and recovering the expressed monoclonal antibody from the culture of the host cell.

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