Anti-t3 antibody mab 68 or antigen binding fragment, method of making and use

Abstract

The application belongs to the technical field of immune detection, and discloses an anti-T3 antibody mAb68 or an antigen binding fragment, which comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the LCDR2 is TAS, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 3; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1-3 are shown as SEQ ID NO. 4-6 respectively. The anti-T3 antibody mAb68 is used for detecting T3 standard antigen, the detection sensitivity is lower than 0.2 ng / ml, the clinical sample is detected by a magnetic chemiluminescence method, and in a sample range of 1-80 pmol / L, the correlation with R clinical comparison is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to anti-T3 antibody mAb68 or antigen-binding fragment and application thereof. BACKGROUND

[0002] Triidothyronine (T3) is an important component of thyroid hormone and a judgment index of thyroid hormone. In the human body, about 99.7% of T3 is specifically combined with thyroid hormone binding protein (TBG) to exist in a combined state; and only about 0.3% of T3 is free T3 (FT3) with metabolic activity. T3 is involved in almost every physiological process of the body, including growth and development, metabolism, regulation of body temperature and heart rate, etc., and it is one of the key factors to maintain normal metabolism of the human body. At the same time, T3 is a specific index for diagnosing hyperthyroidism and has important reference value for estimating whether hyperthyroidism recurs. The increase is seen in hyperthyroidism, triidothyronine type hyperthyroidism crisis in early stage, iodine deficiency goiter, high thyroid binding globulinemia, chronic active hepatitis, primary biliary cirrhosis, etc., and the decrease is seen in hypothyroidism, thyroid tumor, chronic thyroiditis, etc.

[0003] At present, the "gold standard" for T3 detection is mass spectrometry, but mass spectrometry detection requires a complex and time-consuming sample pretreatment process and high automation requirement of clinical, which is difficult to scale application. Therefore, the immunoassay method is usually used for detection in clinic. The immunoassay technology is simple to operate and rapid to detect, and is a practical alternative method for small molecule mass spectrometry detection. Because small molecules lack antigen epitopes, the competitive method is generally used for clinical immunoassay at present. The competitive method has low sensitivity, poor specificity, is prone to false positives, and has complex process.

[0004] Therefore, there is an urgent need to develop a high-specificity and high-sensitivity monoclonal antibody for T3 sandwich method to realize the detection of T3 by double antibody sandwich method. SUMMARY

[0005] In order to overcome the above technical problems, the present application provides an anti-T3 antibody mAb68 or antigen-binding fragment with good specificity and high affinity, which can successfully realize the rapid detection of T3 in an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method, and has important significance in the diagnosis and treatment of thyroid diseases.

[0006] In one aspect, the present application provides an anti-T3 antibody mAb68 or antigen binding fragment, comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is QNINSW (SEQ ID NO. 1), the amino acid sequence of the LCDR2 is TAS, and the amino acid sequence of the LCDR3 is QQGYIGSNVENA (SEQ ID NO. 3).

[0007] In certain embodiments, the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is GFSLSSYA (SEQ ID NO. 4), the amino acid sequence of the HCDR2 is INTGGFP (SEQ ID NO. 5), and the amino acid sequence of the HCDR3 is ARGHTINNNYYFAAYGMDP (SEQ ID NO. 6).

[0008] In certain embodiments, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 7 or an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO. 7; specifically, the VL is 109 amino acids in length, the 4 domains of the FR are 26, 16, 36 and 10 amino acids in length, the 3 domains of the LCDR are 6, 3 and 12 amino acids in length, the regions of the LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 49aa-51aa and 88aa-99aa, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are QNINSW (SEQ ID NO. 1), TAS (SEQ ID NO. 2), QQGYIGSNVENA (SEQ ID NO. 3), respectively.

[0009] In certain embodiments, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 8 or an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO. 8. Specifically, the VH is 125 amino acids in length, the 4 domains of the FR are 25, 17, 38 and 11 amino acids in length, the 3 domains of the HCDR are 8, 7 and 19 amino acids in length, the regions of the HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-114aa, respectively, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are GFSLSSYA (SEQ ID NO. 4), INTGGFP (SEQ ID NO. 5), ARGHTINNNYYFAAYGMDP (SEQ ID NO. 6), respectively.

[0010] In certain embodiments, the antigen binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, diabody and antibody minimal recognition unit; preferably, the antibody rest of the sequence is derived from one or more of the following species: rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey and human.

[0011] In another aspect, the present application also provides a biological material comprising a polynucleotide encoding the anti-T3 antibody mAb68 or antigen binding fragment; a vector carrying the polynucleotide; or a cell carrying the polynucleotide, or containing the vector, or capable of expressing the anti-T3 antibody mAb68 or antigen binding fragment.

[0012] In another aspect, the present application also provides a method for preparing the anti-T3 antibody mAb68 or antigen binding fragment, comprising culturing the cell; optionally, the cell is prepared by transforming a polynucleotide encoding the anti-T3 antibody mAb68 or antigen binding fragment into a cell, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, the transformation comprising co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0013] In certain embodiments, the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a 293 cell or a CHO cell.

[0014] In another aspect, the present application also provides the use of the anti-T3 antibody mAb68 or antigen binding fragment or the biological material in any of the following:

[0015] 1) detecting T3 for non-diagnostic and therapeutic purposes;

[0016] 2) preparing products for detecting T3;

[0017] 3) purifying T3;

[0018] 4) preparing products for purifying T3.

[0019] In another aspect, the present application also provides a detection reagent or a detection kit comprising the anti-T3 antibody mAb68 or antigen binding fragment or the biological material.

[0020] In certain embodiments, the detection kit is a double antibody sandwich ELISA or a chemiluminescence method prepared immunological detection kit.

[0021] The anti-T3 antibody mAb68 or antigen binding fragment of the present application can bind to T3 with high specificity and high affinity, and the affinity constant Ka can reach 8 x 1010 8 L / mol. The anti-T3 monoclonal antibody mAb68 of the present application can also be prepared into various immunoassay kits for detecting T3, especially, applied to the immunoassay kits prepared by double antibody sandwich ELISA or chemiluminescence method. The double antibody sandwich chemiluminescence platform detects T3 standard antigen, and the detection sensitivity is lower than 0.2 ng / ml. The magnetic chemiluminescence method detects clinical samples in the sample range of 1-80 pmol / L, and has good correlation with R clinical comparison. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is an electrophoretogram of the full-length amplified products of the heavy chain and light chain of mAb68, and M is a DNA molecular weight marker.

[0023] Figure 2 It is the detection of cross proteins and antigen samples by mAb68 sandwich ELISA method, and the vertical coordinate is the OD value of the detection.

[0024] Figure 3 It is the detection of T3 standard antigen by mAb68 magnetic particle chemiluminescence method, the horizontal coordinate is the concentration of T3 standard antigen (ng / ml), and the vertical coordinate is the luminescence value of the detection. The R 2 = 0.9951, the linear detection range is 0.2-10 ng / mL, and the sample concentration calculation formula is y = 415501x-238223.

[0025] Figure 4 It is the detection of clinical samples by mAb68 magnetic particle chemiluminescence method, the horizontal coordinate is the free T3 concentration (pmol / L) of the clinical sample, and the vertical coordinate is the luminescence value of the detection. The R 2 = 0.9556, the linear detection range is 1-80 pmol / L, and the sample concentration calculation formula is y = 35177x-142086. DETAILED DESCRIPTION

[0026] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods not specified in the following examples are generally carried out according to the conventional conditions, the conditions described in the laboratory manual or the conditions suggested by the manufacturer.

[0027] Example 1 Preparation of T3 sandwich method rabbit monoclonal antibody

[0028] 1) Preparation of immunogen

[0029] The T3 complex was prepared, and the immunogen purity was more than 90%, which met the purity requirement for preparing monoclonal antibodies.

[0030] 2) Animal immunization

[0031] The prepared T3 complex was emulsified with complete Freund's adjuvant at a volume ratio of 1:1, and a New Zealand white rabbit weighing about 2 kg was immunized by subcutaneous injection at a dose of 800 μg per rabbit. Two weeks later, the second immunization was performed at a dose of 400 μg per rabbit using incomplete Freund's adjuvant emulsified at a volume ratio of 1:1. After two immunizations, the tail blood was collected, and the serum titer was determined by gradient dilution ELISA. According to the standard that the OD450 of ELISA titer 128000 is greater than 1.0, it was determined whether to collect PBMCs or continue immunization, and the rabbit with the highest antibody titer was selected for PBMCs collection.

[0032] 3) PBMCs separation, specific B cell sorting, and recombinant cloning

[0033] The rabbit was fixed on the operating table in a supine position, the fur around the heart was removed, and the skin was disinfected with alcohol. A 50 ml syringe was used to puncture the most obvious part of the heart, and blood flowed into the syringe after the needle was inserted into the heart. After obtaining the required amount of blood, the needle was quickly removed, and the whole blood in the syringe was transferred to a sterile 50 ml tube. After mixing with an equal amount of PBS, it was slowly added dropwise to the top of the lymphocyte separation medium, and centrifuged at 400 x g for 30 minutes at room temperature. After centrifugation, the liquid surface was divided into four layers from top to bottom: yellow plasma layer, white film layer (single nucleus cell layer), separation medium layer, and red blood cell layer. The single nucleus cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium to obtain rabbit PBMCs.

[0034] The antigen-specific B cells were further sorted from the rabbit PBMCs for culture. The supernatant of the cultured B cells was screened for positive clones using an antigen-coated ELISA plate. The cells of the positive clones were collected, lysed, and the RNA was extracted and reverse transcribed into cDNA. The naturally paired rabbit monoclonal antibody light chain and heavy chain full-length sequences were amplified from the cDNA of the corresponding positive clones, and the rabbit monoclonal antibody expression vector was constructed by recombinant cloning method. The sequence was determined by sequencing, and the amplified full-length PCR product results are as follows Figure 1 .

[0035] 4) Preparation and purification of monoclonal antibodies

[0036] In order to obtain a plurality of strains of rabbit monoclonal antibodies recognizing T3 small molecules, the heavy chain and light chain genes of the rabbit monoclonal antibodies are loaded on an expression vector, the plasmid is transfected into Kek293 cells, and the culture supernatant containing the recombinant rabbit monoclonal antibodies recognizing T3 small molecules is obtained 120-144 hours after transfection. The cell suspension is collected, centrifuged to obtain the supernatant, and the antibodies are purified by affinity chromatography. The concentration of the purified monoclonal antibodies is determined by the BCA method, and then they are subpackaged, freeze-dried, and named as rabbit monoclonal antibodies mAb68.

[0037] Example 2 Identification of T3 sandwich method rabbit monoclonal antibodies

[0038] 1) Specific identification of rabbit monoclonal antibodies

[0039] Indirect ELSA method was used for detection. The cross protein and T3 complex antigen were used to coat the enzyme-labeled plate at a concentration of 1 μg / ml, 4°C overnight, and the enzyme-labeled plate was blocked with 1% BSA-PBST. 10 4 fold dilution of purified rabbit monoclonal antibodies, 37°C for 50 min, PBST plate washing 3 times, HRP-goat anti-rabbit IgG secondary antibody, 37°C for 50 min, PBST plate washing 5 times, TMB color development for 10 min, and stop solution, enzyme-labeled instrument measurement A450.

[0040] Figure 2 The results show that the cross protein reacts negatively with the rabbit monoclonal antibody mAb68, and the OD450 is less than 0.1; the T3 complex reacts positively with the mAb68 antibody, and the OD value is much higher than that of the cross protein, indicating that the T3 sandwich method rabbit monoclonal antibody of the application specifically recognizes the T3 complex.

[0041] 2) Determination of the affinity constant of the rabbit monoclonal antibody

[0042] Non-competitive ELISA method was used to determine the affinity constant (Ka).

[0043] Coating: Dilute the antigen with carbonate buffer to a concentration of 1, 0.5, 0.1, and 0.05 μg / mL, and add 100 μL / well to the 96-well enzyme-labeled plate for coating, 4°C for 24 h;

[0044] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h;

[0045] Add monoclonal antibodies: Wash the plate 4 times with PBST, dilute the rabbit monoclonal antibodies with carbonate buffer starting from 100 μg / mL, add 100 μL per well, and incubate at 37°C for 2 h;

[0046] Add enzyme-labeled secondary antibody: wash the plate with PBST for 4 times, 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;

[0047] Color development and termination: wash the plate with PBST for 4 times, add 100 μL of substrate color development solution to each well, and react at 37°C for 15 min in the dark; add 50 μL of 1.0 mol / L H2SO4 termination solution to each well to terminate the reaction;

[0048] Detection: measure the absorbance value (A450nm) at 450 nm.

[0049] Taking the logarithm of the antibody concentration as the abscissa and the OD value as the ordinate, an S-shaped curve is drawn, and the affinity constant Ka of the T3 sandwich method monoclonal antibody mAb68 is calculated to be 8×10 8 L / mol.

[0050] 3) Sandwich method antibody pairing

[0051] In order to select the best combination of coating antibodies and detection antibodies, the T3 complex antibody is coated on the enzyme-labeled plate and placed at 4°C overnight. The next day, the enzyme-labeled plate is taken out, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, washed with PBST for 3 times; add 100 μl of T3 complex to each well, with a concentration of 20 ng / ml, and incubate at 37°C for 1 hour; after incubation, take out the enzyme-labeled plate, wash with PBST for 3 times, and add HRP-labeled rabbit monoclonal antibody mAb68 as the detection antibody, incubate at 37°C for 1 hour. Wash with PBST for 5 times, add TMB substrate, and develop color at 37°C for 10 min. After taking out, add the termination solution, and measure the OD450 reading on the enzyme-labeled instrument. According to the OD value of the sample and the background value of the negative control, the most ideal antibody pair is selected, and the pairing screening results are shown in Table 1.

[0052] Table 1 Antibody pairing experiment screening results

[0053]

[0054] It can be seen that the antibody mAb68 involved in the present application is used for the best sandwich method detection.

[0055] Example 3 Analysis of monoclonal antibody variable region gene and amino acid sequence Take the recombinant plasmid of the antibody as the DNA template, design the light chain variable region and heavy chain variable region sequencing primers according to the 5' end vector sequence of the light chain and heavy chain on the template, and use the sequencer ABI 3730 for sequencing. The nucleotide sequences of the light chain and heavy chain variable regions of the rabbit monoclonal antibody are obtained by sequencing.

[0056] The nucleotide sequence of the light chain variable region and the heavy chain variable region were analyzed using IMGT / V-QUEST analysis software on the Internet at http: / / www.imgt.org, respectively. The amino acid sequence of the light chain variable region of the rabbit monoclonal antibody mAb68 is shown as SEQ ID NO. 7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8.

[0057] The VL is 109 amino acids long, and the 4 domains of the FR are 26, 16, 36 and 10 amino acids, respectively, and the 3 domains of the LCDR are 6, 3 and 12 amino acids, respectively, and the regions of the LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 49aa-51aa and 88aa-99aa, respectively, and the amino acid sequences are QNINSW (SEQ ID NO. 1), TAS (SEQ ID NO. 2) and QQGYIGSNVENA (SEQ ID NO. 3), respectively.

[0058] The VH is 125 amino acids long, and the 4 domains of the FR are 25, 17, 38 and 11 amino acids, respectively, and the 3 domains of the HCDR are 8, 7 and 19 amino acids, respectively, and the regions of the HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-114aa, respectively, and the amino acid sequences are GFSLSSYA (SEQ ID NO. 4), INTGGFP (SEQ ID NO. 5) and ARGHTINNNYYFAAYGMDP (SEQ ID NO. 6), respectively.

[0059] Example 4 T3 sandwich method rabbit monoclonal antibody for magnetic particle chemiluminescence immunoassay

[0060] 1. Detection principle and method

[0061] The magnetic particle chemiluminescence immunoassay technology based on the principle of double antibody sandwich method was used. The biotin-labeled T3 antibody was fixed with SA magnetic beads, and the ALP-coupled T3 complex rabbit monoclonal antibody was placed on the KOSMAY full-automatic magnetic particle chemiluminescence instrument together with T3, ALP substrate and corresponding buffer components, and the instrument program was set for detection. The result was positive according to the signal-to-noise ratio of the luminescence value 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 sample. The standard curve was drawn according to the luminescence value of the measured standard, and the T3 concentration value in the sample to be tested was obtained from the standard curve. Figure 3

[0062] 2. Composition of the magnetic particle chemiluminescence detection kit for detecting T3 ​

[0063] 1) SA magnetic beads combined with biotin-T3 antibody: take 50 μl magnetic beads into a 0.5 ml centrifuge tube, place on a magnetic stand, remove the supernatant after 1 min; wash the magnetic beads with 0.5 ml antibody diluent for 3 times; add a certain amount of biotin-labeled T3 antibody and rotate for 60 min at room temperature; resuspend in magnetic preservation buffer after magnetic separation, and the working concentration is 0.5 mg / ml.

[0064] 2) mAb68 coupled with ALP: first, reduce the antibody mAb68 using 2-IT; second, form ALP-SMCC intermediates, and finally, couple ALP-SMCC with the reduced antibody. After the coupling is completed, dilute to the working concentration using the ALP preservation buffer.

[0065] 3) Washing buffer: a conventional PBST with pH 7.4 containing 0.05% Proclin 300, prepared as a 20-fold concentrated solution.

[0066] 4) Chemiluminescence developing solution: purchased from Avid Biological.

[0067] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, sterilized by filtration.

[0068] 6) Standard: T3 small molecules, diluted to 5 μg / ml using PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin 300, sterilized by filtration and aseptically packaged.

[0069] 3, Detection of T3 clinical samples

[0070] Process different T3 concentrations of clinical samples, use T3 antibody as the coating antibody, mAb68 antibody as the detection antibody, and detect different concentrations of clinical samples by the above detection method, and the detection results are shown in Figure 4 .

[0071] According to the result data, the rabbit monoclonal antibody described in the application is used for a magnetic microparticle chemiluminescence immunoassay reagent, and in the range of 1-80 pmol / L samples, the correlation with clinical comparison is good.

[0072] In summary, the T3 sandwich rabbit monoclonal antibody mAb68 of the application is applied to the double antibody sandwich ELISA or chemiluminescence method prepared immunoassay kit, the double antibody sandwich chemiluminescence platform detects T3 standard antigen, and the detection sensitivity is lower than 0.2 ng / ml, the magnetic chemiluminescence method detects clinical samples, and in the range of 1-80 pmol / L samples, the clinical complex rate is >0.95, which is obviously higher than that of the traditional competition detection method, and the process is simple, which breaks through the limitation of the traditional competition method.

[0073] The embodiments are only illustrative of the present application, and are not intended to limit the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. An anti-T3 antibody mAb 68 or an antigen binding fragment thereof, characterized in that, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO. 7 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 7; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:

8.

2. The anti-T3 antibody mAb 68 or an antigen-binding fragment thereof according to claim 1, characterized in that, The antigen binding fragment of the antibody is one of F(ab')2, Fab', Fab, Fv, scFv or dsFv.

3. The anti-T3 antibody mAb 68 or an antigen-binding fragment thereof according to claim 1, characterized in that, The biological material is a polynucleotide encoding the anti-T3 antibody mAb68 or the antigen binding fragment thereof according to any one of claims 1-3, a vector carrying the polynucleotide, or a cell carrying the polynucleotide, or containing the vector, or capable of expressing the anti-T3 antibody mAb68 or the antigen binding fragment thereof according to any one of claims 1-3.

4. Biomaterial, characterized in that, The cell is a cell into which a polynucleotide encoding the anti-T3 antibody mAb68 or the antigen binding fragment thereof is transformed, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, the transformation comprising co-transformation of the heavy chain expression plasmid and the light chain expression plasmid into the cell.

5. A method for the preparation of the anti-T3 antibody mAb 68 or an antigen binding fragment thereof according to any one of claims 1 to 3, characterized in that, The cell is a eukaryotic cell. The cell is a mammalian cell.

6. The production method according to claim 5, wherein The cell is a 293 cell or a CHO cell.

7. The production method according to claim 6, wherein 9. Use of the anti-T3 antibody mAb68 or the antigen binding fragment thereof according to any one of claims 1-3 or the biological material according to claim 4 in any one of:

8. The preparation method according to claim 7, characterized in that, 1) detecting T3 for non-diagnostic and therapeutic purposes; 2) preparing a product for detecting T3; 3) purifying T3; 4) preparing a product for purifying T3. The detection reagent or the detection kit comprises the anti-T3 antibody mAb68 or the antigen binding fragment thereof according to any one of claims 1-3 or the biological material according to claim 4. The detection kit is a double antibody sandwich ELISA immunoassay kit or a chemiluminescence immunoassay kit.

10. A test reagent or test kit characterized in that, ​ 11. The detection reagent or detection kit according to claim 10, characterized by ​

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