T4 competition method paired antibody and application thereof in chemiluminescence platform

Abstract

The invention provides a T4 competition method pairing antibody. The T4 competition method pairing antibody comprises one of an mAb1 monoclonal antibody, an mAb2 monoclonal antibody, an mAb3 monoclonal antibody, an mAb4 monoclonal antibody, an mAb5 monoclonal antibody, an mAb6 monoclonal antibody, an mAb7 monoclonal antibody, an mAb8 monoclonal antibody and an mAb9 monoclonal antibody. The mAb1 monoclonal antibody, the mAb2 monoclonal antibody, the mAb3 monoclonal antibody, the mAb4 monoclonal antibody, the mAb5 monoclonal antibody, the mAb6 monoclonal antibody, the mAb7 monoclonal antibody, the mAb8 monoclonal antibody and the mAb9 monoclonal antibody are nanometer antibodies, and the amino acid sequences of the nanometer antibodies are shown as SEQ ID NO. 1 to SEQ ID NO. 9 respectively. The invention also provides an application of the T4 competition method pairing antibody in preparation of a chemiluminescence detection kit, and also provides the chemiluminescence detection kit. The antibody can replace the traditional T4 derivative antigen raw material, and only needs to be coupled with acridinium ester during application, so that the operation steps are simplified, the batch-to-batch difference of the raw materials can be reduced to a certain extent, and meanwhile, the research and development cost is saved for process development.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a T4 competitive method paired antibody and an application thereof in a chemiluminescence platform. Background Art

[0002] T4 (Thyroxine) is an important hormone secreted by the thyroid gland and is of great significance in clinical testing. T4 testing is mainly used to evaluate thyroid function and help diagnose and manage thyroid-related diseases. The following are the main significances of T4 clinical testing:

[0003] 1. Thyroid function assessment: including hyperthyroidism and hypothyroidism. Increased T4 level is one of the important indicators of hyperthyroidism. Common symptoms include palpitations, weight loss, anxiety, hand tremors, etc. Decreased T4 level is the main diagnostic basis for hypothyroidism. Common symptoms include fatigue, weight gain, fear of cold, dry skin, etc.

[0004] 2. Diagnosis and differential diagnosis of thyroid diseases: For example, primary and secondary thyroid diseases. Primary thyroid diseases (such as Graves' disease and Hashimoto's thyroiditis) usually show abnormalities in T4 and TSH (thyroid stimulating hormone). Secondary thyroid diseases (such as pituitary or hypothalamic lesions) may show a simultaneous decrease or increase in T4 and TSH. For example, in the case of thyroid nodules or tumors, T4 testing helps to assess whether thyroid nodules or tumors affect thyroid function.

[0005] 3. Treatment monitoring of thyroid disease: After antithyroid drugs, radioactive iodine therapy or surgery for hyperthyroidism, monitor T4 levels to evaluate efficacy and adjust treatment plans. During thyroid hormone replacement therapy (such as levothyroxine) for hypothyroidism, regularly test T4 levels to ensure appropriate dosage.

[0006] 4. Newborn screening: T4 test is an important indicator for newborn congenital hypothyroidism screening. Early diagnosis and treatment can prevent intellectual retardation and growth and development disorders.

[0007] 5. Thyroid function monitoring during pregnancy: Abnormal thyroid function (hyperthyroidism or hypothyroidism) during pregnancy may affect fetal development. T4 testing can help early detection and management of thyroid diseases during pregnancy.

[0008] 6. Auxiliary diagnosis of non-thyroid diseases: Certain systemic diseases (such as severe infection, liver disease, and kidney disease) may affect thyroid function, and T4 testing helps to assess the condition. Certain drugs (such as amiodarone and lithium) may affect thyroid function, and T4 testing helps to monitor drug side effects.

[0009] The total T4 in the serum is called total T4 (TT4), and the free part of T4 is called free T4 (FT4). After T4 enters the blood circulation, about 99.7% of it will bind to the thyroxine binding protein in the blood, of which about 60% will bind to TBG, 30% will bind to TBPA, and the rest will bind to albumin. Only about 0.05% of T4 is in the free state (FT4) in the blood. Under normal circumstances, a dynamic balance is maintained between the two forms. Only FT4 can enter the target cells and bind to the receptors to exert its physiological function. FT4 is the active part of the thyroid hormone in the circulating blood and has biological activity. Since the determination of FT4 is not affected by the content of serum binding proteins and other iodine-containing impurities, it is an important indicator for reflecting thyroid function, and its sensitivity and specificity are significantly higher than TT4. T4 in urine is mainly FT4 filtered out by the glomerulus, so it is also not affected by TBG. Therefore, it is generally believed that FT4 is one of the active components of thyroid hormone. The mechanism of the thyroid gland is closely related to the level of FT4 in the circulation, and can be used as an important indicator to distinguish between hyperthyroidism, hypothyroidism and subclinical states of thyroid function. Increased or decreased thyroid hormone secretion can lead to thyroid dysfunction and endocrine and metabolic disorders. Therefore, the correct detection of thyroid-related hormones is of great significance for the diagnosis and treatment of thyroid diseases.

[0010] In addition, TSH is a sensitive indicator of thyroid function, and the combined detection of TSH and T4 can improve the accuracy of thyroid disease diagnosis. For example, patients with hyperthyroidism have lower TSH and higher T4, while patients with hypothyroidism have higher TSH and lower T4. The clinical detection of T4 is of great significance in thyroid function assessment, disease diagnosis, treatment monitoring, and neonatal screening. Combining TSH with other thyroid-related indicators (such as T3 and thyroid antibodies) can more comprehensively evaluate the thyroid function status and provide an important basis for clinical diagnosis and treatment.

[0011] At present, the conventional method for determining thyroxine (T4) is to coat the biotinylated T4 antibody on a solid phase carrier, i.e., magnetic beads coupled with streptavidin, label acridinium ester and BSA on T4 derivatives (T4 derivatives must be coupled with BSA and then decoupled from acridinium ester), and use the above complex to compete for binding to T4 in the sample. However, the operation of the above method is relatively complicated, and the labeling batch-to-batch difference of T4 derivatives greatly affects its application and promotion in clinical practice. Summary of the invention

[0012] The technical problem to be solved by the present invention is to provide a T4 competitive method paired antibody and its application in a chemiluminescent platform, wherein the antibody can replace the traditional T4 derivative antigen raw material and only needs to be coupled with acridinium ester (without coupling with BSA) when used, which not only simplifies the operation steps, but also reduces the batch difference of raw materials to a certain extent, while saving R&D costs for process development.

[0013] The present invention adopts the following technical solutions to solve the above technical problems:

[0014] A T4 competition method paired antibody comprises one of mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8 and mAb9 monoclonal antibodies; the mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8 and mAb9 monoclonal antibodies are nano antibodies, and the amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.9 respectively.

[0015] As one of the preferred embodiments of the present invention, mAb2 or mAb6 monoclonal antibodies are specifically selected.

[0016] As one of the preferred embodiments of the present invention, the T4 competitive paired monoclonal antibody can specifically bind to the T4 first antibody to form a "competitive antibody pair" for detecting T4 by chemiluminescence.

[0017] An application of the T4 competitive method paired antibody as a detection antibody in the preparation of a chemiluminescence detection kit.

[0018] A chemiluminescence detection kit comprises the above-mentioned T4 competitive method paired antibody and T4 primary antibody.

[0019] As one of the preferred embodiments of the present invention, the T4 competitive paired antibody is also coupled with a chemiluminescent detection signal generating molecule; the chemiluminescent detection signal generating molecule is one of acridinium ester, isoluminol, alkaline phosphatase, horseradish peroxidase, and fluorescein.

[0020] As one of the preferred embodiments of the present invention, the chemiluminescent detection signal generating molecule is specifically an acridinium ester.

[0021] As one of the preferred embodiments of the present invention, when the chemiluminescent detection kit is used for T4 detection, a fully automatic chemiluminescent immunoassay analyzer is used to detect the T4 calibrator, and a standard curve is drawn and built into the computer software; then the clinical samples are tested, and the sample concentration is calculated according to the sample luminescence value; finally, the performance (linearity, clinical relevance) of the T4 chemiluminescent immunoassay kit is evaluated.

[0022] Detection method and principle:

[0023] (1) The T4 in the sample is contacted with a second reagent to form a mixture; the second reagent comprises a second antibody labeled with a chemiluminescent detection signal generating molecule (ie, T4 competitive paired antibody).

[0024] (2) contacting the mixture with a third reagent; the third reagent comprises magnetic particles coated with a first antibody, the first antibody having a specific binding affinity for T4, and the T4 competitive paired antibody in the second reagent can compete with the T4 small molecule in the sample for binding to the first antibody, thereby forming an "acridinium ester-labeled T4 competitive paired antibody / first antibody complex", or a "T4 small molecule / first antibody complex".

[0025] (3) After incubation and washing steps, the concentration of T4 in the sample is determined based on the chemiluminescence signal intensity (RLU) and the calibration curve. Within a certain range, RLU is inversely proportional to the concentration of thyroxine in the sample.

[0026] The advantages of the present invention compared to the prior art are:

[0027] (1) The present invention obtains some T4 competitive method paired antibodies through a large number of screenings. These antibodies can replace the traditional T4 derivative antigen raw materials and be used for chemiluminescent detection of T4; at the same time, when the antibodies are used, they only need to be coupled with acridinium ester once (no need to couple with BSA again), which reduces the number of coupling processes compared with the traditional method, simplifies the operation steps, and can also reduce the batch difference of raw materials to a certain extent, while saving R&D costs for process development;

[0028] (2) The present invention combines chemiluminescence and immune microparticles to provide a nearly homogeneous reaction system with a fast reaction speed and is widely applicable to T4 determination in general chemiluminescence analyzers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the binding activity of the T4 competitive paired antibody of the present invention;

[0030] Figure 2 is the competitive binding activity of the T4 competitive paired antibody of the present invention;

[0031] Figure 3 is a graph showing the clinical relevance of the T4 competitive paired antibody mAb2 of the present invention;

[0032] Figure 4 It is a graph showing the clinical relevance of the T4 competitive paired antibody mAb6 of the present invention. DETAILED DESCRIPTION

[0033] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the following embodiments are conventional reagents or methods in the art unless otherwise specified, and will not be repeated.

[0034] Example 1, preliminary screening of T4 competitive paired antibodies:

[0035] Alpacas were immunized with T4 monoclonal antibody (Anhui Qiancheng Biotechnology Co., Ltd., product number: MC09701, referred to as T4mAb), peripheral blood was taken to isolate PBMC cells, RNA was extracted and reverse transcribed (RNA extraction kit was B518651-0050 from Sangon Biotechnology, and reverse transcription kit was AE311-02 from Quanshijin Biotechnology). According to the alpaca antibody database information provided by the IMGT website, specific primers were designed to amplify light chain and heavy chain fragments. The amplified product was cloned into a phagemid vector and introduced into TG1 competent cells by electroporation, successfully constructing a library with a capacity of approximately 1×10 8 Subsequently, the antibody library strain was used to prepare recombinant phages with a titer of about 1×10 13 The recombinant phage library was used for subsequent alpaca antibody screening.

[0036] The prepared T4 mAb biotin protein (Example 8) was used to incubate the cells with Thermo Dynabeads. TM M-280 streptavidin was combined and T4 small molecule competitive elution method was used to perform multiple rounds of liquid phase screening on the recombinant phage of the alpaca immune library. The screening process was referred to books (Therapeutic antibody engineering, William R. Strohl and Lilam M. Strohl, 2012; Antibody Engineering Methods and Protocols, Patrick Chames, 2012.). Starting from the second round of screening, a large number of monoclonal colonies were picked from the bacterial culture plate and inoculated into 96 deep-well plates for culture. When the bacterial cell density reached OD 600 When the p-value was ≈0.3, IPTG was added and induced overnight at 30°C to prepare soluble antibody supernatant. Finally, monomeric soluble antibody supernatant containing Flag tag was obtained for subsequent experiments.

[0037] Example 2, ELISA screening of positive clones:

[0038] T4 mAb protein (Anhui Qiancheng Biotechnology Co., Ltd., product number: MC09701) was plated and stained with NaHCO 3Dilute it to 1 μg / ml with TPBS (PBS + 0.1% The wells were washed three times with Tween20, 3% skim milk powder dissolved in TPBS was used as a blocking agent, filled each well (about 250 μl), and blocked at 37°C for 1 h; washed three times with TPBS, soluble expression supernatant was added to the experimental group, 100 μl / well, and incubated at room temperature for 1 h; ELISA in the control group was added with soluble expression supernatant mixed with T4 small molecules at a final concentration of 1 μg / mL, 100 μl / well, and incubated at room temperature for 1 h; washed three times with TPBS, 1:5000 diluted Anti-Flag / HRP secondary antibody (purchased from Sigma, product number A8592) was added, 100 μl per well, and incubated at room temperature for 30 min; washed three times with TPBS, 100 μl of OPD o-phenylenediamine (purchased from Sigma, product number 78412) substrate working solution containing 0.1% hydrogen peroxide was added to each well for color development, and 100 μl of 1M sulfuric acid was added after about 10 min to stop, and OD was measured by microplate reader. 450 Absorbance value.

[0039] According to the ELISA results, the monoclone corresponding to the positive well was selected, and the absorbance values ​​of the corresponding wells of the control group ELISA were compared. The smaller the absorbance value of the latter and the larger the ratio of the experimental group to the control group, the more suitable it was to be selected as a positive clone. The plasmid contained in the positive clone bacteria was purified and sequenced. After analyzing the sequencing sequence, a total of 9 nanoantibodies with strong binding activity were obtained. The amino acid sequences are shown in SEQ ID NO.1 to 9, which are the T4 competitive paired antibody sequences required for the target.

[0040] Example 3, Construction of eukaryotic expression plasmids of 9 nanobodies:

[0041] The 9 nanoantibody DNA sequences (SEQ ID NO.10-18) screened in Example 2 were used as templates to design appropriate PCR primers (SEQ ID NO.19, SEQ ID NO.20), and restriction sites BamHI and XbaI were set at both ends. The PCR system refers to the full-form gold FastPfu fast high-fidelity DNA polymerase (Cat. No. AP221-01), including 25 μL 2*FastPfu Mix, 1 μL forward primer (SEQ ID NO.19), 1 μL reverse primer (SEQ ID NO.20), an appropriate amount of gene template, and ddH 2 The PCR reaction program was as follows: 95°C, 2 min; 95°C 20 s, 56°C 20 s, 72°C 15 s, 30 cycles; 72°C, 5 min.

[0042] After recovering the PCR product, pre-mix an appropriate volume of the enzyme digestion system, use BamHI and XbaI enzymes to digest the PCR product, and construct the variable region of the nanobody onto the pcDNA3.4 vector that was also digested by T4 DNA Ligase. The connection system is 10 μl.

[0043] Positive clones were selected, enzyme digestion verified and sequenced to obtain eukaryotic expression plasmids of 9 nanoantibodies. The plasmids of these two clones were transiently transfected into Expi293 suspension cells respectively, and the expression supernatant after transfection was purified using Protein A affinity chromatography gel of Tiandi Renhe Company to obtain the pure monoclonal antibody proteins of the corresponding clones, namely mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, and mAb9, and the amino acid sequences corresponded to SEQ ID NO.1 to 9 respectively.

[0044] Example 4, stable expression method of antibodies (taking mAb2 and mAb6 as examples):

[0045] (1) Construction of stable cell lines of CHO-K1-mAb2 or CHO-K1-mAb6 antibody proteins

[0046] ①Cell recovery

[0047] Take a tube of cells frozen in liquid nitrogen. The number of cells in the frozen cells is about 1×10 7 , quickly thaw it in a 37℃ water bath, and then disinfect the surface of the cryopreservation tube with alcohol cotton. Centrifuge at 300g for 5min, place in a clean bench to discard the supernatant, add 1mL of CHO complete medium preheated to 37℃, and then add to 20mL, resuspend the cells and place in a 125mL shake flask for culture. The culture conditions were set at 37℃, 5% CO 2 , rotation speed 120~130rpm.

[0048] ②Plasmid extraction

[0049] Inoculate the "DH5α strain stably expressing mAb2 antibody nucleic acid molecule" or "DH5α strain stably expressing mAb6 antibody nucleic acid molecule" (obtained by cloning) in advance. Inoculate the strain with 30 mL of 2×YT medium, culture overnight at 37°C with shaking, and extract the stably transfected plasmid pXC-mAb2 or pXC-mAb6 using a commercial plasmid extraction kit.

[0050] ③Cell transfection

[0051] The viability was greater than 95% and the cell density reached 2-3×10 6 The CHO cells without obvious aggregation were centrifuged at 300g for 5 min and the supernatant was discarded. 7Cells were resuspended in 200 μL Celetrix commercial electroporation solution. 25 μg of pXC-mAb2 or pXC-mAb6 plasmid was transfected each time. Transfection was performed on a Celetrix electroporator with a voltage set at 1250V.

[0052] ④Cell recovery and pressurization

[0053] Resuspend the transfected cells in CHO complete medium and place at 37°C, 5% CO 2 After recovery, the cells were centrifuged at 300 g for 5 min, the culture medium was discarded (1 mL was left for measuring the expression level), and CHO complete culture medium and MSX at a final concentration of 25 μM were added to adjust the cell density to 1×10 6 The cells were placed at 37°C and 5% CO 2 Culture in an incubator for 8 to 10 days until the viability exceeds 30%.

[0054] ⑤Monoclonal screening

[0055] Using Kangsheng commercial monoclonal culture medium, cells with a viability of more than 30% were diluted to 2.5 cells / mL, 200 μL per well, and spread over 96-well plates, for a total of 30 plates. The culture conditions were 37°C, 5% CO 2 , until the monoclonal growth completely covers the bottom of the well. The relative expression of supernatant protein was determined by ELISA, and after screening the monoclonal cells with higher expression, they were transferred to shake flasks for large-scale culture. Finally, the CHO-K1 monoclonal working cell bank was constructed and frozen in a liquid nitrogen tank for subsequent antibody expression.

[0056] (2) Expression of mAb2 or mAb6 in CHO-K1 suspension expression system

[0057] ①Cell recovery

[0058] Take out the CHO-K1 working cell line constructed in step (1) from the liquid nitrogen tank and quickly thaw it in a 37°C water bath. After thawing, resuspend the cells in 20 mL of CHO cell culture medium and culture them in a cell culture shaker for 48 hours.

[0059] ②Cell transfer and fed-batch culture

[0060] The revived cells were transferred to the fermentation medium and the cell density was adjusted to 0.5×10 6 / mL. On the 5th day (Day 5), the feeding operation was started, and on the 7th day (Day 7), the culture temperature was appropriately lowered to extend the cell production period.

[0061] ③Supernatant collection

[0062] The culture supernatant was collected on day 15 or when the cell viability dropped below 60%, the cell precipitate was removed by centrifugation, and the culture supernatant was retained for subsequent purification steps.

[0063] (3) Affinity purification of mAb2 or mAb6

[0064] ① Affinity column filling

[0065] Calculate the required amount of Protein A filler according to experimental requirements, load the commercial Protein A filler into the chromatography column, and wash the filler with PBS equilibration buffer to ensure that the column is in equilibrium.

[0066] ② Loading and cleaning

[0067] The cell culture supernatant collected by centrifugation was loaded onto the affinity column at a low flow rate. After loading, the column was washed with equilibration buffer for 10 column volumes. Then, the column was washed again with a pre-elution buffer at pH 5.0 for 10 column volumes to remove non-specific binding substances.

[0068] ③ Elution and neutralization

[0069] The target antibody protein was eluted with a citric acid eluent at pH 3.2. The eluted protein solution was immediately neutralized with 2MTris buffer to maintain protein activity. After the neutralized antibody solution was measured for concentration, it was dialyzed and replaced with PBS buffer and finally stored in aliquots.

[0070] Example 5: ELISA to detect the binding activity of T4 competitive paired antibodies:

[0071] Use of NaHCO 3Dilute T4 mAb to 1 μg / mL with buffer, add 100 μL per well to the ELISA plate, and let stand overnight at 4°C to complete coating. The next day, wash the ELISA plate three times with PBST (PBS + 0.1% Tween 20) cleaning solution, add 250 μL per well of blocking solution prepared with PBST (PBS + 0.1% Tween 20) and 2.5% skim milk powder, and block at 37°C for 1 hour. After blocking, wash the ELISA plate three times with PBST cleaning solution, dilute the 9 proteins mAb1 to mAb9 with blocking solution, with a starting concentration of 1 μg / mL, and continuously dilute in a 3-fold gradient to form 8 different gradients. Add the diluted antibodies to the ELISA plate, add 100 μL to each well, and incubate at 37°C for 2 hours. Wash the ELISA plate three times with PBST cleaning solution, then dilute the HRP-labeled goat anti-human secondary antibody with blocking solution, add it to the ELISA plate, 100 μL per well, and incubate at 37°C for 1 hour. After washing the ELISA plate three times with PBST cleaning solution, add 100 μL of PD colorimetric solution for colorimetric reaction. The colorimetric reaction time is 5 to 15 minutes, and the reaction is stopped according to the colorimetric effect. When the reaction is terminated, add 100 μL of 2M H 2 SO 4 Finally, use a microplate reader at wavelength OD 450 Detect absorbance value.

[0072] Results Figure 1 The analysis results show that mAb1 to mAb9 can bind to T4 mAb.

[0073] Example 6: ELISA to detect the competitive binding activity of T4 competitive paired antibodies:

[0074] Use of NaHCO 3Dilute T4 mAb to 1 μg / mL with buffer, add 100 μL per well to the ELISA plate, and let stand overnight at 4°C to complete coating. The next day, wash the ELISA plate three times with PBST (PBS + 0.1% Tween 20) cleaning solution, add PBST (PBS + 0.1% Tween 20) and 2.5% skim milk powder to prepare blocking solution, 250 μL per well, and block at 37°C for 1 hour. After blocking, wash the ELISA plate three times with PBST cleaning solution, dilute mAb1 and other 9 proteins with blocking solution to a final concentration of 1ug / mL, and then dilute T4 small molecules with the diluents of the above 9 antibodies. The starting concentration of T4 small molecules is 1ng / mL, and dilute continuously in a 3-fold gradient to form 7 different gradients. Add the diluted mixture to the ELISA plate, add 100 μL per well, and incubate at 37°C for 2 hours. Wash the ELISA plate three times with PBST cleaning solution, then dilute the HRP-labeled goat anti-human secondary antibody with blocking solution, add 100 μL to each well of the ELISA plate, and incubate at 37°C for 1 hour. After washing the ELISA plate three times with PBST cleaning solution, add 100 μL OPD colorimetric solution for colorimetric reaction. The colorimetric reaction time is 5 to 15 minutes, and the reaction is stopped according to the colorimetric effect. When the reaction is terminated, add 100 μL 2M H 2 SO 4 Finally, use a microplate reader at wavelength OD 450 Detect absorbance value.

[0075] Results Figure 2 The analysis results show that mAb1 to mAb9 antibodies can compete with T4 small molecules for binding to T4 mAb.

[0076] In addition, when using the competition method to detect the T4 content in the blood, in order to achieve a sufficient linear width, the affinity of the T4 competition method paired antibody should not be too high. Figure 1 , 2 As a result, mAb2 and mAb6 with medium and low affinity were selected for the subsequent preparation of chemiluminescent detection reagents.

[0077] Example 7, Preparation of Streptavidin Magnetic Beads:

[0078] (1) Magnetic bead activation

[0079] 100 mg of 1 μm compliant magnetic beads (from Suzhou Weidu Biotechnology Co., Ltd., catalog number CMP1001CA) were weighed, separated in the magnetic field of a magnetic stand, and washed twice with 10 mL of 0.1 M MES (pH 6.5) buffer. Subsequently, 10 mL of 0.1 M MES buffer (pH 6.5) and 20 mg of EDC were added and reacted at room temperature for 20 min to complete the activation of the magnetic beads.

[0080] (2) Coupling

[0081] 2 mg of streptavidin protein (supplier: Roche, catalog number: 28903727103) was added to the activated magnetic beads, and incubated with stirring at room temperature for 3 h to complete the coupling.

[0082] (3) Closed treatment

[0083] After the coupling is completed, the magnetic beads are separated in a magnetic field, 5 mL of 5% BSA solution is added to resuspend the magnetic beads, and incubated at room temperature for 3 h to complete the blocking.

[0084] (4) Magnetic bead cleaning and storage

[0085] After blocking, the magnetic beads were separated in a magnetic field and washed three times with a buffer containing 50 mM MOPS (pH 8.0), 5% betaine, 0.1% Tween-20, 0.1% BSA and 0.1% PC300. Finally, the magnetic beads were resuspended to a final concentration of 1% and stored for later use.

[0086] Example 8, biotinylation of T4 mAb (T4 mAb biotin):

[0087] (1) Ultrafiltration concentration and liquid replacement

[0088] 1 mg of T4 mAb was concentrated using a 10 kDa ultrafiltration concentrator and exchanged into PBS.

[0089] (2) Coupling

[0090] NHS-PEG12-biotin (Thermo Fisher, catalog number: 21312) was added to the antibody solution at a molar ratio of "antibody:biotin" of "1:10" and coupled at room temperature for 1 hour.

[0091] (3) Ultrafiltration concentration and liquid replacement

[0092] Use a 10kDa ultrafiltration concentrator to concentrate the biotinylated antibody, remove free biotin, and replace the solution with PBS. Adjust the antibody concentration to 0.5 mg / mL, add glycerol and store for later use, which is T4 mAb biotin.

[0093] Example 9, Acridinium ester coupling components of T4-BSA, mAb2 and mAb6:

[0094] (1) Coupling

[0095] 1 mg T4-BSA (thyroxine T4 and bovine serum albumin complex), mAb2 or mAb6 was dissolved in 20 mM MES6.5, and NSP-SA-NHS (Suzhou Yake, catalog number: Y0080) was added at a "protein: acridinium ester" molar ratio of "1:20" and incubated at room temperature for 3 h.

[0096] (2) Fluid exchange

[0097] Use a 3kDa ultrafiltration concentrator or a 3KDa dialysis bag to replace the protein after coupling with acridinium ester into 20mM MES6.5 150mM NaCl, adjust the protein concentration to 0.5mg / mL, add 0.1% BSA and 40% glycerol to a final concentration, and store at low temperature and away from light for later use, which is the chemiluminescent marker T4-BSA-acridinium ester, mAb2-acridinium ester or mAb6-acridinium ester.

[0098] Embodiment 10, chemiluminescent reagent:

[0099] When the detection antigen is FT4, the chemiluminescent reagent of this embodiment includes an M component, an R1 component and an R2 component.

[0100] Wherein, the M component is the streptavidin magnetic beads prepared in Example 7; the working concentration of the magnetic beads is 0.75 mg / mL, and the buffer solution is composed of 50 mM Tris-HCl PH7.4, 150 mM NaCl, 0.1% Tween 20, 0.5% BSA, and 0.1% proclin 300. According to the content of each component of the reagent M component, each component is mixed in the same container and mixed evenly to obtain the product.

[0101] The R1 component is composed of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% BSA, 1% trehalose, 0.1% proclin 300, and 2.3 ug / ml biotinylated antibody T4 mAb biotin (prepared in Example 8). According to the content of each component of the reagent R1 component, each component is mixed in the same container and mixed evenly to obtain the product.

[0102] The R2 component is composed of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% BSA, 1% trehalose, 0.1% proclin 300 and 0.2 ug / mL T4-BSA-acridinium ester, mAb2-acridinium ester or mAb6-acridinium ester (prepared in Example 9). According to the content of each component of the reagent R2 component, each component is mixed in the same container and mixed evenly to obtain the obtained product.

[0103] In the above, the reagent containing T4-BSA is referred to as T4-BSA kit, the reagent containing mAb2 is referred to as mAb2 kit, and the reagent containing mAb6 is referred to as mAb6 kit.

[0104] Test Example 1

[0105] This test example uses the components M, R1, and R2 in Example 10 to form a chemiluminescent detection kit for detecting TT4 or FT4, and tests its related performance.

[0106] 1. Chemiluminescence analyzer detection method:

[0107] Take the Feipeng Shine i1000 fully automatic chemiluminescence immunoassay as an example: first add 50μL R1 component, add 50μL sample, add 50μL M component, add 50μL R2 component, and incubate at 37℃ for 5min. Use a magnetic separator to separate and remove the supernatant, wash twice with cleaning solution, add substrate to react and detect the luminescence value; within a certain range, RLU is inversely proportional to the thyroxine concentration.

[0108] The system will analyze the set concentration of the calibration material and the measured luminescence value to automatically generate a calibration curve. When measuring a sample, the instrument will automatically calculate the corresponding concentration value of the sample through the calibration curve based on the luminescence signal of the sample.

[0109] 2. Sensitivity detection:

[0110] Using the zero concentration calibrator as a sample, mAb2 reagent and mAb6 reagent were used to repeat the measurement 20 times according to the above-mentioned fully automatic chemiluminescence immunoassay detection method, and the relative luminescence value (RLU) of each 20 measurement results was obtained, and the average value M and standard deviation SD were calculated, and the RLU values ​​X1 and X2 corresponding to M+2SD were obtained respectively. According to the concentration-RLU value between the zero concentration calibrator A and the adjacent calibrator B, two-point regression fitting was performed to obtain a linear equation, and the concentration value corresponding to X1 or X2 was obtained, which was the minimum detection limit. The minimum detection limit determined by the mAb2 reagent in the present invention is 0.07ng / dL, and the minimum detection limit determined by the mAb6 reagent is 0.08ng / dL.

[0111] 3. Linear range detection:

[0112] A high concentration sample close to 300 nmol / L was added with an appropriate amount of ANS to dissociate serum total T4, and then gradiently diluted with normal saline, with the dilution multiples being 1 / 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32, to prepare a total of 6 sample solutions of different concentrations, namely 300 nmol / L, 150 nmol / L, 75 nmol / L, 37.5 nmol / L, 18.75 nmol / L, and 9.37 nmol / L. The concentration of each dilution gradient sample was determined using a fully automatic chemiluminescence immunoassay analyzer, and each concentration was measured three times, and the average value of each measurement was calculated.

[0113] The linear regression analysis was performed with the dilution concentration as the independent variable and the average value of the measurement results as the dependent variable, and the regression equation and correlation coefficient r were calculated. The test data are shown in Tables 1, 2 and 3. The results showed that the regression equations of the luminescent reagent prepared by mAb2 and the luminescent reagent prepared by mAb6 were y=1.002x-0.2009 and y=1x-0.4341, respectively, and the correlation coefficients were R 2 =0.9998, R 2 =0.9999. From the experimental results in the table, compared with the control example using T4-BSA reagent, the sensitivity of mAb2 reagent and mAb6 reagent is better, and there is no significant difference in the curves of the three kits.

[0114] Table 1. Linear range of T4-BSA kit

[0115]

[0116] Table 2. mAb2 kit linear range detection

[0117]

[0118] Table 3. mAb6 kit linear range detection

[0119]

[0120]

[0121] 4. Repeatability and precision:

[0122] A quality control product with a FT4 concentration of 3.2 ng / dL was used as a sample and the measurement was performed according to the fully automatic chemiluminescence immunoassay detection method. The three reagents were used to repeat the measurement of the quality control product 10 times, and the measurement means and standard deviations were calculated respectively. The results are shown in Table 4.

[0123] It can be seen from Table 4 that the repeatability and accuracy of the detection kit prepared by mAb2 or mAb6 can meet the requirements of clinical application.

[0124] Table 4. Repeatability and precision test of the kit of Experimental Example 1

[0125]

[0126]

[0127] 5. Clinical relevance testing:

[0128] Ten clinical samples of FT4 with Siemens electrochemiluminescence values ​​were used, and the reagents in Example 10 were used to perform correlation tests on the clinical samples using a fully automatic chemiluminescence immunoassay analyzer. The test results are shown in Figure 3 , Figure 4 The results show that the luminescence kit prepared with mAb2 has a good clinical relevance for FT4 sample detection, with a correlation coefficient of R 2 =0.965. The luminescence kit prepared using mAb6 has a reasonable clinical relevance for FT4 sample detection, with a correlation coefficient of R 2 =0.9779.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A T4 competitive method paired antibody, characterized in that: It includes one of mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8 and mAb9 monoclonal antibodies; the mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8 and mAb9 monoclonal antibodies are nano antibodies, and the amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.9 respectively.

2. The T4 competitive paired antibody according to claim 1, characterized in that: Specifically select mAb2 or mAb6 monoclonal antibodies.

3. The T4 competitive paired antibody according to claim 1, characterized in that: The T4 competitive paired monoclonal antibody can specifically bind to the T4 first antibody to form a "competitive antibody pair" for detecting T4 by chemiluminescence.

4. Use of the T4 competitive paired antibody as claimed in any one of claims 1 to 3 as a detection antibody in the preparation of a chemiluminescence detection kit.

5. A chemiluminescent detection kit, characterized in that: The method comprises the T4 competitive method paired antibody according to any one of claims 1 to 3 and the T4 first antibody.

6. The chemiluminescent detection kit according to claim 5, characterized in that: The T4 competitive method paired antibody is also coupled with a chemiluminescent detection signal generating molecule; the chemiluminescent detection signal generating molecule is one of acridinium ester, isoluminol, alkaline phosphatase, horseradish peroxidase, and fluorescein.

7. The chemiluminescent detection kit according to claim 6, characterized in that: The chemiluminescent detection signal generating molecule is specifically acridinium ester.

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