Interleukin 12 p70 antibodies and interleukin 12 p70 detection products containing the same

Abstract

The application belongs to the technical field of biological detection, and relates to an interleukin 12p70 antibody and an interleukin 12p70 detection product containing the same. The amino acid sequence of the heavy chain complementarity determining region 1 of the interleukin 12p70 antibody or an antigen binding fragment thereof is shown as SEQ ID NO. 1, the amino acid sequence of the heavy chain complementarity determining region 2 is shown as SEQ ID NO. 2, the amino acid sequence of the heavy chain complementarity determining region 3 is shown as SEQ ID NO. 3, the amino acid sequence of the light chain complementarity determining region 1 is shown as SEQ ID NO. 4, the amino acid sequence of the light chain complementarity determining region 2 is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain complementarity determining region 3 is shown as SEQ ID NO. 6. The interleukin 12p70 antibody or the antigen binding fragment thereof of the application can specifically and highly affinitively recognize and combine with interleukin 12p70, and has good stability. The interleukin 12p70 detection product of the application containing the interleukin 12p70 antibody or the antigen binding fragment thereof of the application can be used for simply and conveniently operating, rapidly reacting, highly sensitively and specifically detecting interleukin 12p70.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to interleukin-12p70 antibodies and interleukin-12p70 detection products containing them. Background Technology

[0002] Interleukin-12p70 (IL-12p70) is a heterodimeric cytokine composed of p35 and p40 subunits. It is primarily secreted by monocytes, macrophages, dendritic cells, and neutrophils, and is one of the core molecules in the body's immune regulatory network. IL-12p70 can induce the proliferation and differentiation of T cells and NK cells, promote the secretion of interferon-gamma (IFN-γ), and enhance the body's cellular immune response, playing a crucial role in anti-infection, anti-tumor activity, and the development of autoimmune diseases.

[0003] The detection of IL-12p70 is of great significance for the auxiliary diagnosis, disease monitoring and prognosis assessment of related diseases. Currently, commonly used detection methods include enzyme-linked immunosorbent assay (ELISA), immunochromatography, chemiluminescence immunoassay, etc. Among these detection methods, the specificity and sensitivity of the antibody are the core factors that determine the accuracy of the detection method.

[0004] Existing IL-12p70 antibodies have several shortcomings: some antibodies have poor specificity and are prone to cross-reaction with IL-23 or p35 / p40 monomers, leading to false positive results; some antibodies have low affinity and insufficient detection sensitivity, failing to meet the detection requirements of low-concentration IL-12p70 samples; at the same time, detection products based on existing antibodies suffer from poor stability, complex operation, and long detection cycles, making them difficult to meet the needs of rapid clinical testing.

[0005] Therefore, developing an IL-12p70 antibody with high specificity, strong affinity, and good stability, as well as an IL-12p70 detection product with simple preparation process, accurate and rapid detection, and high stability, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] The primary objective of this invention is to provide an antibody against interleukin 12p70 or its antigen-binding fragment, which can recognize and bind to interleukin 12p70 with high specificity and high affinity, and with good stability.

[0007] To achieve this objective, in a basic embodiment, the present invention provides an interleukin-12p70 antibody or an antigen-binding fragment thereof, said interleukin-12p70 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region includes a heavy chain complementarity determination region and a heavy chain framework region, and the light chain variable region includes a light chain complementarity determination region and a light chain framework region. The heavy chain complementarity-determining region (LCD) includes LCD 1, LCD 2, and LCD 3. The amino acid sequence of LCD 1 is shown in SEQ ID NO.1, the amino acid sequence of LCD 2 is shown in SEQ ID NO.2, and the amino acid sequence of LCD 3 is shown in SEQ ID NO.3. The light chain complementarity-determining region includes light chain complementarity-determining region 1, light chain complementarity-determining region 2, and light chain complementarity-determining region 3. The amino acid sequence of light chain complementarity-determining region 1 is shown in SEQ ID NO.4, the amino acid sequence of light chain complementarity-determining region 2 is shown in SEQ ID NO.5, and the amino acid sequence of light chain complementarity-determining region 3 is shown in SEQ ID NO.6.

[0008] In a preferred embodiment, the present invention provides an interleukin 12p70 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0009] In a preferred embodiment, the present invention provides an interleukin-12p70 antibody or an antigen-binding fragment thereof, wherein the interleukin-12p70 antibody is a rabbit-derived antibody.

[0010] The second objective of this invention is to provide an interleukin-12p70 detection product that is easy to operate, has a rapid response, high sensitivity, and high specificity for detecting interleukin-12p70.

[0011] To achieve this objective, in a basic implementation, the present invention provides an interleukin-12p70 detection product, which is based on the principle of immunoassay and includes an interleukin-12p70 antibody or its antigen-binding fragment as described above.

[0012] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be called "VH," and the variable domain of the light chain can be called "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The variable region of the light or heavy chain consists of a frame region interrupted by three hypervariable regions called "complementarity-determining regions" or "CDRs." The frame region (FR) of the antibody, which is the combination of the light and heavy chains, plays a role in locating and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0013] The “framework” or “FR” region refers to the region outside of those areas defined as CDRs for antibody variable domains. Each antibody variable domain frame can be further subdivided into adjacent regions FR1, FR2, FR3, and FR4 separated by CDRs.

[0014] Typically, the variable regions VH / VL of heavy and light chains can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0015] In this invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three complementary determining regions (i.e., high-variability regions) of the heavy chain variable region, and correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three complementary determining regions (i.e., high-variability regions) of the light chain variable region.

[0016] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the interleukin-12p70 detection product is a labeled antibody test strip, an enzyme-linked immunosorbent assay (ELISA) kit, or a chemiluminescence immunoassay kit. All of these detection products are developed based on the specific reaction between antigen and antibody, and all employ the "sandwich" principle of double antibody sandwich detection of antigen.

[0017] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the interleukin-12p70 detection product is a labeled antibody test strip. The labeled antibody test strip includes a base plate and a sample pad, a labeling pad, a detection pad, and a sample absorption pad that are sequentially stacked on the base plate. The labeled pad is coated with a label containing an interleukin-12p70 antibody or its antigen-binding fragment. The test pad is equipped with a test line and a control line. The test line is coated with another interleukin-12p70 antibody or its antigen-binding fragment. The interleukin-12p70 antibody or its antigen-binding fragment, or the other interleukin-12p70 antibody or its antigen-binding fragment, is the interleukin-12p70 antibody or its antigen-binding fragment as described above.

[0018] In a preferred embodiment, the present invention provides an interleukin 12p70 detection product, wherein the marker is selected from one or more of colloidal gold, colored microspheres, time-resolved fluorescent microspheres, and quantum dot microspheres.

[0019] This invention, based on the principle of antigen-antibody reaction, utilizes colloidal gold, colored microspheres, time-resolved fluorescent microspheres, and / or quantum dot microspheres to label interleukin-12p70 monoclonal antibodies or their antigen-binding fragments, and then immobilizes the labels on a glass fiber membrane. Another monoclonal antibody or its antigen-binding fragment of interleukin-12p70 is coated onto a detection pad (e.g., an NC membrane). Based on the principle of antigen-antibody reaction, detection can be performed visually or using a compatible instrument within the detection time. If interleukin-12p70 is present in the sample, a double-antibody sandwich structure is formed, resulting in a visible band or a light intensity signal on the instrument; if interleukin-12p70 is absent in the sample, no band appears on the NC membrane or no light intensity signal is detected on the instrument. The presence or absence of the signal allows for a negative / positive determination, or the intensity of the light signal allows for a prediction of the analyte load.

[0020] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the colloidal gold particle size is 40-100 nm, and can be selected as 40, 50, 60, 80, 100 nm, etc.

[0021] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the mass ratio of an interleukin-12p70 antibody or its antigen-binding fragment to colloidal gold is (0.04-0.32):1, optionally 0.04:1, 0.08:1, 0.12:1, 0.16:1, 0.20:1, 0.24:1, 0.28:1, or 0.32:1.

[0022] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the particle size of the colored microspheres is 100-300 nm, and can be selected from 100, 200, 300 nm, etc.

[0023] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the mass ratio of an interleukin-12p70 antibody or its antigen-binding fragment to colored microspheres is (0.1-0.4):1, optionally 0.1:1, 0.2:1, 0.3:1, or 0.4:1.

[0024] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the particle size of the time-resolved fluorescent microspheres is 100-300 nm, optionally 100, 200, or 300 nm.

[0025] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the mass ratio of an interleukin-12p70 antibody or its antigen-binding fragment to time-resolved fluorescent microspheres is (0.1-0.4):1, optionally 0.1:1, 0.2:1, 0.3:1, or 0.4:1.

[0026] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the particle size of the quantum dot microspheres is 100-300 nm, optionally 100, 200, or 300 nm.

[0027] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the mass ratio of an interleukin-12p70 antibody or its antigen-binding fragment to quantum dot microspheres is (0.1-0.4):1, optionally 0.1:1, 0.2:1, 0.3:1, or 0.4:1.

[0028] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the control line is coated with an anti-antibody, the anti-antibody being an antibody from another species against the interleukin-12p70 antibody or its antigen-binding fragment.

[0029] In a preferred embodiment, the present invention provides an interleukin 12p70 detection product, wherein the detection line is coated with 0.5-2 mg / mL (optionally 0.5, 1.0, 1.5, 2.0 mg / mL) of another interleukin 12p70 antibody or its antigen-binding fragment; and / or the control line is coated with 0.5-2 mg / mL (optionally 0.5, 1.0, 1.5, 2.0 mg / mL) of goat anti-rabbit IgG polyclonal antibody.

[0030] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein the diluent for one type of interleukin-12p70 antibody, another type of interleukin-12p70 antibody, and the goat anti-rabbit IgG polyclonal antibody is a 10-50 mM PB buffer containing trehalose, wherein the concentration of trehalose is 10-50 mM (optional 10, 20, 30, 40, 50 mM), that is, each 100 mL of diluent contains 0.3423-1.7115 g (optional 0.3423, 0.6846, 1.0269, 1.3692, 1.7115 g) of trehalose.

[0031] In a preferred embodiment, the present invention provides an interleukin-12p70 detection product, wherein: The interleukin-12p70 antibody or its antigen-binding fragment described above is the interleukin-12p70 antibody or its antigen-binding fragment as described above. Furthermore, the amino acid sequence of the heavy chain complementarity-determining region 1 of the other interleukin-12p70 antibody or its antigen-binding fragment is shown in SEQ ID NO. 9, the amino acid sequence of the heavy chain complementarity-determining region 2 is shown in SEQ ID NO. 10, the amino acid sequence of the heavy chain complementarity-determining region 3 is shown in SEQ ID NO. 11, the amino acid sequence of the light chain complementarity-determining region 1 is shown in SEQ ID NO. 12, the amino acid sequence of the light chain complementarity-determining region 2 is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain complementarity-determining region 3 is shown in SEQ ID NO. 14.

[0032] In a preferred embodiment, the present invention provides an interleukin 12p70 detection product, wherein the amino acid sequence of the heavy chain variable region of another interleukin 12p70 antibody or its antigen-binding fragment is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16.

[0033] This invention also provides an interleukin-12p70 detection kit, which comprises the interleukin-12p70 detection product and a housing as described above, wherein the interleukin-12p70 detection product is disposed inside the housing. Preferably, the housing comprises a detachably connected upper cover and a lower cover, wherein the upper cover is provided with an observation window and a sample application port. The shapes of the observation window and the sample application port are not specifically limited; the observation window may be, for example, square, located above the test line and control line of the test strip, for observing the test results; the sample application port may be, for example, a circular hole with a diameter of 0.5-1 cm, located above the sample pad.

[0034] The beneficial effects of the present invention are that the interleukin 12p70 antibody or its antigen-binding fragment of the present invention can recognize and bind interleukin 12p70 with high specificity and high affinity, and has good stability; the interleukin 12p70 detection product of the present invention, including the interleukin 12p70 antibody or its antigen-binding fragment of the present invention, can detect interleukin 12p70 with simple operation, rapid reaction, high sensitivity and high specificity.

[0035] Therefore, using the interleukin-12p70 detection product of this invention for sample testing can monitor the body's immune status and provide auxiliary diagnosis for inflammatory diseases. In particular, using the labeled antibody test strip of this invention, the entire process can yield test results in 10-20 minutes, which is rapid and efficient, helping medical personnel to obtain test results in a timely manner and make comprehensive judgments and timely treatments based on the test results. Attached Figure Description

[0036] Figure 1The image shows the SDS-PAGE electrophoresis results of the interleukin 12p70 monoclonal antibody prepared in Example 1. Lane 1 is the protein molecular weight standard, lane 2 is interleukin 12p70 monoclonal antibody 1, and lane 3 is interleukin 12p70 monoclonal antibody 2.

[0037] Figure 2 This is a schematic diagram of the top cover of the interleukin 12p70 detection kit in Example 2, which includes an observation window 1 and a sample application well 2.

[0038] Figure 3 This is a schematic diagram of the bottom cover of the interleukin 12p70 detection kit in Example 2, which includes the detection card strip area 3.

[0039] Figure 4 This is a schematic diagram of the structure of the interleukin 12p70 test strip in the interleukin 12p70 detection kit in Example 2. It includes a base plate 4, a detection pad 5, a sample absorption pad 6, a labeling pad 7, a sample pad 8, a control line 9, and a detection line 10. Detailed Implementation

[0040] The following examples further illustrate specific embodiments of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; unless otherwise specified, the materials and reagents used in the examples are commercially available; unless otherwise specified, the diluents for the interleukin-12p70 antibody and goat anti-rabbit IgG polyclonal antibody used in the examples are 20mM PB buffer containing trehalose, with each 100mL of diluent containing 0.6846g of trehalose.

[0041] Example 1: Preparation and identification of interleukin-12p70 monoclonal antibody 1 and interleukin-12p70 monoclonal antibody 2 I. Experimental Materials Interleukin 12p70: Self-made. Based on the interleukin 12p70 data published in the NCBI database (NCBI sequence numbers, NM_000882.3 and NM_002187.3), the encoding nucleotide sequence was synthesized by Nanjing Genscript Biotech Co., Ltd. Nickel ion affinity chromatography column, Protein A and Protein G affinity chromatography column: purchased from GE Healthcare; Freund's adjuvant: purchased from SIGMA; White Rabbit from New Zealand: Purchased from the China National Institutes for Food and Drug Control.

[0042] II. Test Methods (1) Preparation of interleukin-12p70: A: Using IL12A (NM_000882.3) and IL12B (NM_002187.3) as templates, the target fragment was amplified. Then, the vector (pcDNA 3.1-IRES) was used, and the amplified IL12A, IL12B, and the vector were ligated using enzyme digestion. Positive clones were identified and screened to obtain the vector containing the target fragment. Subsequently, with the assistance of PEI, the plasmid containing the target fragment was transfected into HEK293T cells (approximately 1 × 10⁻⁶ cells). 6 Using the cell’s own secretion system and processing modification mechanism, the IL-12p70 dimer with the natural design was expressed and secreted. About 72 hours after transfection, the cell supernatant was collected. B: Protein purification: 1) Centrifuge the cell supernatant (10,000 rpm, 10 minutes) to remove cell debris, and filter with a 0.22 μm filter to further remove impurities; 2) Load the supernatant into a Protein A affinity chromatography column. After loading, wash the column with washing buffer (0.02 mol / L PBS, 0.5 mol / L NaCl, 20 mmol / L imidazole, pH 7.4), then elute the target protein with elution buffer (0.02 mol / L PBS, 0.5 mol / L NaCl, 250 mmol / L imidazole, pH 7.4). Collect the elution peak and desalt it by dialyzing (0.02 M PBS, pH 7.4). Then, detect the protein concentration and determine the protein purity by SDS-PAGE electrophoresis (two clear bands of 35 kDa and 40 kDa should appear under reducing conditions).

[0043] (2) Animal immunization: A: The interleukin-12p70 immunogen prepared by the above method (diluted to 1 mg / mL) was thoroughly mixed and emulsified with Freund's adjuvant, and then New Zealand white rabbits were immunized by subcutaneous multi-point injection. Each New Zealand white rabbit was injected with 500 μg of immunogen, once every two weeks, for a total of 7 immunizations, and a total of 5 New Zealand white rabbits were immunized. B: One week after the last immunization, blood was collected from the marginal ear vein of New Zealand white rabbits, the serum was separated, and the antibody titer in the serum was tested by ELISA (enzyme-labeled plate coated with interleukin-12p70). The OD values ​​of different New Zealand white rabbit serums after different dilutions are shown in Table 1.

[0044] Table 1. OD values ​​of different New Zealand white rabbit serums after different dilutions According to Table 1, rabbits #2, #3, #4, and #5 had lower antibody titers and were therefore eliminated; rabbit #1 had the best titer and was selected for monoclonal antibody cell preparation.

[0045] (3) Preparation of monoclonal antibodies: A: Screening of hybridoma cells a) Aseptically remove the spleens of the selected New Zealand white rabbits, and separate the spleen cells (cell concentration approximately 1×10⁻⁶). 7 (cells / mL) and myeloma cells (cell concentration approximately 1×10⁻⁶) were respectively compared with myeloma cells (cell concentration approximately 1×10⁻⁶). 6 The cells were fused in equal volumes (cells / mL) using the polyethylene glycol method, and the fused cells were screened and plated using the limiting dilution method. b) Screening and culturing monoclonal cell wells, detecting the supernatant after cell culture by ELISA, and selecting the four monoclonal cell wells with the highest OD values ​​in the cell culture plate as target hybridoma cells (1#, 2#, 3#, 4#) for culture and amplification, and retaining them.

[0046] B: Preparation of monoclonal antibodies The antibody variable region gene was isolated from hybridoma cells using RT-PCR, reverse transcribed to obtain cDNA, and then amplified by PCR to obtain the antibody gene sequence. The obtained antibody gene sequence was used to construct an expression vector (pCDNA3.4), resulting in paired pCDNA3.4-antibody heavy chain plasmid and pCDNA3.4-antibody light chain plasmid. The paired monoclonal antibody heavy and light chain expression plasmids were transfected into 293T cells at a 1:1 ratio. The cell culture supernatant was then collected to obtain the crude monoclonal antibody extract. The crude extract was purified using affinity purification (Protein A). The specific steps are as follows: 1) Centrifugation to remove impurities: Centrifuge at 10000×g at room temperature for 30 min; 2) The supernatant after centrifugation was purified using a Protein A affinity purification column; 3) The purified monoclonal antibody was concentrated using a centrifuge column, PBS was used as the antibody preservation buffer, and the concentration of the concentrated antibody was measured.

[0047] Monoclonal antibodies Ab1#, Ab2#, Ab3#, and Ab4# were thus obtained.

[0048] III. Testing Section 1. Screening of monoclonal antibodies (1) Enzyme-linked immunosorbent assay (ELISA) to verify antibody function Interleukin 12p70 was coated onto an ELISA plate (0.1 μg / well). The concentration of four monoclonal antibodies (Ab1#, Ab2#, Ab3#, Ab4#) was adjusted to 10 mg / mL. The four antibodies were then serially diluted. The dilution buffer for each monoclonal antibody was added to the ELISA plate (100 μL per well). The plate was incubated at 37°C for 1 h. After washing the plate, the ELISA-labeled secondary antibody dilution buffer (5000-fold dilution, 100 μL per well) was added and incubated at 37°C for 0.5 h. TMB was added for color development for 15 min, and the process was terminated. The absorbance (OD) was measured at 450 / 620 nm wavelengths (the OD of the detection result was read at 450 nm, and the OD of the interference was read at 620 nm. The OD of 450 nm was subtracted from the OD of 620 nm when calculating the result). The results are shown in Table 2.

[0049] Table 2. Results of absorbance measurement (I) Based on the analysis of Table 2, the recognition effect of different concentrations of monoclonal antibodies on interleukin-12p70 can be seen that when monoclonal antibodies Ab#1, Ab2#, Ab3#, and Ab4# are diluted 2,560,000 times, they still have a good recognition effect on interleukin-12p70.

[0050] (2) Verify antibody affinity Coat each well with interleukin-12p70 (0.1 μg / well) and adjust the concentration of four monoclonal antibodies (Ab1#, Ab2#, Ab3#, Ab4#) to 20 ng / mL. Mix 50 μL of diluted monoclonal antibody with 50 μL of 2 μg / mL interleukin-12p70 solution (Ab# + interleukin-12p70), and mix 50 μL of diluted monoclonal antibody with 50 μL of 0.02M PBS solution (Ab#). Incubate each solution at 37°C for 1 h. Then add 100 μL of the mixture to each well of the ELISA plate and incubate at 37°C for 1 h. After washing, add 100 μL of ELISA-labeled secondary antibody dilution buffer (5000-fold dilution) to each well and incubate at 37°C for 0.5 h. Add TMB for color development for 15 min and then stop the reaction. The absorbance value (OD) was measured at a wavelength of nm (the OD of the detection result was read at 450nm, and the interference OD was read at 620nm. When calculating the result, the OD of 450nm was subtracted from the OD of 620nm). The results are shown in Table 3.

[0051] Table 3. Results of absorbance measurement (II) As shown in Table 3, except for Ab#3, the affinity constants kd of the other three monoclonal antibodies are between 1.2 and 2.5 nM, indicating good affinity.

[0052] 2. Confirmation of antibody pairing combinations The alternative monoclonal antibodies were combined using a sandwich method, and the samples were detected by fluorescence immunochromatography (coating conditions: 0.1 M Tris buffer, coating concentration 1.0 mg / mL, drying temperature 50℃, drying time 16 h; labeling conditions: 0.02 M PBS buffer, antibody to fluorescent microspheres (particle size about 150 nm) mass ratio 2:1, labeling for 16 h; sample loading volume was 100 μL, and the results were read 15 min after loading). The test samples were: 30 serum samples (P001-P030) that were positive for interleukin 12p70 and 30 serum samples (N001-N030) that were negative for interleukin 12p70 collected from relevant hospitals, as well as borderline samples (interleukin 12p70 diluted with purified water to a concentration of 5 pg / mL). The ratio (I) of the sample T / C value to the borderline sample T / C value was calculated. This parameter was used as the borderline value (I≥1 for positive, I<1 for negative). Antibody combinations with high positive and negative concordance rates and large sample gradients were screened. The results are shown in Table 4.

[0053] Table 4. Results of antibody combination screening As shown in Table 4, the positive and negative concordance rates of samples were best when Ab1# antibody coating was combined with Ab4# antibody detection and Ab4# antibody coating was combined with Ab1# antibody detection.

[0054] 3. Verification of cross-reactivity of monoclonal antibodies Two candidate monoclonal antibody pairs were combined using a sandwich method, and different concentrations of hemoglobin, bilirubin, triglycerides, interleukin-23, P40 homodimer, P35 homodimer, interleukin-6, and tumor necrosis factor were detected by fluorescence immunochromatography. The anti-interference ability of different monoclonal antibody combinations against these easily cross-reacting substances was observed. The results of cross-reaction are shown in Table 5.

[0055] Table 5 Results of monoclonal antibody cross-reactivity The results in Table 5 show that the monoclonal antibody has the best detection effect on Ab1# + Ab4# with no cross-reactivity. Therefore, monoclonal antibodies Ab1# and Ab4# were selected for interleukin-12p70 detection.

[0056] 4. Monoclonal antibody sequencing The prepared interleukin-12p70 monoclonal antibody 1 (Ab1#) and interleukin-12p70 monoclonal antibody 2 (Ab4#) were sequenced, and the results are shown in Tables 6 and 7 below.

[0057] Table 6 Sequencing results of the prepared interleukin-12p70 monoclonal antibody 1 and interleukin-12p70 monoclonal antibody 2 (I) Table 7 Sequencing results of the prepared interleukin-12p70 monoclonal antibody 1 and interleukin-12p70 monoclonal antibody 2 (II) A 12% SDS-PAGE gel was prepared using standard methods. The prepared interleukin-12p70 monoclonal antibody 1 and interleukin-12p70 monoclonal antibody 2 were loaded at 5 μg each. SDS-PAGE electrophoresis was performed using protein molecular weight standards as a reference. The results showed that both interleukin-12p70 monoclonal antibodies exhibited two characteristic bands of approximately 25 KD and 50 KD, representing the light and heavy chains of IgG, respectively. Figure 1 The antibody content of all bands was above 90% after scanning and analysis.

[0058] Example 2: Preparation of Interleukin-12p70 Detection Kit An interleukin-12p70 detection kit was prepared using interleukin-12p70 monoclonal antibody 1 and interleukin-12p70 monoclonal antibody 2 prepared in Example 1. The structure of the kit is as follows: Figure 2 , Figure 3 , Figure 4 As shown, the kit includes an interleukin-12p70 test strip and a housing, with the test strip housed inside the housing. The kit includes an observation window 1, a sample application well 2, a strip loading area 3, a base plate 4, a test pad 5, an aspiration pad 6, a labeling pad 7, a sample pad 8, a control line 9, and a test line 10. Specifically, the interleukin-12p70 test strip includes a base plate 4 and a sample pad 8, a labeling pad 7, a test pad 5, and an aspiration pad 6 stacked sequentially on the base plate 4; the test pad 5 has a test line 10 and a control line 9; the housing includes a detachably connected upper and lower cover; the upper cover has an observation window 1 and a sample application well 2, and the lower cover has a strip loading area 3. This kit uses a double-antibody sandwich method to quantitatively detect interleukin-12p70 antigen in samples.

[0059] The interleukin-12p70 detection kit is, for example, an interleukin-12p70 detection kit prepared by time-resolved fluorescent microsphere labeled antibody, and its preparation method is as follows.

[0060] (a) Main materials used Interleukin-12p70 monoclonal antibody 1 prepared in Example 1 was used to label and coat sample pad 8, and interleukin-12p70 monoclonal antibody 2 prepared in Example 1 was used to coat detection line 10. Goat anti-rabbit IgG antibody was purchased from Shenzhen Feipeng Biotechnology Co., Ltd., and was used to coat the quality control line 9 on the nitrocellulose membrane (i.e., NC membrane, purchased from Sartorius). Other consumables such as PVC board were purchased from Biken Labs, and commonly used reagents were all analytical grade reagents.

[0061] (II) Preparation process Preparation of time-resolved fluorescent microsphere-labeled antibody: Take 1 mL of 1% carboxyl time-resolved fluorescent microspheres (purchased from Suzhou Weidu Biotechnology Co., Ltd.), add 9 mL of MES buffer, then add 25 μL of EDC solution (10 mg / mL) and 25 μL of NHS solution (10 mg / mL), and react with shaking at room temperature for 30 min. Centrifuge to collect the precipitate. Add HEPES reconstitution solution, sonicate to disperse evenly, then add 1 mL of 1 mg / mL interleukin 12p70 monoclonal antibody 1 prepared in Example 1, react with shaking at room temperature for 120 min, and centrifuge to collect the precipitate. Add 1 mL of blocking buffer (0.02 M PBS solution containing 1% BSA), react with shaking at room temperature for 120 min. Centrifuge to collect the microsphere precipitate, and reconstitute with reconstitution solution (0.02 M PBS solution containing 0.5% trehalose).

[0062] Preparation of fluorescently labeled pads: The labeled time-resolved fluorescent microspheres were diluted with a microsphere reconstitution solution (0.02M PBS solution containing 0.5% trehalose) and sprayed onto the fluorescent pads using a gold spraying spectrometer at a density of 3 μL / cm and a spraying spacing of 6 mm. After spraying, the pads were dried at 37℃ with low humidity (<30%) for 2 hours.

[0063] NC membrane coating of CT lines: T lines were prepared using interleukin-12p70 monoclonal antibody 2 at a concentration of 1.5 mg / mL in Example 1, and C lines were prepared using goat anti-rabbit IgG antibody at a concentration of 1 mg / mL. The lines were streaked at 1 μL / cm and then dried at 37°C with low humidity (<30%) for 24 h.

[0064] Sample pad preparation: The sample pad preparation solution consists of buffer salts, slow-release agents, solubilizers, and blocking agents. The specific formulation is 20mM Tris buffer, with each 100mL of Tris buffer containing 1g BSA, 0.5g Tween 20, and 2g sucrose. Apply according to a 20cm... 2 Use 1 mL of sample processing solution for treatment. After thorough treatment, place the sample in a 37°C, low humidity (<30%) environment for 2 hours to dry.

[0065] Test strip assembly: Attach the NC film, sample absorption pad, fluorescent pad, and sample pad sequentially to a PVC board. The sample absorption pad and fluorescent pad should each overlap the NC film by 1-2 mm, and the sample pad should overlap the fluorescent pad by 1-2 mm. After assembly, cut the test strip to a width of 4±0.4 mm and insert it into a cartridge. Place the cartridge and desiccant together in an aluminum foil bag and seal it. Affix labels and box to obtain the finished test card.

[0066] Example 3: Sample detection using the interleukin-12p70 detection kit (time-resolved fluorescent microsphere labeling) prepared in Example 2 Place the test card on a clean, flat surface, and add 80-100 μL of the processed sample to the sample dispensing end of the test card. At the same time, set up a PBS control group.

[0067] Import the standard curve of the test strip. After 10 minutes, use a fluorescence immunoassay analyzer to scan the detection area to obtain the fluorescence signal. After detection, the concentration value corresponding to the interleukin-12p70 antigen will be displayed. Exemplary measurement results are shown in Tables 8 and 9 below.

[0068] Table 8 Test results of diluent samples Table 9 Results of Recombinant Interleukin-12p70 Antigen Test Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention is also intended to include these modifications and variations. The above embodiments or implementations are merely illustrative examples of this invention, and it can also be implemented in other specific ways or forms without departing from its gist or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of this invention should be defined by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of this invention.

Claims

1. An interleukin-12p70 antibody or its antigen-binding fragment, characterized in that: The interleukin-12p70 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes a heavy chain complementarity determination region and a heavy chain framework region, and the light chain variable region includes a light chain complementarity determination region and a light chain framework region. The heavy chain complementarity-determining region (LCD) includes LCD 1, LCD 2, and LCD 3. The amino acid sequence of LCD 1 is shown in SEQ ID NO.1, the amino acid sequence of LCD 2 is shown in SEQ ID NO.2, and the amino acid sequence of LCD 3 is shown in SEQ ID NO.

3. The light chain complementarity-determining region includes light chain complementarity-determining region 1, light chain complementarity-determining region 2, and light chain complementarity-determining region 3. The amino acid sequence of light chain complementarity-determining region 1 is shown in SEQ ID NO.4, the amino acid sequence of light chain complementarity-determining region 2 is shown in SEQ ID NO.5, and the amino acid sequence of light chain complementarity-determining region 3 is shown in SEQ ID NO.

6.

2. The interleukin-12p70 antibody or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The interleukin-12p70 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that: The interleukin-12p70 antibody is a rabbit-derived antibody.

4. Interleukin-12p70 detection product, characterized by: The interleukin-12p70 detection product is based on the principle of immunoassay and includes the interleukin-12p70 antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. The interleukin-12p70 detection product according to claim 4, characterized in that: The interleukin-12p70 detection products mentioned above are labeled antibody test strips, enzyme-linked immunosorbent assay (ELISA) kits, or chemiluminescence immunoassay kits.

6. The interleukin-12p70 detection product according to claim 5, characterized in that: The interleukin-12p70 detection product is a labeled antibody test strip. The labeled antibody test strip includes a base plate and a sample pad, a labeling pad, a detection pad, and a sample absorption pad that are sequentially stacked on the base plate. The labeled pad is coated with a label containing an interleukin-12p70 antibody or its antigen-binding fragment. The test pad is equipped with a test line and a control line. The test line is coated with another interleukin-12p70 antibody or its antigen-binding fragment. The interleukin-12p70 antibody or its antigen-binding fragment described herein, or the other interleukin-12p70 antibody or its antigen-binding fragment described herein, is the interleukin-12p70 antibody or its antigen-binding fragment as described in any one of claims 1-3.

7. The interleukin-12p70 detection product according to claim 6, characterized in that: The marker is selected from one or more of colloidal gold, colored microspheres, time-resolved fluorescent microspheres, and quantum dot microspheres.

8. The interleukin-12p70 detection product according to claim 6, characterized in that: The quality control line is coated with anti-antibody, which is an antibody from another species that is an anti-interleukin 12p70 antibody or its antigen-binding fragment.

9. The interleukin-12p70 detection product according to claim 8, characterized in that: The detection line is coated with 0.5-2 mg / mL of another interleukin-12p70 antibody or its antigen-binding fragment; and / or the control line is coated with 0.5-2 mg / mL of goat anti-rabbit IgG polyclonal antibody.

10. The interleukin-12p70 detection product according to claim 6, characterized in that: The interleukin-12p70 antibody or its antigen-binding fragment described herein is the interleukin-12p70 antibody or its antigen-binding fragment as described in any one of claims 1-3. Furthermore, the amino acid sequence of the heavy chain complementarity-determining region 1 of the other interleukin-12p70 antibody or its antigen-binding fragment is shown in SEQ ID NO. 9, the amino acid sequence of the heavy chain complementarity-determining region 2 is shown in SEQ ID NO. 10, the amino acid sequence of the heavy chain complementarity-determining region 3 is shown in SEQ ID NO. 11, the amino acid sequence of the light chain complementarity-determining region 1 is shown in SEQ ID NO. 12, the amino acid sequence of the light chain complementarity-determining region 2 is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain complementarity-determining region 3 is shown in SEQ ID NO. 14.

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