[0059] Example 1
[0060] In this example, the monoclonal antibody of mouse interleukin 6 contains a first antibody and a second antibody, the first antibody secretes by hybridoma cell line 20-A4-D5, the second antibody from hybridoma cells The strain 10-B8-C9 secretes, the hybridoma cell lines 20-A4-D5 and the hybridoma cell lines 10-B8-C9 were prepared as follows:
[0061] S1, artificially synthetic mice interleight dielectric interleukin 6 gene, recombinantly group the mouse interleukin 6 gene into expression vector plasmid PATX1 to obtain an IL6-PATX1 expression vector, electrophoresis verification of the carrier, figure 1 Shown in figure 1 It can be seen that the cloned site is EcoRI / XHOI, the sequence of mouse interleukin 6 gene is shown in SEQ ID NO.13; the IL6-PATX1 expression vector is transfected into a 293F cell line for 6 days, collecting the supernatant The liquid was purified by nickel column to give mice interleukin 6 (IL6); purified IL6 for SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis), figure 2 As shown, from figure 2 It can be seen that the purification of IL6 has reached more than 95%;
[0062] S2, using mouse interleukin 6 immunization 10 purchased SD rats from Hubei Experimental Animal Research Center, per rats immunized at least 4 times, take the third, 4 times of immunized serum titer test qualified small Cell fusion was carried out in a rat, and 12 of the 12 mean tumor cell lines were obtained, as shown in Table 1 below;
[0063] Table 1 Inter-ELISA screening obtained hybridoma cell line
[0064] 2-A9-F6 6-H3-B12 10-B8-C9 20-A4-D5 35-D9-E12 65-F1-A6 68-B4-H12 77-G3-E3 80-H5-G8 95-D1-C5 101-C2-D12 150-i6-D1
[0065] S3, the 12 strains of hematoma cells obtained by step S2 are separated by acridine ester labels and biotin labels to obtain acridine-labeled antibodies and biotin-labeled antibodies, and formulated mice interpolain 6 standards and Quality control;
[0066] The method of labeled antibodies with acridine includes the steps of:
[0067] S311, dissolved acridine esters in N, N-dimethylformamide, and a concentration of 3 mg / ml acridine ester solution;
[0068] S312, the antibody secreted in the plurality of hybridoma cells obtained in step S2 is dissolved in a phosphate buffer, and is adjusted to pH = 8.8 with a carbonate buffer, and the final concentration of the antibody is 3 mg / ml to obtain an antibody solution. I;
[0069] S313, the proportion of 20 μl of the acridine ester per mg of antibody is mixed with the acridine solution and the antibody solution 1 after 27 ° C to prevent light by 2.5 hours;
[0070] S314, the addition of lysine is terminated, and stirring at 27 ° C for 1 hour, the reaction is collected using phosphate buffer dialysis overnight, and the phosphate buffer is replaced 4 times to obtain an acridine-labeled antibody;
[0071] The method of biotin-labeled antibody includes the following steps:
[0072] S321, the biotin is dissolved in N, N-dimethylformamide, and a biotin solution having a concentration of 15 mg / ml is formed;
[0073] S322, the antibody secreted in the plurality of hybridoma cells obtained in step S2 is dissolved in a phosphate buffer, and is adjusted to pH = 8.8 with a carbonate buffer, and the final concentration of the antibody is 3 mg / ml to obtain an antibody solution. Ii;
[0074] S323, the biotin solution and the antibody solution II were mixed at room temperature for 2.5 hours after mixing of 10 μl of the biotin solution per mg antibody.
[0075] S324, collecting reactants using phosphate buffer dialysis overnight, and replacing phosphate buffer 4 times in the middle;
[0076] S325, adding the solution obtained by step S324 to embed a parentin-free magnetic bead solution, and stirring at 27 ° C for 2.5 hours to obtain biotin-labeled antibodies; the magnetic bead used is obtained by purchase, the item number is JSR, MAGNOSHPHERE- MS160 / streptavidin;
[0077] S4, the antibody of any acridine-labeled antibody obtained by step S3, any biotin-labeled antibody, two two pairs, and the following reagents need to be diluted into a working concentration before the start of the pairing experiment;
[0078] Biotin-labeled antibodies, the working concentration is 0.2 μg / ml ~ 2 μg / ml; the antibody dilution buffer used is R1: configure 20 mmol / L HEPES buffer, pH = 7.4, then add BSA 30g, sucrose 50g, glycine 2g, respectively , TWEEN-20 5ml, nan 3 0.5g, after fully dissolving, to 1 L using HEPES buffer;
[0079] A acridine-labeled antibody, a working concentration of 0.1 μg / mL to 1 μg / ml. The antibody dilution buffer used is R2: configure 20 mmol / L HEPES buffer, pH = 5.5, then add BSA 30g, sucrose 20g, glycine 20g, TWEEN-20 5 mL, nan 3 0.5g, after fully dissolving, to 1 L using HEPES buffer;
[0080] Preparing the magnetic beads, the magnetic bead diluent is: configure 50 mmol / l PBS solution, pH = 7.4, joined BSA 2G, Nan 3 0.5g, Tritonx-100 5ml, after sufficient dissolution, to 1 L 1L with PBS buffer; the final concentration of the magnetic bead is 1 to 5 mg / ml;
[0081] Mouse interleukin 6 standard and quality control, a phosphate buffer having a dilution of pH = 7.4;
[0082] The above four reagents are placed in the corresponding reagent bin in accordance with the fully automated chemiluminescence instrument, and automatically perform the preparation of the double antibody sandwich complex in the automatic chemistime instrument and screening the best pairing. Amia-labeled antibody and biotin-labeled antibodies;
[0083] The 12 x 12-co-144 group double antibody sandwich composite obtained after the packet is screened, for example, after the antibody secreted by the hybridoma cell line 20-A4-D5 is labeled, with all 12 strains of hybrid tumor cell lines. Secreted biotin-labeled antibodies (including biotin-labeled antibodies secreted by hybridoma cell lines 20-A4-D5) for dual-resistant sandwich pairing; finally screened to obtain 4 pairs of antibodies and capture antibodies can be used as double Anti-sandwich complex detection mouse interleukin 6 standard product, then the four pairs of pairs of antibodies were detected in 10 serum samples, respectively, and each sample repeatedly detected the average value of the detection result, calculated 3 detection variation coefficients The results are shown in Table 2, Table 3, Table 4, and Table 5.
[0084] Table 2 Coefficient of variation of the first pair of assays
[0085]
[0086] Table 3 Second pair coefficient of variation of antibody detection results
[0087]
[0088]
[0089] Table 4 Third pair coefficient of differential antibody detection results
[0090]
[0091] Table 5 Fourth pair coefficient of variation of antibody detection results
[0092]
[0093]
[0094] By the above comparison, the acridine-labeled antibody secreted by the hybridoma cell line 20-A4-D5 and the biotinoid-labeled antibody secreted by the hybridoma cell line 10-B8-C9 detects the positive serum signal value. Small variation, thus hybridoma cell lines 20-A4-D5 and 10-B8-C9 are optimal pairs, which secreted monoclonal antibodies are first antibodies and second antibodies.
[0095] The heavy chain and light chain complete sequence of the first antibody and the second antibody are obtained by sequencing, and then according to Kabat Rules (Reference: Kabat EA, WU TT, Bilofsky H.attempts to Locate Residues) of Antibody Combining Sites Thatmakecontact with antigen .Proc.natl.acad.sci.usa.1976; 73: 617-619) Sequence of the heavy chain and light chain CDR zone can be obtained.
[0096] The amino acid sequence of the heavy chain of the resulting first antibody such as SEQ ID NO.14, the amino acid sequence of the light chain of the resulting first antibody is shown in SEQ ID NO.15.
[0097] The amino acid sequence of the heavy chain variable region HcDR1, HCDR2, and HCDR3 was finally obtained, as shown in SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO.3.
[0098] The amino acid sequence of the light chain variable region LCDR1, LCDR2, and LCDR3 of the first antibody is shown in sequence, such as SEQ IDNO, SEQ ID NO. 5, SEQ ID NO.6.
[0099] The amino acid sequence of the heavy chain of the obtained second antibody is as shown in SEQ ID NO.16, and the amino acid sequence of the light chain of the obtained second antibody is shown in SEQ ID NO.17.
[0100] The amino acid sequence of the heavy chain variable region of the second antibody HCDR4, HCDR5, and HCDR6 sequentially, as shown in SEQ IDNO 7, SEQ ID No.8, SEQ ID NO.9.
[0101] The amino acid sequence of the light chain variable region LCDR4, LCDR5, and LCDR6 of the second antibody sequentially, such as SEQ IDNO.10, SEQ ID NO.11, SEQ ID NO.12.
[0102] The second antibody labeled secondary antibody labeled with acridine-labeled first antibody and biotin is applied to the chemiluminescence kit of mouse interleukin 6, and the kit also includes mouse interleukin 6 standard, mouse white cells. Medicine, phosphate (PBS) buffer, acridine ester labeled carbonate (CBS) buffer, biotin-labeled carbonate (CBS) buffer, diluted biotin-labeled antibody reagent buffer Liquid R1, diluted acridine labeled antibody reagent buffer R2, magnetic bead dilution, standard dilution and quality control dilution.
[0103] 1. Detection of sensitivity
[0104] The experimental scheme of the "Clinical Laboratory Test Procedure Detection Capability Evaluation Guide (2nd Edition)" (EP17A) released by the American Clinical Laboratory Standardization Committee (CLSI) is calculated to calculate the chemiluminescence kit for quantitative detection of mouse interleukin 6 content. The sensitivity, the sensitivity to be <1.5pg / ml, which has very high sensitivity.
[0105] 2, linear range detection
[0106] The selected concentration of 0,6.25 pg / ml, 12.5pg / ml, 25pg / ml, 50 pg / ml, 100 pg / ml, 200 pg / ml, 400 pg / ml IL6 standard; the standard dilution used is: 900ml deion Water, join NA 2 HPO 4 11.45g, NAH 2 PO 4 2.28g, sucrose 30g, glycine 1g, Tritonx-100 5ml, nan 3 0.5 g, fully dissolved, to 1 L using deionized water.
[0107] The above-described chemiluminescence kit was used to perform linear analysis, and the linear correlation coefficient, R = 0.999 was calculated, and the linear range of the chemiluminescence kit on the detection of mouse interleukin 6 was 6.25 to 400 pg / ml.
[0108] 3, precision detection
[0109] A low concentration IL6 sample and 300 pg / ml high concentration IL6 sample were taken at a concentration of 10 pg / ml. Each sample was performed three parallel tests, and the above chemiluminescence kit was calculated and calculated. The difference between the difference, indicating that the above chemiluminescence kit is <3%.
[0110] 4. Relevance to the R & D kit to detect clinical samples:
[0111] The same batch of samples were detected by the above chemiluminescence kit and the R & D Mouse IL-6Quantikine ELISA KIT (Double-Antibody Sandwich) kit, and the correlation between the resulting results was compared, and the results are shown in Table 6 and image 3.
[0112] Table 6 Example 1 of the kit and the R & D kit detected clinical sample correlation results
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