A hybridoma cell line secreting anti-aluminum monoclonal antibody and its application
A hybridoma cell line, monoclonal antibody technology, applied in analytical materials, microorganism-based methods, instruments, etc., can solve the problems of long experimental period, unsuitable for rapid detection of large numbers of samples, complicated processing process, etc., and achieve good detection sensitivity. Effect
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Embodiment 1
[0020] Example 1 Preparation of Complete Antigen Al-ITCBE-KLH
[0021] Synthesis of complete antigen: Weigh 2 mL of KLH (10 mg) solution into a sterile plastic bottle, add 3 mL of HBS solution (0.1 M, pH 9.0) to dissolve. Slowly add 48.4 µL ITCBE (10 mg / mL) in DMSO solution, maintain the pH at 9.0, and stir the reaction at room temperature for 24 h. After ITCBE-KLH coupling was successful, 0.5 mg aluminum solution (1 mg / mL aluminum standard solution) was added dropwise, and 1 M NaOH was added dropwise after each drop to keep the pH of the solution at 8.0, and the reaction was carried out at room temperature for 6 h. After the reaction was completed, centrifuge at 8000 rpm for 20 min with an AmiconUltra-4Ultracel-3K ultrafiltration centrifuge tube with a cutoff of 3000. After each ultrafiltration, dissolve and resuspend with 3mL HBS solution (0.01 M, pH 7.4). Repeated ultrafiltration for 3 times, after the final ultrafiltration, add HBS solution (0.01M, pH 7.4) to wash out th...
Embodiment 2
[0022] Example 2 Animal Immunization
[0023] After mixing and emulsifying the complete aluminum antigen with an equal amount of Freund's adjuvant, BALB / c mice were immunized by subcutaneous injection at multiple points on the back of the neck (except for sprint immunization). The complete Freund's adjuvant was used for the first immunization, and the dose was 100 μg / rat; the incomplete Freund's adjuvant was used for multiple booster immunizations, and the dose was halved to 50 μg / rat; no adjuvant was used for the sprint immunization, and it was directly diluted with normal saline For intraperitoneal injection, the dose was halved to 25 μg / monkey. The interval between the first immunization and the second booster immunization is one month, the interval between multiple booster immunizations is 21 days, and the interval between sprint immunization and the last booster immunization is 18-21 days. Observing the immune effect of mice by indirect competitive enzyme-linked immunoas...
Embodiment 3
[0024] Example 3 Cell Fusion and Screening
[0025] (1) Cell fusion: Three days after the sprint immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps are as follows:
[0026] a. Remove eyeballs from mice to take blood. After the mice were sacrificed by cervical dislocation, they were immediately sterilized in 75% alcohol, soaked for about 5 minutes, and the spleens of the mice were aseptically removed, moderately ground with a syringe rubber tip and passed through 200 mesh cells Sieve to obtain splenocyte suspension, collect, centrifuge (1200rpm, 8 min), wash splenocytes with RPMI-1640 medium three times, after the last centrifugation, dilute splenocytes to a certain volume, count, and set aside;
[0027] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were incubated with RPMI-1640 medium containing 10% FBS (fetal bovine serum) in 5% CO 2 cultured in an incubato...
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