A kind of antibody combined with strep-tag II label and application thereof
An antibody and labeling technology, applied to the antibody combined with the Strep-Tag II label and its application field, can solve the problems of false positives, long time-consuming, high cost, etc., and achieve the effect of strong affinity
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Embodiment 1
[0068] Example 1 Antigen, mouse immunization and hybridoma preparation
[0069] 1. Antigen
[0070] The immune antigen was Strep-Tag II (NWSHPQFEK-KLH) coupled with hemocyanin (Keyhole Limpet Hemocyanin, KLH); the detection antigen was Strep-Tag II (NWSHPQFEK-OVA) coupled with chicken ovalbumin (Ovalbumin, OVA), All were purchased from Hangzhou Zhongpei Biochemical Co., Ltd.
[0071] immunity
[0072] The immune antigen protein (Strep-Tag II (NWSHPQFEK-KLH) polypeptide coupled with KLH) was dissolved in physiological saline at a concentration of 0.5 mg / mL. Mix 100 μL of the above antigen polypeptide solution with an equal volume of Freund’s complete adjuvant (Sigma; Cat#: 1001646446), and immunize Balb / c mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) to generate an immune response, and then dilute the above-mentioned antigenic protein with normal saline to a concentration of 0.25 mg / mL, and add 100ul The diluted ...
Embodiment 2
[0076] Example 2 Screening of positive hybridoma cells
[0077] The existence of anti-Strep-Tag II antibody in the supernatant of hybridoma culture was detected by ELISA test and flow cytometry, and finally the hybridoma cells that could not only proliferate indefinitely but also secrete antibodies were screened out. details as follows:
[0078] 1. ELISA screening
[0079] Add 1 μg / mL OVA-coupled Strep-Tag II (NWSHPQFEK-OVA) to a 96-well plate (purchased from Corning) overnight at 4°C, then discard the supernatant and wash with phosphoric acid containing 0.05% Tween-20 Wash with salt buffer solution (PBST) 5 times. Plates were blocked with 1% bovine serum albumin for 2 hours at room temperature and washed 5 times with PBST. Add 100 μL / well hybridoma supernatant, incubate at room temperature for 1 hour, and then wash 5 times with PBST. Add 100 μL / well of HRP-labeled goat anti-mouse secondary antibody (Invitrogen, Cat#: 31430), and incubate at room temperature for 1 hour aft...
Embodiment 3
[0086] Example 3 Sequencing
[0087] Positive single clones were selected, and total RNA was extracted by TRIZOL method. RT-PCR generated cDNA, and then the heavy and light chains were amplified by PCR respectively (RT-PCR kit was purchased from Quanshijin, Cat#: AE311-03, and GXL high-fidelity DNA polymerase was used in PCR, purchased from TaKaRa , Cat#: R050A, see product manual for specific operation). Then the PCR product was cleaned with a cleaning kit (AXYGEN, Cat#: 155. Please refer to the product manual for specific operation) and then sequenced, and the sequence was delivered to Beijing Ruiboxingke Co., Ltd. For the amino acid sequences of the light chain variable region and the heavy chain variable region, see Table 1 and Table 2, and the sequence listing. The antibody clone numbers screened are 8A882 and 8F8D1, respectively.
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