Monoclonal antibody to pes1 and its application
A monoclonal antibody and kit technology, applied to the PES1 monoclonal antibody and its application field, can solve the problems of poor specificity, high color background, difficult clinical application and promotion of polyclonal antibodies, and achieve high sensitivity and specificity. The effect of improving sensitivity and specificity
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Embodiment 1
[0049] Embodiment 1, preparation of anti-human PES1 monoclonal antibody
[0050] 1. Preparation of PES1 antigen
[0051]The 5' end sense primer 5'-CGCGGATCCTTCCGCCTTTACCAGTTGC-3' (SEQ ID NO: 1) and the 3' end antisense primer 5'-CCGGAATTCTCACTCCGGCCTTGCCTTCTTG-3' (SEQ ID NO: 2) (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.), and BamH I and EcoR I restriction sites were introduced at both ends of the primers. Total RNA of human gastric cancer cell AGS was extracted with Trizol (purchased from Invitrogen), reverse-transcribed into cDNA, and the PES1 cDNA fragment was amplified by PCR using the above primers, and the product length was 1062bp (SEQ ID NO: 3).
[0052] The PCR product and the pGEX-4T1 vector (purchased from Amersham Biosciences) were digested with BamH I and EcoR I and then ligated, and the ligated product was transformed into Escherichia coli BL21 strain (purchased from Tiangen Biochemical Technology Co., Ltd.) and Single clones were pick...
Embodiment 2
[0081] Example 2, identification of the purification of 1B7 antibody, 1H5 antibody and 3B1 antibody
[0082] The subtypes of 1B7 antibody, 1H5 antibody and 3B1 antibody were identified by conventional ELISA method (the antibody subtype kit was purchased from Sigma Company), and it was confirmed that they were all IgG1, so ProteinG Sepharose-4B column was selected for purification. Take 10 mL of ascitic fluid collected in Example 1 and centrifuge at 12,000 × g, avoid grease and absorb the supernatant, dilute 5 times with the balance buffer, filter through a 0.45 μm filter, and purify using a Purify protein purifier (purchased from GE Company). The diluted ascitic fluid was loaded at a flow rate of 1 mL / min, after the impurities were washed away by equilibration buffer (PBS), it was eluted with pH 2.6 acidic eluent (100 mmol / L Glycin-HCl (pH 2.6)), and collected For the components with OD280>0.5, add 100 μl 2M Tris-base neutralizing solution to each ml for neutralization to keep...
Embodiment 3
[0083] Embodiment 3, determine the specificity analysis of 1B7 antibody, 1H5 antibody and 3B1 antibody
[0084] The purified 1B7 antibody, 1H5 antibody and 3B1 antibody were dialyzed with PBS buffer, and the concentration of the antibody was measured at 280 nm with an ultraviolet spectrophotometer.
[0085] Add 1B7 antibody, 1H5 antibody or 3B1 antibody at a concentration of 2 μg / ml to the GST-PES1 protein-coated microtiter plate, and add the same concentration of antibody to the GST protein-coated microtiter plate as a negative control, and Normal mouse IgG (purchased from Zhongshan Biological Co., Ltd.) was used as the antibody positive control, and incubated at room temperature for 1 hour; after that, the reaction well was washed 3 times with 0.05% Tween-20 / PBS, and the reaction well was washed 2 times with PBS; then add horseradish Peroxidase-labeled goat anti-mouse IgG antibody (purchased from Jackson Research Laboratories), incubated at room temperature for 1 hour; then ...
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