Establishment and Preliminary Application of Double Antibody Sandwich ELISA Method for Detecting Tyrophagus putrescentiae
LI Huiping
SUN Jiaqi
NIU Mengna
YUAN Lianyan
XU Ping
LIN Jinsheng
HOU Lijuan
JIANG Ning
MA Lin
QU Shaoxuan
Abstract：To establish an enzyme linked immunosorbent assay(ELISA)detection method for Tyrophagus putrescentiae,New Zealand white rabbits were immunized with total mite protein to prepare polyclonal antibodies.BALB/c mice were immunized with the mite allergen Tyr p10 protein to prepare monoclonal antibodies.Using the polyclonal antibody as the coating antibody and the monoclonal antibody as the enzyme-labeled antibody,a double-antibody sandwich ELISA detection method for Tyrophagus putrescentiae was established,and its detection sensitivity and reliability in the culture medium and mycelium of Pleurotus ostreatus were verified.The results showed that the purity of the rabbit-sourced mite polyclonal antibody was over 85%,the concentration was 1.70 mg/mL,and the titer was approximately 870.4×103.One cell line(AntiTput-10)was screened from 38 hybridoma cell lines.The purity of the antibody after purification was over 90%,the concentration was 3.1 mg/mL,and the affinity constant was 2.71×1010.The concentration of the coating antibody was determined to be 100μg/mL and the dilution ratio of the enzyme-labeled antibody was 1∶2000 by the checkerboard titration method.The established ELISA detection method can detect 4 Tyrophagus putrescentiae mites per gram of edible fungus culture medium,which has high sensitivity.The proposed method could be applied to mite detection in different scenarios.
Keywords：Tyrophagus putrescentiaeELISApolyclonal antibodymonoclonal antibodiesTyr p10 protein
Publication Date：2025-12-30
Online Publishing Date：2025-12-10(First online date of this platform, not the publication date of the document)
Pages：7( 470-475,503 )