Development Of Immunomagnetic Bead Double Antibody Sandwich ELISA For The Detection Of Trace Fibroin

Enzyme-linked immunosorbent assay（ELISA）based on immunological principles is an effective method to identify fibroin.In this study,five mouse monoclonal antibodies against fibroin were prepared and used for the indirect ELISA detection of fibroin,and a double antibody sandwich ELISA method based on magnetic beads as solid-phase carriers was developed and optimized.（1）Five monoclonal,1A2-6-5,1A3-5-7,1A5-5-2,1A4-1-6 and 1A6-5-5,were prepared by direct immunization for mice with fibroin vaccine.Splenocytes from mice after four immunizations were taken out and fused with SP2/0 cells.The positive fused cells were screened,then the supernatant cryopreserved cells were co llected.Afterwards,the hybridoma cells were injected into the abdominal cavity of mice to get ascites which were collected for titer detection,and purified into the antibody.The results showed that the serum of mice after four immunizations was up to standard（1:10000>0.6）.In antibody subtype detection,the light chains of five monoclonal antibodies,were k chain,and heavy chains were respectively 1A2-6-5 Ig A2b,1A3-5-7 Ig G1,1A5-5-2 Ig G1,1A4-1-6 Ig A and 1A6-5-5 Ig G1.Tested by indirect ELISA,the titer of murine monoclonal antibody 1A2-6-5 was the highest.（2）1A2-6-5 antibody was used to develop of a double antibody sandwich ELISA method for the detection of fibroin.The antibody was coated in 96-well microporous for 24 h,subsequently fibroin was added and incubate together.The rabbit polyclonal antibody and HRP-labeled sheep anti-rabbit antibody were as first and second antibody to check the optical densities(OD_450nm).The results showed that when the concentration of fibroin was 0～1000 ng/m L,the linear equation was y=0.0003x+0.1492,R²=0.9931,and the concentration of fibroin had a good linear relationship with absorbance.The standard curve had a good linearity,and the detection limit was about 40 ng/m L.The double-antibody sandwich ELISA was applied to detect fibroin on magnetic beads.The certain amount of 1A-2-6-5 antibody was chelated with magnetic beads to prepare the immunomagnetic beads（IMBs）.The next steps were same as the above double antibody sandwich ELISA method.The results showed that the linear equation y=0.0022x+0.2248,R²=0.9998,and the detection limit was about 20 ng/m L when the fibroin concentration was 0～1000 ng/m L.The difference was significant（P<0.05）,and the reproducibility was good（RSD<5%）.In the standard addition recovery experiment,10 m L of 5-10μg/m L fibroin solution was mixed into 1 mg of soil.When 200μL fibroin-soil mixture was added to 50μg of immunomagnetic beads,the recovery rate of fibroin was about 70%.（3）Carboxy agarose immunomagnetic beads were prepared to absorb fibroin.The80%methanol solution was used to elute the reacted immunomagnetic beads twice for5 min each time.The eluent was collected on a magnetic frame and dried with nitrogen freezing,then was examined by indirect ELISA.The absorbing fibroin by immunomagnetic beads could be eluted and proved by the ELISA,which provides a good basis for detecting the trace fibroin.