Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte.
Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24h were able to detect the analyte RAG-IgG at a concentration as low as 3.75ng mL(-1) with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3microg mL(-1) and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1microL of human blood was sufficient to perform the assay with the modified fibers.
Jain
Swati
S
Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016, India. swatijain.iitd@gmail.com
Chattopadhyay
Sruti
S
Jackeray
Richa
R
Singh
Harpal
H
D000180:Acrylic Resins / Q000737:chemistry*; D000588:Amines / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D000818:Animals; D055910:Antibodies, Immobilized / Q000276:immunology / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006041:Goats; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D015203:Reproducibility of Results; D017550:Spectroscopy, Fourier Transform Infrared
D000180:Acrylic Resins; D000588:Amines; D055910:Antibodies, Immobilized; C051538:IGG-horseradish peroxidase; D007074:Immunoglobulin G; C010504:polyacrylonitrile; D006735:Horseradish Peroxidase