Preparation, Characterization And Application Of Immunolatex

Latex immunochromatography technology has been widely used in rapidimmunodetection recently, due to easy operation, excellent stability and high sensitivity.However, the deficiencies such as false negative and positive always appeared as it was usedin rapid immunodetection, which showed adverse impact in the application ofimmunochromatography technology. It is, therefore, very important to research the factorsaffecting the application of immunochromatography, and find the main reason leading to theappearance of false negative and positive phenomenon.In this paper, immunolatex was prepared using covalent coupling, and the stability ofimmune-latex was also studied by TEM, DLS and Zeta potential, results showed that thecoupling ratio was the highest when activator was EDC/NHS, active time was15min, thereaction buffer was PBS, coupling time was2.5h, pH value was6.0, and the antibodyconcentration was800μg/mL. The stability of latex was enhanced significantly after it wascovalent coupled with antibody protein, based on the data of TEM, DLS and Zeta potentialtests.The secondary structure of antibody protein on immunolatex, prepared in differentconditions, was detailed using FT-IR combining with computer aided analysis technology.Results showed that content of ordered structure of antibody protein increased along with theincreasing of pH, latex concentration and antibody protein concentration, which attributed tothe increasing of repulsive force and steric hindrance between latex microsphere and antibodyprotein.The interaction between latex microsphere and antibody protein was revealed byfluorescence spectrum technology. Results showed latex microsphere has a significantquenching effect on the intrinsic fluorescence spectrum of antibody protein. The quenchingmechanism might be static quenching. The main interaction between latex microsphere andantibody protein is electrostatic interaction which changed the tertiary structure of antibodyprotein to some extent and made a significant change in the hydrophobicity of antibodyprotein.The latex immunochromatography assay strip was prepared, and the relationships of the structure of antibody protein on immunolatex-sensitivity and the structure of antibody proteinon immunolatex-specificity were detailed. Results showed that the highest sensitivity ofantibody protein of immune-latex was observed as the pH6, latex concentration1%andantibody protein concentration0.8mg/mL. However, false positive phenomenon could beappeared as pH, latex concentration and antibody protein concentration were lower than6,0.75%and0.6mg/mL, respectively. This result also indicated that the sensitivity andspecificity was improved when the content of ordered structure of antibody protein increased.