ABSTRACT
AIM: To prepare the monoclonal antibodies (mAbs) against human Apo B100 and establish a double-antibody sandwich ELISA system for human Apo B100 detection. METHODS: The BALB/c mice were immunized with human Apo B100 antigen. After cell fusion and screening, the hybridoma cells were cultured in serum-free medium and the supernatant was purified to obtain mAbs using protein A. The affinity, subtype, specificity and epitope of the mAbs were characterized and the sandwich ELISA system was established. RESULTS: Four hybridoma cell lines 4-1-2, 4-2-2, 4-3-2 and 4-6-3 were obtained. The affinity of the anti-Apo B100 mAbs was up to 1×109 L/mol and nearly had no cross reaction with other relevant proteins. Linear detection of the sandwich ELISA system using 4-3-2 and 4-6-3 covered a range from (1.3-80) ng/mL, and its sensitivity was 1.24 ng/mL. The intra-assay coefficient of variation (CV) and inter-assay CV were less than 10% and 15%, respectively, and the recovery rate was above 90%. CONCLUSION: The mAbs against human Apo B100 have been prepared and a sandwich ELISA system for human Apo B100 detection has been established successfully, which provide a basis for human Apo B100 detection and disease diagnosis.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Apolipoprotein B-100/immunology , Enzyme-Linked Immunosorbent Assay , Animals , Antibody Affinity/immunology , Antibody Specificity/immunology , Cross Reactions/immunology , Humans , Hybridomas/cytology , Hybridomas/immunology , Male , Mice , Mice, Inbred BALB C , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To assess the value of two-color infrared fluorescence imaging system in detecting protein phosphorylation in comparison with chemiluminescent detection. METHOD: The lung tissue homogenate of mice treated with lipopolysaccharide (LPS) for different time lengths were prepared to separate the proteins by SDS-polyacrylamide gel electrophoresis followed by transfer of the proteins onto PVDF membranes. The membranes were incubated with the antibodies against total p42/44 MAPK/phospho-p42/44 MAPK, and then with goat anti-mouse or anti-rabbit secondary antibodies conjugated to Alexa Fluor 680, IRDye 800 or horseradish peroxidase. The blotted proteins were detected and quantified using Odyssey infrared imaging system or chemiluminescent detection. RESULTS: LPS treatment rapidly induced p42/44 MAPK phosphorylation, which reached the peak level 1 h after the treatment and resumed the baseline level at 12 h. Consistent results were obtained by the two detection methods, but two-color infrared fluorescence imaging system showed better sensitivity in detecting the target protein, and was easy to manipulate with good efficiency and capable of analyzing two proteins simultaneously. CONCLUSION: Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins.
Subject(s)
Blotting, Western/methods , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Animals , Blotting, Western/instrumentation , Fluorescence , Fluorescent Dyes/chemistry , Lipopolysaccharides , Luminescent Measurements/instrumentation , Luminescent Measurements/methods , Lung/drug effects , Lung/enzymology , Lung/pathology , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 3/chemistry , Phosphorylation , Reproducibility of Results , Shock, Septic/chemically induced , Shock, Septic/enzymologyABSTRACT
In nature, most self-incompatible flowering plants (angiosperms) show gametophytic self-incompatibility. Although gametophytic self-incompatibility functions can ultimately prevent self-fertilization, flowering plants have adopted different signal transduction pathways to reject self pollen. At present, there are mainly two pathways of signal transduction on gametophytic self-incompatibility. One is the S-RNase-based signal transduction in Solanaceae, Scrophulariaceae, and Rosaceae. The other is the cytosolic free Ca2+ acting as a second messenger in pollen of Papaveraceae. This review highlights the recent progress made towards understanding the signal transduction on gametophytic self-incompatibility.
