ABSTRACT
<p><b>OBJECTIVE</b>To assess the binding activity of polypeptide containing human Na+, K+-ATPase alpha1 subunit M1-M2 extracellular segment (HES1 derivative).</p><p><b>METHODS</b>HES1 derivative was synthesized by Fmoc method and purified by high-performance liquid chromatography-mass spectrometry, and its binding activity was identified by radioligand binding assay.</p><p><b>RESULTS</b>3H-ouabain and synthetic HES1 derivative showed some binding activity with the equilibrium dissociation constant (KD) of 24.58 nmol/L, with the the receptor density of 492.43 fmol x mg(-1) pro. and IC50 of 3.078 x 10(-7) mol/L.</p><p><b>CONCLUSION</b>HES1 derivative can bind to ouabain and has the potential of becoming an effective therapeutic agent.</p>
Subject(s)
Humans , Binding Sites , Extracellular Space , Metabolism , Ouabain , Chemistry , Pharmacology , Peptides , Chemistry , Protein Binding , Sodium-Potassium-Exchanging ATPase , Chemistry , Genetics , MetabolismABSTRACT
<p><b>AIM</b>To improve specificity and accuracy of endogenous ouabain measurement assay.</p><p><b>METHODS</b>Anti-ouabain polyclonal antibody egg yolk (IgY) and anti-ouabain rabbit antibody (IgG) were prepared respectively. In the presence of two kinds of antibody, then the specificity and accuracy of enzyme-linked immunosorbent assay (ELISA) were compared.</p><p><b>RESULTS</b>The ELISA, in the presence of IgY, provided a sensitivity of the average intraassay coefficient of variation(CV) was 2.03%, and the inter-assay CV was 2.34% respectively. In contrast, IgG were 2.83% and 3.29%. No significant interferences were observed with hydrocortisone and dexamethasone. There was 3.45% vs. 5.95%, 3.20% vs. 5.20% of crossreaction with cedilanid and digoxin.</p><p><b>CONCLUSION</b>The specificity and accuracy of ELISA, in which IgY was used, were more better than IgG.</p>
Subject(s)
Animals , Male , Rabbits , Antibody Specificity , Chickens , Allergy and Immunology , Cross Reactions , Egg Yolk , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Methods , Immunoglobulin G , Allergy and Immunology , Immunoglobulins , Allergy and Immunology , OuabainABSTRACT
<p><b>OBJECTIVE</b>To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues.</p><p><b>METHODS</b>Ouabain-BSA compound was used to immunize hens, and the eggs were collected one week after the first immunization. The IgY antibodies in the egg yolk were separated and purified by PEG-6000 Method, and analyzed by 12% SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) for titration. The IgY antibodies obtained were applied subsequently in ELISA and immunohistochemistry.</p><p><b>RESULTS</b>The IgY titer increased rapidly after the second immunization, with the highest titer of 1:10240 that lasted for at least 4 weeks. Competitive ELISA for IgY detection showed an average intraassay coefficient of variation (CV) of 2.03% and an inter-assay CV of 2.34%. Immunohistochemistry visualized the location of the endogenous ouabain mainly in the cytoplasm of the zona reticularis of rat adrenal cortex.</p><p><b>CONCLUSION</b>Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.</p>