Development Of An Immunochromatographic Assay For The Detection Of Copper Ions

Copper was first conjugated to a carrier protein （BSA） by a bi-functional chelator（p-SCN-Bn-DOTA） to make the complete antigen （Cu-p-SCN-Bn-DOTA-BSA） andcontrol antigen （p-SCN-Bn-DOTA-BSA）. The SDS-PAGE, detection copper amountsin complete antigen and Ultraviolet Spectrum Scanning were used to characterizethem. According to the results of SDS-PAGE, the relationship of their migration ratein gel was: BSA＞p-SCN-Bn-DOTA-BSA＞p-SCN-Bn-DOTA-BSA. The results ofgraphite furnace atomic absorption spectroscopy （GFAAS） showed that there were nocopper ions in BSA solution, buffer solution and p-SCN-Bn-DOTA-BSA solution, butin the complete antigen solution, the concentration of copper ions was pretty high.Results from ultraviolet spectrum scanning indicated their absorption peak weredifferent. All results reavealed one thing: the complete antigen was synthesissuccessfully.Recovery the hybridoma cell line against copper which was observed by our laband culture it in an incubator at37℃. Then cells were injected to abdominal cavity ofBalb/c mice to harvest ascites. Caprylic-ammonium sulfate and HiTrap Protein G HPchromatography were used in conjunction to purify ascites. Then antibody IgG1washarvested. The purity of the antibody was analyzed by SDS-PAGE. The concentrationof it was analyzed by BCA method. And its activity was characterized byindirect-ELISA. The results showed that there were only heavy chain and light chainand no other protein in gel by SDS-PAGE. Its concentration was1.45mg·mL^–1and itsactivity was1:51200. All results indicated that the antibody was in good conditions.Colloidal gold particles with a mean diameter of20nm were made to label themonoclonal antibody. A competitive format was used to establish animmunochromatographic assay for the detection of copper ions. The test strip wascombined with a strip reader to test standard copper samples and spiked samples. The sensitivity of the assay was found to be236.59±2.18ng·mL^–1and the detection rangewas89.51-625.34ng·mL^–1. The test strip only had3.66%cross-reactivity with Zn2+and minimal cross-reactivity （<0.24%） with other metals. The recovery for spikedsamples was from97.90%to100.46%and showed a good correspondence with theresult of graphite furnace atomic absorption spectroscopy （GFAAS） in parallelanalysis. This assay can be completed within10min and it not only can be used forqualitative analysis by visual conveniently but also can be used for quantitative andsemi-quantitative analysis by a membrane strip reader.