Development Of Indirect Competitive ELISA Kit And Novel One Step Competitive ELISA For Detection Of Hexavalent Chromium

Objectives: To develop ELISA kits for the detection of Cr⁶⁺in environmentalwater sample based on the optimal indirect competitive ELISA and the newsample pretreatment procedure. To develop more convenient and sensitivedirect competitive ELISA based on MAb-HRP/Ag-HRP complexes for fasterdetection of Cr⁶⁺.Methods: For optimization of the immunoassay, test conditions, such aspH and ionic strength, were studied and selected. In the pretreatment procedure,the Cr³⁺existing in samples were removed firstly by adding nanometer titaniumdioxide （TiO₂）. Second, the original Cr⁶⁺of samples were reduced to by treating withan optimized protocol, and the original concentration of Cr⁶⁺in samples werecalculated as Cr³⁺detected by ELISA. On those basis, the half maximuminhibition concentration （IC50）, the limit of detection （LOD）, the limit ofquantitation （LOQ）, the cut-off value, the degree of accuracy of the ELISA kitwere comprehensive evaluated. For develping rapid direct competitive ELISAs,MAb-HRP/Ag-HRP complexes were prepared by using colloidal gold or NaI04methods. And then the new developed direct competitive ELISAs based onMAb-HRP/Ag-HRP complexes were developed and evaluated.Results: The optimum reaction buffer of the ELSIA was15mM, pH7.4phosphate buffered saline containing0.15M NaCl. The IC50, the LOD, theLOQ of the ELISA kit were6.2,0.95,4.68ng/mL, respectively, the linear rangeof the kit was1-80ng/mL, the cross-reactivities with other heavy metals wereall less than1%, the shelf life was12months, the coefficient of variations wereall less than12%. The pretreatment procedure was established, Cr³⁺and Cr⁶⁺could be separated at pH9.0by using nano-TiO₂and then the collectedsupernatant containing Cr⁶⁺was mixed with60mg/L of NaHSO3solution, theCr⁶⁺was reduced to Cr³⁺at pH3.0and reacted for15min. The correlationcoefficient between the ELISA and ICP-AES was0.9887. A total of5differentdirect competitive ELISAs were developed, the IC50s were from2.74to6.4 ng/mL, the recoveris were from88.8%to109.8%and the correlationcoefficients were all more than96%.Conclusions: A specific, stable, reliable and accurate indirect competitiveELISA kit was developed, it’s suitable for detection of Cr⁶⁺in water samples.Five sensitive, simple and convenient direct competitive ELISAs were primarydeveloped.