| | Re: antigen not binding to the plates
Passive adsorption has many advantage but also several drawbacks, that include desorption, binding capacity, and nonspecific binding. Because of the non covalent nature of the plastic/protein interactions, desorption may take place during the stages of the assay. But if the conditions of assay are standardized, leaching does not affect the viability of the majority of tests. For standardization is need having wide knowledge on coating target, not limited to molecular weight but also on other greater chemical characteristics such as, pI value, carbohydrate content, et cetera. From a theoretical point of view, the isoelectric point (pI) value of the used protein will influence buffer and pH choice. The best is to use a buffer with a pH value 1 to 2 units higher than the isoelectric point (pI) value of the protein being attached. This is, of course, not easy to determine in complex mixtures of proteins. The capacity for proteins to attach to microplate wells is influenced by the exact nature of the protein adsorbed to the specific plate used. Protein with high carbohydrate content is associated to bind poorly to plastic and, to clear hurdle, is need chemical activate plate, to realize antigen being covalently bound to prevent desorption (Elisadeveloper). On the last, I've realized easy chemical protocol, that can be furnished on request. The same attention, go observed to : a) ionic strength (µ) of the coating buffer solution in combination with an optimal pH, give better results for the attachment of various peptides; b) lower pH coating when to use acidic protein to neutralize repulsive forces between protein and the solid phase; c) capacity of the plastic surfaces for adsorption molecule is finite, and influenced by the exact nature of the protein adsorbed to the specific plate used. Saturation levels of between 50 and 500 ng per well have been found valid for a variety of proteins when added as 50 ul volumes. This capacity is improved a lot when to use plate at high bound capacity (Danfive); d) appropriate post coating wash for ionic strength, pH, surface tension and washing force prevent desorpition of the coated molecule.