Nonspecific binding procedure

An overview of the procedure for an ELISA is as follows Fix cells onto the well of the plate block the cells and well surface to prevent nonspecific binding incubate the cells in the presence of the primary antibody rinse away any unbound antibody incubate the cells in the presence of the enzyme-linked secondary antibody rinse away unbound antibody add enzyme subshate read the color reaction with a plate reader. 

These results show that in a MISPE procedure one may encounter substances with different patterns of behavior. In the ideal case the template (or the template and a group of its analogs which need to be carried on for further analysis) are retained 100% during the sample application and the wash step. All other substances (or at least those which interfere with further analysis) should be fully removed. In the non-ideal case some interferents will not be removed by a particular protocol. In this case it is useful to observe the behavior of these interferents on the NIP. If they are retained by the NIP, too, then their retention is likely due to nonspecific binding. In this case a better choice of the solvents in the protocol may help to eliminate the interferent. The danger here is that the new protocol may also reduce the retention of the template and reduce its recovery. It is therefore interesting to note that even in this good paper it has not been shown that the addition of 1 % methanol to the dichloromethane sample was really necessary with the real sample matrices. 

Lasne, F. (2001) Double-blotting a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures. J. Immunol. Methods 253, 125-131. 

Nicotinic receptor. [3H]-Nicotine (85 Ci/mmol Amersham) was used to assay specific binding of alkaloids to the nicotinic ACh receptor (nAChR). The membrane preparation was incubated for 40 min with differing concentrations of alkaloids or 1 mM nicotine as a positive control. The GF/C filters were presoaked with polyethylene glycol 8000 (5% in water) for 3 h to reduce nonspecific binding of [3H]-nicotine. Further procedures were the same as described above for mAChR. 

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