• Use freshly prepared substrate A and substrate B (in 2-reagent substrate systems)
• Do not hold substrate solution longer than 1 hour.
• Follow procedure of working substrate solution.
• The temperature of solution is important because it effect s rate of colour reaction.
• Do not add fresh substrate to reagent bottle containing old substrate.
• Clean old substrate solution bottle with H SO and thoroughly rinse with distilled water.
• Store at recommended temperature.
• Never store excessively diluted conjugate for use at some later time.
• Always make up the working dilution of conjugate just before you need it.
• Never leave conjugate on the bench for excessive time.
3. Addition of Samples
• Problems are usually caused by failure to put sample into buffer in well, leaving it on the side of the plate. Pay attention to the proper addition of samples.
4. Stopping Reagents
• Stopping reagents are added to prevent further enzyme reaction in ELISA.
• The stopping is usually made at a time when the relationship among the enzyme-substrate product is in the linear phase.
• Molar concentration of strong acids or strong bases stops enzyme activity by quickly denaturing enzymes. Some stopping reagents are enzyme-specific. Sodium azide is a potent inhibitor of HRPO, whereas EDTA inhibits Alkaline phosphatase by the chelation of metal ion cofactors.
• Since addition of stopping agents may alter the absorption spectrum of the product, the absorption peak must be known.
5. Laboratory conditions
• The laboratory should be devoid of any acid fumes.