1. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.
2. The most common method of preparing the antibody is to dialyze the isolated antibody into 0.1 M NaHCO3, pH 8.3/0.5 M NaCl buffer and adjust the concentration to give 50 mg/20 mL of buffer for cyanogen bromide Sepharose conjugation. It is important to measure the concentration of the solution so the total amount of antibody exposed to the gel is known in order to calculate the efficiency of coupling. The antibody must not be dialyzed or reconstituted into buffers that contain primary or even secondary amines such as Tris-HCl, which will react with the activated group on the gel, thereby reducing the efficiency of IgG immobilization. Inactivation of activated gels occurs for other buffer and gel combinations as well, most manufacturers will discuss specific incompatible combinations and the scientist must be aware of the chemistry involved in the activation of the gels.
3. The amount of antibody reacted with the gel is critical for good binding of the analyte. If too little antibody is used only nonspecific bind ing will be observed (use more than 1 mg antibody per mL of gel). If too much antibody is used the gel will show poor binding efficiency presumably because of steric hindrance. An appropriate amount of the antibody for the immobilization step is 5-10 mg of IgG/mL of gel.
4. Sample preparation is important to the longevity of column. Centrifuge the sample to eliminate any precipitate, after which the samples should be passed through 0.45-micron filters (and/or 0.22 micron) to prevent column fouling.
5. The analyte binding efficiency is matrix dependent. Some matrices, such as urine and tissue extracts, can be directly loaded onto the column, other matrices such as milk may need sample processing prior to loading onto an immunoaffinity column. The simplest sample preparation method is dilution; this method has been applied to serum, liver, and kidney extracts after removal of particulates. Sometimes dilution alone is not sufficient to eliminate the matrix effect and classical sample preparation techniques (solvent/solvent extraction, solid phase extraction, etc.) will be necessary prior to immunoaffinity chromatography. We found milk often needs this type of treatment.
6. The conditions required to elute the analyte from the IAC are empirically determined. Several common methods can be examined:
a. Change the pH of the elution buffer; we have often used 50 ml glycine-HCl, pH 2.8. Low pH solutions have been utilized as an analyte elution solution more often than high pH solutions.
b. Increase ionic strength, most often by increasing the salt concentration, 1M NaCl has commonly been used.
c. Use of a competitive eluent that contains structural elements of the analyte can often selectively elute the analyte.
7. When a radiolabeled form of the analyte is available, the amount in the wash and sample elution steps is easily measured, thereby facilitating optimum development. Other analytical techniques can be used, although care must be taken to avoid interference from the sample matrix. The ability of IAC to separate analyte from interferences can be assessed through either LC or GC determination for small molecule determination or SDS-PAGE for large molecule determination where co-elution of other substances can be easily observed.
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