Caprylic acid precipitation

For best results, plates should be coated with the IgG fraction of an antiserum, this can be conveniently prepared using caprylic acid precipitation [see Chapter 10 and ref. 15). Where this is not possible, indirect capture may be used, such as antispecies antibody on the plate, or the streptavidin-biotin system. Any indirect capture system must be compatible with the final label, e.g, labeled antigen or different species antisera with no crossreaction with die indirect coating antibody 11. Safety data (from ref. 16)... 

Temponi, M., Kekish, U., and Ferrone, S. (1988). Immunoreactivity and affinity of murine IgG monoclonal antibodies purified by caprylic acid precipitation. /. Immunol. Methods 115, 151-152. 

Soaps are composed of sodium salts of various fatty acids. These acids include those with the general structure CH3-(CH2) -COOH where n = 6 (caprylic acid), 8 (capric acid), 10 (lauric acid), 12 (myristic acid), 14 (palmitic acid), and 16 (stearic acid). Oleic acid (CH3-(CH2)7-CH=CH-(CH2)7-COOH) and linoleic acid (CH3-(CH2)4-CH=CH- H2-CH=CH-(CH2)7-COOH) are also common soap ingredients. These sodium salts readily dissolve in water, but other metal ions such as Ca2+ and Mg2+ form precipitates with the fatty acid anions. For example, the dissolution of the sodium salt of lauric acid and the subsequent formation of a precipitate of the lauric acid anion with calcium ion is given by... 

Addition of appropriate amounts of salts, such as ammonium or sodium sulfate (2), or other chemicals, such as PEG, cause precipitation of IgG from serum. Caprylic acid can also be used to fractionate proteins from serum Although such IgG is usually contaminated with other proteins, the ease of these precipitation procedures coupled with the high yield of IgG produced has led to them being very widely used to produce enriched IgG preparations suitable for many immunochemical procedures, e.g., production of immunoaffinity columns, and as a starting point for further purification. The precipitated IgG is usually very stable and such preparations are ideally suited for long-term storage or distribution and exchange between laboratories. 

Caprylic (octanoic) acid can be used to isolate mammalian IgG from serum, plasma, ascites fluid, and hybridoma culture supernatant by precipitation of non-IgG protein (3) (see Note 6). Other methods have been described in which caprylic acid has been used to precipitate immunoglobulin depending on the concentration used. The concentration of caprylic acid required to purify IgG varies according to species (see step 2 below). 

As with serum IgG (see Chapter 10), enrichment of MAbs by precipitation is a useful starting point for further purification. The use of ammonium sulfate, sodium sulfate, PEG, and caprylic acid, as described in Chapter 10, is applicable to hybridoma culture supernatants and ascitic fluid. 

At moderately acidic pH values, the short chain fatty acid, caprylic acid, is an effective protein precipitant. It has found an application in immunoglobulin purification as conditions have been developed that result in the precipitation of the majority of non-immunoglobin proteins in the serum leaving the antibodies in solution. The concentration of caprylic acid required differs for the serum of different species as shown in Table 1 (see Note 12). 

Caprylic acid (also called octanoic acid) can also be used for the precipitation of antibodies from serum, ascites, or hybridoma culture supernatant.60"63 In terms of antibody purity, precipitation with caprylic acid is significantly less efficient than the chromatographic methods. Note also that precipitation with caprylic acid is associated with a reduction in the affinity properties of some antibodies and is not suitable to purify murine IgA and IgG3. 

Ultrafiltration is conducted to remove precipitating agents such as ethanol, PEG, and caprylic acid through diafiltration and to concentrate the protein to the desired level. It has replaced lyophilization for ethanol removal from albumin and immunoglobulins and size exclusion chromatography for buffer exchange. 

Other water miscible solvents (ethanol, methanol, and acetone), and other salts can also be used to differentially precipitate out proteins. Caprylic acid can precipitate proteins as effectively as... 

Fractional precipitation methods can have damaging effects on certain mAbs. Pilot studies can examine this possibihty. Methods classically involve the use of ammonium sulfate, sodium sulfate, caprylic acid, Rivanol, and polyethylene glycol. These separate mAh from the majority of other proteins in ascites. Following dialysis, this procedure may well be sufficient for use in ELISA. 

A number of other precipitating agents have been used in the purification of proteins. In some instances, the use of sodium sulfate can result in a purer antibody preparation, but generally it does not offer advantages over ammonium sulphate. Caprylic (octanoic) acid, however, offers a different approach and also has a long history of use (5). Conditions can be created where this short chain fatty acid will effectively precipitate the majority of serum proteins with the exception of the immunoglobulins. 

Sodfum Caprylate. Sodium caprylate is prepared from opiylic acid, which is a component of coconut and palm lUls The salt precipitates as cream-colored granules that are 4iluble in water and sparingly soluble in alcohol. 

Most starches contain 20-30% amylose (Table 4.24). New corn cultivars (amylomaize) have been developed which contain 50-80% amylose. The amylose can be isolated from starch, e. g., by crystallization of a starch dispersion, usually in the presence of salts (MgS04) or by precipitation with a polar organic compound (alcohols, such as n-butanol, or lower fatty acids, such as caprylic or capric), which forms a complex with amylose and thus enhance its precipitation. 

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