Haptens containing carboxyl groups or which can be carboxylated

Haptens containing carboxyl groups or which can be carboxylated 

If a hapten does not contain free carboxyl, such group(s) may often be introduced. Alkylation of oxygen or nitrogen substituents with halo-esters, followed by hydrolysis of the ester, is frequently used for this purpose (Kabakoff, 1980). For steroids, carboxyl groups may be introduced to the hydroxyl or keton groups, through the formation of hemisuccinate esters or (carboxymethyl)oximes, respectively. 

Widely used methods to conjugate carboxyl group-containing molecules to proteins are (i) the mixed anhydride method (ii) the CDI method and, (iii) the NHS ester method. 

Separate conjugates from unreacted compounds by gel nitration on Sephadex G-25, equilibrated with 50 mM Tris-acetic acid buffer, pH 7.5, containing 100 mM NaCI, 10 mM MgCl2, and 10 mM 2-ME. Pool enzyme fractions, purify by methods discussed in Section 12.4, and add 0.1 % (w/v) BSA and 0.02% (w/v) NaNj. 

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