Antibodies through carbohydrate

Different methods are used to immobilize covalently the proteins to HPLC supports. The coupling procedures imply various activation chemistries via epoxide-, diol-, or aldehyde-silica [51,52], Generally, the immobilized antibodies are randomly oriented. Dihydrazide-silica supports are used for an oriented immobilization of the antibody through its carbohydrate residues [53]. Another approach is to bind the antibody to protein A or protein G surfaces [17]. The antibody will bind through the Fc portion, leaving the antigen combining sites oriented away from the support. 

Three forms of purified FSH are available. Urofollitropin, also known as uFSH, is a purified preparation of human FSH that is extracted from the urine of postmenopausal women. Virtually all the LH activity has been removed through a form of immunoaffinity chromatography that uses anti-hCG antibodies. Two recombinant forms of FSH (rFSH) are also available follitropin alfa and follitropin beta. The amino acid sequences of these two products are identical to that of human FSH. They differ from each other and urofollitropin in the composition of the carbohydrate side chains. The rFSH preparations have a shorter half-life than preparations derived from human urine but stimulate estrogen secretion at least as efficiently and, in some studies, more efficiently. The rFSH preparations are considerably more expensive. 

The enzymes commonly used as labels in ELISA and other immunochemical reactions include horse radish peroxidase (HRP) and alkaline phosphatase (AP). The enzyme can be covalently coupled to the antibody using glutaraldehyde conjugation to reactive amino groups on the enzyme (lysines) in a phosphate buffered aqueous solution at neutral pH, as shown in Fig. 19 (103). Alternatively, carbohydrates present in the immunoglobulin structure can be cleaved by periodate treatment (see Fig. 20) and bound to free amino groups on the enzyme through a Schiff base reaction (103). 

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