EDC conjugation

Section 3 for additional information on the properties of EDC conjugation using small peptides coupled to carrier proteins. 

Figure 18.25 Amino-PEG -carboxylate compounds contain a primary amine on one end and a carboxylate group on the other end. They can be used to add water-soluble spacer arms to molecules or surfaces. Using an amine-reactive group, the amino-PEG -carboxylate compound can be coupled via an amide bond, thus leaving the carboxylate end free for further conjugation reactions. Avoid the use of single-step EDC conjugation reactions, as this will polymerize the amino-PEGK-carboxylate by reacting with both ends.

Figure 19.11 The EDC conjugation of tyrosyl-lysine to KLH is illustrated by the gel filtration pattern on Sephadex G-25 after the reaction. The first peak is the carrier protein and the second peak is the peptide. A blank containing no EDC is also shown to provide baseline peak heights that would be obtained if no crosslinking occurred. When more EDC was added, more peptide was conjugated, as evidenced by peptide peak depletion.

Figure 19.12 EDC conjugation reactions can be extraordinarily consistent using the same peptide crosslinked to two carrier proteins. This figure shows the gel filtration pattern on Sephadex G-25 after completion of the crosslinking reaction. Conjugation of tyrosyl-lysine to BSA and KLH are shown. The first peaks represent eluting carrier, while the second peaks are the excess peptide. Note the consistency of conjugation using the same levels of EDC addition.

Most references to the use of EDC describe the optimal reaction medium to be at a pH between 4.7 and 6. However, the carbodiimide reaction occurs effectively up to at least pH 7.5 without significant loss of yield. See Chapter 9, Section 3 for additional information on the properties of EDC conjugation using small peptides coupled to carrier proteins. 

II NHS ester intermediates in EDC conjugation procedures. For specific applications of this technology, the amount of each reagent and unconjugated species may have to be adjusted to obtain an optimal conjugate. 

Figure 276 shows the results of an EDC conjugation study comparing a reaction done at pH 4.7 (A) to one done at pH 7.3 (B and C), with and without added sulfo-NHS (see Chapter 3, Section 1.2). The graphs show the elution profiles of a gel filtration separation after conjugation. In each case, a blank run done without the addition of EDC illustrates the separation of the protein carrier (the first peak) from... 

To illustrate the similarity of an EDC conjugation reaction using a different carrier protein, but the same peptide, Fig. 279 shows the gel filtration separation after conjugation of [Met5]-enkephalin to OVA. The uptake of peptide on addition of EDC is almost identical to that observed when conjugating to BSA. This is logical, since on a per mass basis, there is very little difference between these proteins in the amount of amines or carboxylates available for conjugation. 

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