Conjugation using

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

DSC also is used to activate hydroxylic particles for coupling to amine-containing ligands (Miron and Wilchek, 1993). For methods involving particle conjugation using this homobifunctional compound, see Chapter 14. 

Difluorobenzene derivatives are small homobifunctional crosslinkers that react with amine groups. Conjugation using these compounds results in bridges of only about 3 A in length, potentially providing information concerning very close interactions between macromolecules. 

The following protocol illustrates the use of SIAB in preparing antibody-enzyme conjugates using P-galactosidase. 

Figure 5.13 M2C2H can be used to crosslink a sulfhydryl-containing molecule with an aldehyde-containing compound. Glycoproteins may be conjugated using this reagent after treatment with sodium periodate to form reactive aldehyde groups.

For instance, a dendrimer easily can be coupled with a large number of fluorescent dyes and still provide additional coupling sites for biotinylation. The only limitation to the number of fluorescent modifications is if fluorescence quenching starts to take place, in which case no further modifications will result in increased signal. A series of such conjugates using different levels of fluorophore modification should be done to determine the optimal level of dye-to-dendrimer before quenching occurs. 

Add a quantity of the DyLight 649 dye to the dendrimer solution to provide at least a 1.25-fold molar excess of dye over the amount of dendrimer present (for nonaqueous reactions) or a 6-15-fold molar excess for aqueous reactions. Mix well to dissolve. The optimal amount of dye added should be determined experimentally by preparing a series of conjugates using different molar ratios of dye-to-dendrimer. 

Figure 14.8 Amine-containing particles can be conjugated using alkylation or acylation reactions to result in secondary or tertiary amine linkages or amide bonds.

Native chemical ligation also can be extended to the conjugation of peptides or proteins to other molecules or surfaces. For instance, Reulen et al. (2007) prepared liposomes that contained cysteine-PEG-phospholipid derivatives and then coupled thioester-modified peptides or proteins to form a protein-liposome conjugate. Using this procedure, approximately 100 molecules of a collagen binding protein could be coupled to the cysteine-containing liposomes. 

Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents.

Figure 18.10 NHS-PEG-azide compounds can be used to modify amine-containing proteins or other molecules for subsequent conjugation using either the click chemistry reaction or Staudinger ligation.

This chapter describes the design, preparation, and use of hapten-carrier conjugates used to elicit an immune response toward a coupled hapten. The chemical reactions discussed for these conjugations are useful for coupling peptides, proteins, carbohydrates, oligonucleotides, and other small organic molecules to various carrier macromolecules. The resultant conjugates are important in antibody production, immune response research, and in the creation of vaccines. 

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.

The yield of conjugation using the Mannich reaction is dependent on the reactivity of active hydrogens within the hapten molecule. It is often difficult to predict the relative reactivity of any given compound in this reaction. Thus, trial and error may be necessary to determine the suitability of the Mannich procedure. 

Pool the fractions containing antibody and immediately mix with an amount of maleimide-activated enzyme to obtain the desired molar ratio of antibody-to-enzyme in the conjugate. Use of a 4 1 (enzymerantibody) molar ratio in the conjugation reaction usually results in high-activity conjugates suitable for use in many enzyme-linked immunoassay procedures. Higher molar ratios also have been used with success. 

The following protocol should be compared to the method described for SATA thiolation in Chapter 1, Section 4.1. Although the procedures are slightly dissimilar, the differences indicate the flexibility inherent in the chemistry. For convenience, the buffer composition indicated here was chosen to be consistent throughout this section on enzyme-antibody conjugation using SMCC. Other buffers and alternate protocols can be found in the literature. 

Mix the antibody solution with the enzyme solution at a ratio of 1 1 (v/v). Since an equal mass of antibody and enzyme is present in the final solution, this will result in a 3.75 molar excess of HRP over the amount of IgG. For conjugates consisting of greater enzyme-to-antibody ratios, proportionally increase the amount of enzyme solution as required. Typically, molar ratios of 4 1 to 15 1 (enzyme antibody) give acceptable conjugates useful in a variety of ETISA techniques. 

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