Antibody conjugation

Somatostatin. Figure 1 Somatostatin-like im mu noreactivity in neurons of the periventricular hypothalamic nucleus of the rat. Coronal brain cryostat sections have been processed for im mu nohistochemistry and sequentially incubated with a primary monoclonal mouse anti human somatostatin antibody and secondary antimouse antibody conjugated with the fluorescence-dye Cy-3. Images have been taken with a Zeiss Axioplan fluorescence microscope. Scale bar, 100 pM. 

Pissuwan, D., Cortie, C.H., Valenzuela, S. and Cortie, M.B. (2007) Gold nanosphere-antibody conjugates for therapeutic applications. Gold Bulletin, 40,... 

El-Sayed, I.H., Huang, X. and El-Sayed, M.A. (2005) Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanopartides in cancer diagnostics Applications in oral cancer. Nano Letters, 5, 829-834. 

Most of the molecules introduced in this chapter are hydrophobic. Even those molecules that have been functionalized to improve water-solubility (for example, CCVJ and CCVJ triethyleneglycol ester 43, Fig. 14) contain large hydrophobic structures. In aqueous solutions that contain proteins or other macromolecules with hydrophobic regions, molecular rotors are attracted to these pockets and bind to the proteins. Noncovalent attraction to hydrophobic pockets is associated with restricted intramolecular rotation and consequently increased quantum yield. In this respect, molecular rotors are superior protein probes, because they do not only indicate the presence of proteins (similar to antibody-conjugated fluorescent markers), but they also report a constricted environment and can therefore be used to probe protein structure and assembly. 

Fig. 24 HER2/neu antibody-conjugated poly(amino acid)-coated iron oxide nanoparticles for breast cancer cell imaging. The part labeled as 1 is shown enlarged. (Adapted from [79])...

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

These Ru(H)bpy32+ fluorescent silica nanoparticles were used to detect single bacterial cells using antibodies conjugated to the surface after functionalization with trimethoxysilyl-propyldiethylenetriamine followed by succinylation to create carboxylates. Specific antibody molecules against E. coli 0157 then were coupled to this modified fluorescent particle using the carbodiimide method with EDC and NHS (Zhao et al., 2004). 

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

In addition, the PEG-based heterobifunctional crosslinkers described in Chapter 18, Section 2, provide enhanced water-solubility for antibody conjugation applications. Conjugation of antibody molecules using a maleimide-PEG -NHS ester compound actually increases the solubility of the antibody and may help to maintain stability for certain sensitive monoclonals better than the traditional aliphatic crosslinkers. The methods described below for SMCC may be used with success for PEG-based reagents or other maleimide-NHS ester heterobifunctionals. 

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. 

With the great diversity of targeted toxic agents being developed for cancer therapy, it would be difficult to characterize this section strictly as antibody conjugation. While many,... 

For instance, if toxin A chain-antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of... 

Add 10 mg of the protein to be coupled to the dextran solution. Other ratios of dextran-to-protein may be used as appropriate. For instance, if more than one protein or a protein plus a smaller molecule are both to be conjugated to the dextran backbone, the amount of protein added initially may have to be scaled back to allow the second molecule to be coupled latter. Many times, a small molecule such as a drug will be coupled to the dextran polymer first, and then a targeting protein such as an antibody conjugated secondarily. The optimal ratio of components forming the dextran conjugate should be determined experimentally to obtain the best combination possible. 

Nevertheless, HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

Due to the relatively high-molecular-weight of the enzyme, conjugates formed with antibodies and P-gal tend to be much bulkier than those associated with AP or horseradish peroxidase. For this reason, antibody conjugates made with P-gal may have more difficulty penetrating tissue structures during immunohistochemical staining techniques than those made with the other enzymes. 

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