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Antibodies SMCC activated

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. [Pg.357]

Figure 20.5 Antibodies may be modified with 2-iminothiolane at their amine groups to create sulfhydryls for conjugation with SMCC-activated enzymes. The maleimide groups on the derivatized enzyme react with the thiols on the antibody to form thioether bonds. Figure 20.5 Antibodies may be modified with 2-iminothiolane at their amine groups to create sulfhydryls for conjugation with SMCC-activated enzymes. The maleimide groups on the derivatized enzyme react with the thiols on the antibody to form thioether bonds.
Conjugation of SMCC-Activated Antibody with Thiolated Toxin... [Pg.852]

Mix the thiolated toxin with SMCC-activated antibody at a ratio of 2.25 mg of antibody per mg of toxin. Protect the solution from light. [Pg.852]

Dissolve the enzyme to a concentration of 1 - 2 mg/ml in Soln. A (if it is delivered in another buffer, dialyze against Soln. A). Mix 1 mg -galactosidase in Soln. A with 0.25 mg SMCC activated antibody (concentration about 1 mg/ml for activation see Protocol 4.1.3). Shake at RT for 2 h, dialyze or desalt on a Sephadex G-25 column against Soln. B and concentrate to about 2 mg/ml. Mix 9 vol. of the concentrated conjugate with 1 vol. Soln. C and store without further purification at 4 °C. [Pg.134]

SMCC frequently is used to prepare hapten-carrier or antibody-enzyme conjugates. In both applications, one of the molecules is activated (usually the carrier or the enzyme) with the... [Pg.283]

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. [Pg.285]

Figure 20.3 The reaction of SMCC with the amine groups on enzyme molecules yields a maleimide-activated derivative capable of coupling with sulfhydryl-containing antibody molecules. Figure 20.3 The reaction of SMCC with the amine groups on enzyme molecules yields a maleimide-activated derivative capable of coupling with sulfhydryl-containing antibody molecules.
Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin components. An intact A-B toxin molecule can be modified to contain sulfhydryls by treatment with 2-iminothiolane. Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conjugation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through thioether bond formation. Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin components. An intact A-B toxin molecule can be modified to contain sulfhydryls by treatment with 2-iminothiolane. Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conjugation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through thioether bond formation.
Dialyze an antibody solution against Soln. C and concentrate to 20-30 mg/ml. Add 3 mg of sulfo-SMCC (3-sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-caboxylate, Mr 436.7) or of sulfo-GMBS (N-(y-maleimidobutyryloxy)-3-sulfo-N-hydroxysuc-cinimide ester, Mr 382.3), dissolved in ddH20 to 60 mg/ml, to 10 mg of antibody, rock at RT for 15 min and add further 3 mg of coupling reagent. Desalt on a Sephadex G-25 column, equilibrated with Soln. A, after total incubation time of 30 min and use the activated antibody (protein) for conjugation immediately (see above). [Pg.133]

One note should be mentioned before proceeding when conjugating antibody molecules with (B-galactosidase, the antibody usually is activated with sulfo-SMCC first to take advantage of the indigenous sulfhydryl groups on the enzyme. Therefore, if (3-gal is being used, substitute the antibody for the enzyme mentioned in this protocol,... [Pg.481]

Antibody conjugates with GO can be made using the cross-linking agents glu-taraldehyde (Chapter 10, Section 1.2) or SMCC (Chapter 10, Section 1.1). The heterobifunctional reagent SMCC provides the best control over the conjugation process and usually results in high-activity preparations. [Pg.655]


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See also in sourсe #XX -- [ Pg.832 ]




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