Activation antibody

Disorders with enhanced sensitivity of the CaR to CaQ+. In contrast to inactivating mutations of the CaR, activating antibodies produce a syndrome of autosomal... 

Sondi, L Siiman, O. Koester, S. and Matijevic E. (2000). Preparation of Aminodextran-CdS Nanoparticle Complexes and Biologically Active Antibody-Aminodextran-CdS Nanoparticle Conjugates, Langmuir, 16, 3107-3118. 

An alternative pharmacotherapy for cocaine dependence currently under investigation is use of a cocaine vaccine to blunt the reinforcing effects of cocaine.51 60 The basis of this pharmacotherapy is to decrease the rate of entry of cocaine into the CNS (and therefore the onset of action), by either binding cocaine with antibody generated by active immunization with a stable cocaine conjugate or by using an enzymatically active antibody specific for cocaine. 

Add P-galactosidase to the activated antibody solution at a ratio of 4 mg of enzyme per mg of antibody. 

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. 

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form a sulfhydryl-reactive derivative. Toxin molecules containing disulfide-linked A-B chains may be reduced with DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with the reduced A chain to effect the final conjugate by disulfide bond formation.

Figure 21.7 An intact A-B subunit toxin molecule may be activated with 2-iminothiolane with good retention of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the immunotoxin conjugate through a disulfide bond.

Concentrate the fractions containing SPDP-activated antibody from the gel filtration step to lOmg/ml using a centrifugal concentrator (MW cutoff of 10,000). 

Protocol for the Conjugation of SPDP-activated Antibodies with 2-Iminothiolane-Modified Toxins... 

Conjugation of SPDP-Activated Antibody with Thiolated Gelonin... 

Mix SPDP-activated antibody with thiolated gelonin in equal mass quantities (or equal volumes if they are at the same concentration). This ratio results in about a 5-fold molar excess of toxin over the amount of antibody. 

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A-B toxin molecule with DTT can facilitate subsequent isolation of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-activated antibody causes immunotoxin formation through disulfide bond linkage. The hindered disulfide of an SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

The following method calls for mixing activated antibody with ricin A chain at a ratio of 2 mg antibody per mg of A chain. Adjustments to the amount of antibody and A chain initially dissolved in the reaction buffers should be done to anticipate this ratio. 

Remove unreacted SMPT and reaction by-products by gel filtration on a desalting resin. Pool fractions containing SMPT-activated antibody (the first peak eluting from the column) and concentrate them to lOmg/ml using centrifugal concentrators with a MW cutoff of 10,000. 

Mix the reduced A-chain solution with activated antibody solution at a ratio of 2mg of antibody per mg of A chain. Sterile filter the solution through a 0.22 pm membrane, and react at room temperature under nitrogen for 18 hours. 

Antibody containing, nu,if1v rfithin PDTP-activated antibody... 

Note This protocol requires mixing activated antibody with thiolated toxin at a ratio of 2.25 mg of antibody per mg of toxin. This ratio should be taken into account before starting the reactions. 

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.

Collect the peak containing the activated antibody (eluting first) and concentrate to 10 mg/ml using centrifugal concentrators. Use immediately for conjugating to a thiolated toxin. 

Conjugation of SMCC-Activated Antibody with Thiolated Toxin... 

Roffler, S.R., and Tseng, T.-L. (1994) Enhanced serum half-life and tumor localization of PEG-modified antibody-enzyme conjugates for targeted prodrug activation. Antibody Engineering Conference. San Diego, California. 

RDIg, when administered within 72 hours of delivery of a full-term infant, reduces active antibody formation from 12% to between 1% and 2%. 

Lymphocyte proliferation NK cell activity Antibody formation IL-2 system receptors Cytokine production Apoptosis Cell signaling... 

NE NK activity Antibody-Dependent Cell Cytotoxicity (ADCC) Decrease Decrease... 

Weidle, U.H., Borgya, A., Mattes, R., Lenz, H., and Buckel, R, Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells. Gene, 51, 21-29, 1987. 

Sheeja K, Kuttan G. (2007) Modulation of natural killer activity, antibody dependent cellular toxicity, and antibody dependent complement mediated cytotoxicity by andrographolide in normal and Ehrlich ascites carcinoma bearing mice. Integr Cancer Ther 6 66-73. 

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). 

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. 

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