Reduced Antibodies

Antibody reduction usually is done in the presence of EDTA to prevent re-oxidation of the sulfhydryls by metal catalysis. In phosphate buffer at pH 6-7 and 4°C, one report stated that the number of available thiols decreased only by about 7 percent in the presence of EDTA over a 40-hour time span. In the absence of EDTA, this sulfhydryl loss increased to 63-90 percent 

A final consideration is to provide adequate desalting of the reduced antibody molecule from excess reducing agent. If even a small amount of a thiol-containing reductant remains, subsequent conjugation with a maleimide-activated enzyme will be inhibited. 

Dissolve the IgG to be reduced at a concentration of l-10mg/ml in 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, containing 10 mM EDTA. 

Add 6 mg of MEA to each ml of antibody solution. Alternatively, add DTT or TCEP to a final concentration equal to 3 mole equivalents per mole equivalent of antibody present. Mix to dissolve. 

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. 

Add 6 mg of 2-mercaptoethyIamine (Pierce) to each milliliter of antibody solution. Mix to dissolve. 

Reduction in Hinge Region Yielding Two Half Antibody Molecules Containing Sulfhydtyls 

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The use of a 500-fold molar excess of 2-mercaptoethylamine over the concentration of antibody presence was found to result in a partially reduced antibody in which two disulfides were reduced to yield four thiols (Sun et al., 2005). This strategy can be used to retain a biospecific antibody construct for subsequent discrete conjugation at the hinge region between the heavy chains. 

DPDPB has been used to study the endocytosis of cadherin from intracellular junctions (Troyanovsky et al., 2006), the subunit arrangement in the flagellar rotor assembly (Lowder et al., 2005), and the disease-associated mutations in myelin proteolipid protein in the endoplasmic reticulum (ER) (Swanton et al., 2005). DPDPB can be used to conjugate reduced antibody molecules to p-D-galactosidase using essentially the same protocol as that described by O Sullivan et al. (1979). 

The reduced antibody can be used immediately to label with a maleimide-based cyanine dye using the following protocol. 

Labeling of Reduced Antibody with Thiol-Reactive Cyanine Dye... 

Figure 20.4 Reduction of the disulfide bonds within the hinge region of an IgG molecule produces half-anti-body molecules containing thiol groups. Reaction of these reduced antibodies with a maleimide-activated enzyme creates a conjugate through thioether bond formation.

Purify the thiolated antibody by gel filtration using a desalting resin. Perform the chromatography using 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, containing lOmM EDTA as the buffer. To obtain efficient separation between the reduced antibody and excess reductant, the sample size applied to the column should be at a ratio of no more... 

Figure 21.14 Activation of an intact A-B toxin molecule with MBS with subsequent conjugation with a reduced antibody fragment to produce an immunotoxin.

The following protocol is adapted from Myers et al. (1989). It involves activation of ricin with MBS and conjugation with a partially reduced antibody (Figure 21.14). 

Purify the reduced antibody using gel filtration on a column of Sephadex G-25. Concentrate the protein to lOmg/ml using centrifugal concentrators. 

Conjugation of MBS-Activated Ricin with Partially Reduced Antibody... 

Mix the MBS-activated ricin with the partially reduced antibody in a molar ratio of 15 1 (or 6.24mg activated ricin per mg of reduced antibody). This represents a volume ratio (at lOmg/ml for both proteins) of 1ml ricin solution mixed with 160pi antibody solution. 

Heilmann, C. et al., Reduced antibody responses to vaccinations in children exposed to polychlorinated biphenyls. PLOS Medicine, 3, 1352, 2006.. 

Humoral immunity is characterized by the production of antigen-specific antibodies that enhance phagocytosis and destruction of microorganisms through opsonization. Thus, deficiencies of humoral immunity (B lymphocytes) may lead to reduced antibody titers and are typically associated with acute gram-positive... 

Cadmium cbloride Reduced antibody responses (mouse, rabbit)... 

Polychlorinated biphenyls (PCB) Increased susceptibility for infection (duck) Reduced antibody responses (guinea pig, rabbit)... 

Immunotoxicity spleen weight decrease Offspring, might lead to increased early pup mortality due to reduced antibody concentration in milk... 

Mice treated for 30 days with FireMaster BP-6 in the diet at levels of approximately 1.3 mg/kg/day had a significantly reduced antibody-mediated response to SRBC (p<0.001) (Fraker 1980 Fraker and Aust... 

Fig. 3. SDS-PAGE of reduced antibody-toxin conjugate. (A) Antibody (starting material). (B) Abrin A chain conjugate (as in Fig. 2). Samples were prepared in the presence of 5% w/v DTT and run on a 2-27% gradient polyacrylamide gel.

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