COMPATIBLE CHELATORS
is able to donate electrons and combine by coordinate bonding with a metal ion to form structures called chelates or chelation complexes is suitable for attachment to an antibody or antibody fragment without loss of ability to chelate metal ions or destruction of the immunoreactivity or immunospecificity of the antibody or antibody fragment.
PREPARATION OF ANTIBODY-CHELATOR CONJUGATES The antibody-chelator conjugates (or antibody-metal ion complexes when pre-chelated metal ion is attached to the chelator prior to reaction of the chelator with antibody) of the invention may be produced by reacting the oxidized antibody with a compatible chelator having an available amine group selected from the group consisting of primary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups. The immediately resulting products (antibody-chelator conjugates or antibody-metal ion complexes) contain a carbon-nitrogen double bond resulting from elimination of a molecule of water from the initial addition products: Antibody-CH=O + NH2-R Antibody-CH=N-R + H2O
A solution of the oxidized antibody at a concentration of from about 0.5 to 20 mg/ml is mixed with the compatible chelator (molar ratios of reactive chelator group to antibody aldehyde ranging from about 1 to about 10,000) and the solution incubated for from about 1 to 18 hours. Suitable temperatures are from 0 to 37C and pH may be from about 6 to 8.
HYDRAZIDE-DIETHYLENETRIAMINEPENTAACETIC ACID
HYDRAZIDE-FICOLL
153Gd-DTPA-HYDRAZIDE-THIOACETYLAMINOBENZOICHYDRAZIDE-FICOLL-ACETYLTHIOIgG
For example, glycyl-tyrosyl-lysinediethylenetriaminepentaacetic acid (GYK-DTPA) was coupled to oxidized antibody by incubating an aliquot of the antibody with a 200-fold molar excess of GYK-DTPA in PBS (pH 6.0) for 30 minutes at room temperature. To stabilize the product sodium cyanoborohydride (NaCNBH3) was added to a final concentration of 10mM, and the reaction mixture was maintained at room temperature for a period ranging from 2 hours to overnight. The sample was then passed through a Sephadex G-50 column (Pharmacia Fine Chemicals, Piscataway, NJ) and the protein fractions were pooled.
In other experiments, oxidized antibody was coupled either to p-aminoaniline-diethylenetriaminepentaacetic acid (ADTPA) or polyethyleneiminediethyenetriaminepentaacetic acid (PEI-DTPA) by the same method except that a 2000-fold molar excess of ADTPA or PEI-DTPA was utilized.
. For instance, 111Indium may be attached by incubation of 111InCl3 with the antibody-chelator conjugate in acetate buffer, pH 6.0, at 37C for 1 hour. Such method of attachment advantageously allows purification of the antibody-chelator intermediate without exposing the preparer to excessive handling of radioactive materials, and without necessity of lengthy storage of radioisotopes in the work areas.
More specifically, the purified complexes of the invention contain metal ion chelated to the chelator, but are substantially free of metal ion adventitiously bound to antibody, that is, metal ion associated with the antibody protein or carbohydrate and not complexed to the chelator. The purification can be accomplished by any suitable purification method, including but not limited to high performance gel permeation liquid chromatography.
Expression host Rosetta PlysS Buffer 40mM HEPES, 30mM NaCl, pH 7.5, 3% Glycerol Elution buffer 500mM Imidazole in 40mM HEPES, 30mM NaCl, pH 7.5, 3% Glycerol Mol. Wt of Hom - ~51.3kDa