Preparation of Labeled Antibodies

In addition to labeling immunoglobulins with enzymes to provide detectability through their catalytic action on a substrate, antibody molecules also can be labeled or tagged with small 

The following three sections describe the preparation and properties of fluorescent, radio-labeled, and biotinylated antibodies. 

Chromatograpi Support ContailHijI-Nicket-Chelatedl Iminodiacetic Groups 

Wash the column with 10 vol of water, then equilibrate the support with 2 vol of 10 mM sodium phosphate, 0.15 M NaCl, pH 7 (binding buffer). 

Dissolve or dialyze the conjugate into binding buffer. Apply the conjugate solution to the column while collecting 2-ml fractions. 

Continue to wash the gel with 0.15 M NaCl (saline solution) until the absorbance at 280 nm is down to baseline. The protein eluting from the column at this point is unconjugated enzyme. 

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For preparation of labeled antibody for storage the calculated amount of immunoadsorbent (see below) is washed and added to one or both (pooled) pH 2.0 washes and allowed to react with gentle mixing overnight at 4°. The antibody-bound immunoadsorbent is then washed and resuspended in a total volume of 8 ml of NIGP buffer. The aliquot size for storage is determined by the size and number of assays to be performed, the radioactivity incorporated, and the total counts required per assay tube. 

In immunoradiometric assays the use of labeled antibody as the reagent for antigen measurement imposes the need for a very high degree of purity of the antibody. This situation is the converse of that posed by specificity in a radioimmunoassay. In the latter the antiserum used may be markedly heterogeneous provided only that the purity of the labeled antigen selects from the mixture of antibodies present an immune reaction that has the specificity required. Therefore, in an immunoradiometric assay in order to achieve sufficient purity of the antibody it is essential to prepare immunoadsorbent from a highly purified polypeptide. 

The following procedure details the preparation of labeled anti-IgG. Modification of the procedure for the labeling of other antibodies can... 

G. M. Addison, Ph.D. Thesis Preparation and Properties of Labeled Antibodies, University of Cambridge, 1971. 

Antibodies. — A new volume of Methods in Enzymology covers the preparation and use of affinity-labelled immunoglobulins. The specialized procedures required for the preparation of ferritin-antibody conjugates have been reviewed and the use of the conjugates for the localization of enzymes in mitochondrial membranes discussed. 

Although colloidal gold is readily conjugated to proteins, preparation of direct antibody/gold reagents is not often done. The simplest approach to labelling with colloidal gold is an indirect one, where the primary antibody is detected... 

Detection of Toxin on Fixed Preparations with Labeled Antibodies... 

However, problems are encountered in production of rhenium radionuclides and work is being done to increase the yields of the radionuclides to meet urgent demands for their use in therapy. Moreover, rhenium is not as reactive as technetium. This situation makes rhenium chemistry somewhat specific - optimum conditions in the preparation of rhenium complexes or in antibody labeling using bifunctional ligands must be identified. 

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