Radiolabelled conjugate

Determination of the receptor binding affinity of the radiolabelled conjugates under study by a direct binding assay using cell membranes, cells and antigen. 

A binding of 13.2 0.8% ( = 3) was observed with 5 x 10 MCF-7 cells for 1 gg of the Lu-p-NCS-benzyl-DOTA-estradiol complex. With SepPak purification, the cell uptake improved to 17.1 1.6% (n = 3). No further increase in cell uptake was observed with HPLC purification. It was observed that the tracer uptake in the cells decreased to 8.3 2.0% (n = 3) with the addition of 100 gg of cold estradiol. Similar results were observed when these experiments were carried out on 24 well plates where cells were plated 1 d prior to the experiments. The decrease in cell uptake with the addition of estradiol indicated the specificity of the radiolabelled conjugate for the MCF-7 cell lines. Blank experiments were carried out with Lu labelled BFCA under similar experimental conditions. No retention of the activity in the cell pellet was observed, ruling out the possibility of carrier mediated uptake. 

The labelled conjugate is purified on a CIS SepPak cartridge after conditioning with 2 mL of 0.05M phosphate buffer at pH7.5. The column is eluted with 5 mL of 0.05M phosphate followed by 0.5 mL of ethanol. While free iodide is eluted in the phosphate buffer, the conjugate is obtained in pure form in the ethanol fraction. The ethanol fraction is evaporated under nitrogen and the radiolabelled conjugate is reconstituted in saline. 

Using the same methodology these radiolabelled conjugates were then used clinically in Phase I studies. Julian et. al. used whole body imaging... 

Hapten density is important for both immunization and assay performance, and hence the extent of conjugation or hapten density should be confirmed by established methods. A characteristic ultraviolet (UV) or visible absorbance spectrum that distinguishes the hapten from the carrier protein or use of a radiolabeled hapten can be used to determine the degree of conjugation. If the hapten has a similar A. iax to the protein, the extent of incorporation can still be estimated when the concentration of the protein and the spectral characteristics of the hapten and protein are known. The difference in absorbance between the conjugate and the starting protein is proportional to... 

A monoclonal antibody-based ELISA has been utilized to determine ceftiofur levels in milk. The authors noted that matrix interference occurred, but a 1 100 dilution lowered the interference, and a 1 1000 dilution eliminated the matrix interference. Because of the high dilution, samples could not be measured below l.Opgkg The assay measured ceftiofur, its major metabolite desfuroylceftiofur, and ceftiofur protein conjugates and has been utilized to measure residues in milk from cows treated with therapeutic doses of the drug. The results from the incurred residue correlated well with a previous study using radiolabeled ceftiofur, confirming the detection of a metabolite that was not detected by HPLC. 

Antibody. Rat monoclonal antibody 34A was purified from nu/nu mouse ascites fluid as described (79). The 34A was radiolabeled with 125I using IDO-GEN (Pierce, Rockford, IL) method to a specific activity of 2 to 4 x 105 cpm/pg, and conjugated with NGPE as previously described (7). 

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble products that can be accurately quantified by their absorbance or fluorescence. Alternatively, substrates are available which form insoluble, highly colored precipitates, excellent for localizing antigens in blots, cells, or tissue sections. The flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques. 

Kobayashi, H., Sato, N., Saga, T., Nakamoto, Y., Ishimori, T., Toyama, S., Togashi, K., Konishi, J., and Brechbiel, M.W. (2000) Monoclonal antibody-dendrimer conjugates enable radiolabeling of antibody with markedly high specific activity with minimal loss of immunoreactivity. Eur. J. Nucl. Med. 27, 1334-1339. 

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