Conjugates antifluorescein/horseradish peroxidase

The fluorescein is detected, after membrane blocking, using the antifluorescein/horseradish peroxidase conjugate and ECL detection. Full details of the detection procedure can be found in Chapter 21. 

DNA probes can be labeled using the modified nucleotide, fluorescein-11-dUTP, and subsequently detected with enhanced chemiluminescence (1) using an antifluorescein antibody conjugated to horseradish peroxidase (2). The use of enhanced chemiluminescence avoids the fading of results associated with some colormetric detection procedures, used with nonradioactive labeling and detection systems. Hard copy results showing excellent resolution are produced. 

Dilute the antifluorescein/horseradish peroxidase conjugate 1000-fold in freshly prepared antibody binding buffer. The final volume should be at least equivalent to that used for hybridization. Incubate the blots in diluted conjugate with gentle agitation at room temperature for 60 min see Note 9). 

Enzyme conjugate Antifluorescein antibody conjugated to horseradish peroxidase. Conjugate should be stored at 2-8°C. 

Antifluorescein antibody/horseradish peroxidase conjugate. Available from Amersham International (RPN 3022). 

Antibodies may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase, or to other tags such as fluorescein or biotin (55). The fluorescein is measured by antifluorescein antibodies coupled with an enzyme, and biotin is measured by an avidin-enzyme conjugate, which binds with high affinity to biotin. Michalik et al.(47) used human albumin to tag their second antitoxin, and then used an anti-albumin antibody conjugated to peroxidase for detection of botulism toxins Aand B. They reported a sensitivity for this method of 100 and 300 MLD for toxin types A and B, respectively. 

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