Haptenated antibodies for improved specificity or generalized application

Haptenated antibodies for improved specificity or generalized application 

Another successful method is the haptenation of IgG with azoben-zenearsonate residues (Table 12.11). Ricardo and Cebra (1977) re- 

IgG is suspended in distilled water or dissolved in PBS at a concentration of 20 mg/ml. K2CO3 is added to a final concentration of 20 mg/ml and the suspension stirred until complete dissolution. The subsequent procedures are shielded from light as much as possible, e.g., by wrapping the container in aluminum foil, since dinitrophenyl (DNP) derivatives are light sensitive. 

Dinitrobenzene-sulfonate (DNBS) is recrystallized from ethanol by dissolving its sodium or potassium salt (15 g) in 950 ml ethanol at 70 C with vigorous stirring. Norit A (2 g) is added, the mixture is stirred and then filtered rapidly through several layers of filter paper (Whatman No. 3) on a preheated funnel. Cooling causes the formation of small crystals. This procedure is repeated 2-4 times. The crystals (mp 300 C) are dried at 37 C and stored at 4 C in a dark container. 

The mixture is stirred until the desired degree of haptenation is obtained. This can be determined by measuring the absorbance at 360 nm. For simplicity, this absorbance can be assumed to be due solely to DNP-lysine groups, the molar extinction coefficient of which is 17530. Thus, the (moles of DNP)/(moles of protein) ratio can be calculated from the expressions (using the law of Lambert Beer Section 9.2.3.4)  

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