Immunolatex conjugates

Procedure Immunolatex conjugates are prepared by a method adapted from that used by Cuatrecasas.iS. An aqueous suspension of latex spheres (20-50 mg/ml) adjusted to pH 10.5 with NaOH is activated at 25°C with 10 mg of CNBr per ml of suspension. The reaction mixture is maintained between pH 10 and 11 by the slow addition of 2N NaOH. After 15 minutes, the suspension is diluted with an equal volume of cold O.IM borate buffer at pH 8.5 and equilibrated at 1+°C. A solution of purified antibody in O.IM borate buffer pH 8.5 is added to the latex suspension resulting in a final protein concentration of 0.5 mg/ml. The reaction is allowed to proceed at 1+°C for at least 1+ hours after which it is terminated by the addition of an eaual volume of O.IM glycine buffer at pH 8.5 ... 

Immunolatex conjugates prepared by this procedure have been shown to be active in their capacity to bind antigens. However, some aggregation of the conjugates has been observed during the... 

The following procedure has been routinely used in our laboratories to prepare immunolatex conjugates of high immunochemical activity for applications in cell surface labeling. 

A scanning electron micrograph of human RBC labeled with Immunolatex spheres of diameter 60 nm (600 A) and at a concentration of 20 mg/ml Is shown In Figure 6a. The same dense distribution of spheres was observed when the conjugate concentration was reduced ten fold. Markers of this size are readily seen In the SEM and can be used to Identify cells exhibiting specific surface euitlgens In either mixed cell populations or tissue specimens. 

The specificity of the immunolatex markers can be demonstrated in control experiments designed to measure the amount of nonspecific binding to cell surfaces. RBC, which have not been sensitized with rabbit antihuman RBC antibodies, are treated with goat anti-rabbit Ig antibody-latex conjugates (20 mg/ml) and washed in the usual manner. As shown in Figure 7b, only a few spheres adhere to the surface of the red blood cells. 

The polymer particles formed in Problem 7.11 are suspended in water adjusted to pH 10.5 with NaOH and activated at 25°C with 10 mg of cyanogen bromide per mimiiter of suspension. After 15 min, it is diluted with equal volume of eold 0.1 Af borate buffer at pH 8.5 and 4°C. The immunolatex conjugates thus prepared are known to bind antigens. Explain why this occurs. 

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