Immunolatex spheres

The identification of mouse spleen lymphocytes (B T cells) by SEM and laboratory light microscopy using immunolatex spheres as specific markers. 

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. 

As shown in Figure 8, AKR/j thymocytes are densely labeled with immunolatex spheres 110 nm in diameter. Over 951 ot the cells were labeled. C3H thymocytes, as well as thymocytes from a number of other strains of mice which do not display the AKR specificity, were not labeled with the spheres. In the reverse... 

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 ... 

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. 

---