Simultaneous blockade of Fcγ receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype-restricted epitopes on the kappa chain of rat monoclonal antibodies
✍ Scribed by Péter Balogh; John G. Tew; Andras K. Szakal
- Book ID
- 102135464
- Publisher
- John Wiley and Sons
- Year
- 2002
- Tongue
- English
- Weight
- 76 KB
- Volume
- 47
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
Abstract
Background
Incubation of mouse hemopoietic cells with rat monoclonal antibodies (mAbs) of the IgG class sometimes results in Fc‐region mediated binding of immunoglobulins by Fc‐receptors. This unwanted binding can be prevented by preincubation of target cells with the rat anti‐mouse anti‐CD16/32 (2.4G2) mAb.
Methods
To avoid the cross‐reactivity of fluorochrome‐conjugated secondary anti‐rat antibodies with the Fc‐receptor blocking 2.4G2, direct fluorochrome‐conjugated immunoglobulins need to be used. However, we report that a mouse mAb (MRC OX12) with a strong rat Igk^a^ allotype preference can be used for flow cytometric measurements in conjunction with unlabeled rat mAbs with the simultaneous blockade of Fcγ receptors by the 2.4G2 mAb.
Results and Discussion
This lack of reactivity of OX12 against the 2.4G2 mAb is remarkable, as it could efficiently detect another Sprague‐Dawley–derived rat mAb. This staining procedure (unlabeled rat mAb of the appropriate strain detected by OX12 mAb in the presence of 2.4G2 IgG) is an attractive alternative to using direct antibody conjugates, while satisfying the need for an effective Fcγ‐receptor blockade. Cytometry 47:107–110, 2002. © 2002 Wiley‐Liss, Inc.