Simultaneous blockade of Fc gamma receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype-restricted epitopes on the kappa chain of rat monoclonal antibodies

P. Balogh, John G. Tew, Andras K. Szakal

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 Fc gamma 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 Fc gamma-receptor blockade.

Original languageEnglish
Pages (from-to)107-110
Number of pages4
JournalCytometry
Volume47
Issue number2
Publication statusPublished - Feb 1 2002

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IgG Receptors
Epitopes
Monoclonal Antibodies
Lymphocytes
Fc Receptors
Fluorescent Dyes
Immunoglobulins
Immunoglobulin G
Immunoglobulin Isotypes
Sprague Dawley Rats
Anti-Idiotypic Antibodies
Staining and Labeling
Antibodies

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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title = "Simultaneous blockade of Fc gamma receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype-restricted epitopes on the kappa chain of rat monoclonal antibodies",
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 Fc gamma 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 Fc gamma-receptor blockade.",
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AU - Tew, John G.

AU - Szakal, Andras K.

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N2 - 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 Fc gamma 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 Fc gamma-receptor blockade.

AB - 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 Fc gamma 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 Fc gamma-receptor blockade.

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