Antigen-independent binding of rat immunoglobulins in a radioimmunoassay. Solutions to an unusual background problem

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Antigen-independent binding of rat immunoglobulins in a radioimmunoassay. Solutions to an unusual background problem. / Nilsson, Rune; Sjögren, Hans Olov.

I: Journal of Immunological Methods, Vol. 66, Nr. 1, 1984, s. 17-25.

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T1 - Antigen-independent binding of rat immunoglobulins in a radioimmunoassay. Solutions to an unusual background problem

AU - Nilsson, Rune

AU - Sjögren, Hans Olov

PY - 1984

Y1 - 1984

N2 - A high non-specific binding of immunoglobulins to plastic surfaces was noted with a number of rat sera, when tested in an indirect 125I-labeled protein-A assay for detection of cell-surface-bound rat immunoglobulins of various classes and IgG subclasses. This type of non-specific binding was found with all types of Ig. The degree of binding varied with the type of test plate used and fluctuated with time among sera drawn sequentially from the same donors. Coating test wells with fetal calf serum supplemented with BSA, gelatin or fibrinogen did not eliminate the reactions. The immunoglobulins bind directly to the polystyrene, and not to antigens present in fetal calf serum or autoantigens in rat serum. Two different approaches were used to eliminate the nonspecific reaction. When living cells were used as target antigens, exclusively cell-bound radioactivity was eluted with the non-ionic detergent Nonidet P40, which solubilizes the cell membrane without breaking the protein-A/rabbit IgG, rabbit IgG/rat Ig, or rat Ig/plastic interactions. When rat serum antibodies are tested on target antigens adsorbed on non-tissue culture grade plates, non-specific binding may be avoided by including 0.05% Tween 20 in the incubation mixture.

AB - A high non-specific binding of immunoglobulins to plastic surfaces was noted with a number of rat sera, when tested in an indirect 125I-labeled protein-A assay for detection of cell-surface-bound rat immunoglobulins of various classes and IgG subclasses. This type of non-specific binding was found with all types of Ig. The degree of binding varied with the type of test plate used and fluctuated with time among sera drawn sequentially from the same donors. Coating test wells with fetal calf serum supplemented with BSA, gelatin or fibrinogen did not eliminate the reactions. The immunoglobulins bind directly to the polystyrene, and not to antigens present in fetal calf serum or autoantigens in rat serum. Two different approaches were used to eliminate the nonspecific reaction. When living cells were used as target antigens, exclusively cell-bound radioactivity was eluted with the non-ionic detergent Nonidet P40, which solubilizes the cell membrane without breaking the protein-A/rabbit IgG, rabbit IgG/rat Ig, or rat Ig/plastic interactions. When rat serum antibodies are tested on target antigens adsorbed on non-tissue culture grade plates, non-specific binding may be avoided by including 0.05% Tween 20 in the incubation mixture.

KW - plastic binding of Ig

KW - non-specific Ig binding in RIA

KW - cell surface antigens

KW - binding test

KW - rat Ig (sub)classes

U2 - 10.1016/0022-1759(84)90243-6

DO - 10.1016/0022-1759(84)90243-6

M3 - Article

VL - 66

SP - 17

EP - 25

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 1872-7905

IS - 1

ER -