Monoclonal antibody-based immunoenzymetric assays for quantification of human igg and its four subclasses

Robert G Hamilton, Raymond W. Wilson, Thomas Spillman, Marianne Roebber

Research output: Contribution to journalArticle

Abstract

A panel of 5 immunoenzymetric assays (IEMAs) has been developed for quantification of the total and four subclasses of immunoglobulin G (IgG) in human serum using IUIS/WHO documented monoclonal antibodies (MoAb). Human IgG specific MoAb was adsorbed to microtiter plates and used to capture IgG from serum. Peroxidase conjugated forms of polyclonal mouse anti-human IgG Fc or a mixture of 4 MoAb (anti-kappa, anti-lambda, anti-IgG Fc PAN and anti-IgG Fd PAN) were used as detection antibodies. Use of monoclonal antibody in chromatographically purified form was required for acceptable assay sensitivity (S) and working ranges (WR). All 5 IEMAs displayed good precision (intra-assay %CV <5%, inter-assay %CV <12%) and parallelism (inter-dilutional CV <20%). Both HP6069 or HP6070 (anti-IgG1 Fc) worked well alone or together as capture antibodies in the IgGI IEMA: WR - 20–1250 ng/ml, S = 15 ng/ml. HP6002 (anti-gG2 Fc) alone or in combination with HP6014 (anti-IgG2 Fd) produced an IgG2 IEMA with a WR of 5–200 ng/ml and S of 5 ng/ml. HP6047 (anti-IgG3 hinge) alone generated a sensitive IgG3 IEMA with a narrow working range: WR - 2–50 ng/ml, S = 1.6 ng/ml. Both HP6025 and HP6023 (anti-IgG4 Fc) worked equally well alone and together to produce a useful IgG4 IEMA: WR = 8–250 ng/ml, S = 7.8 ng/ml. HP6017 (anti-IgG PAN Fc) was combined in an equal molar ratio with HP6046 (anti-IgG PAN Fd) to produce a total IgG PAN IEMA with a WR of 5–530 ng/ml and a sensitivity of 5 ng/ml. All 5 IEMAs fulfilled requirements for robust clinical immunoassays that permit the quantitation of human IgG and its 4 subclasses.

Original languageEnglish (US)
Pages (from-to)275-296
Number of pages22
JournalJournal of Immunoassay
Volume9
Issue number3-4
DOIs
StatePublished - Sep 1 1988
Externally publishedYes

Fingerprint

Assays
Immunoglobulin G
Monoclonal Antibodies
Antibodies
Hinges
Serum
Immunoassay
Peroxidase

Keywords

  • Human IgG subclasses
  • immunoassay
  • monoclonal antibodies
  • quality control
  • quantification

ASJC Scopus subject areas

  • Immunology
  • Pharmacology

Cite this

Monoclonal antibody-based immunoenzymetric assays for quantification of human igg and its four subclasses. / Hamilton, Robert G; Wilson, Raymond W.; Spillman, Thomas; Roebber, Marianne.

In: Journal of Immunoassay, Vol. 9, No. 3-4, 01.09.1988, p. 275-296.

Research output: Contribution to journalArticle

Hamilton, Robert G ; Wilson, Raymond W. ; Spillman, Thomas ; Roebber, Marianne. / Monoclonal antibody-based immunoenzymetric assays for quantification of human igg and its four subclasses. In: Journal of Immunoassay. 1988 ; Vol. 9, No. 3-4. pp. 275-296.
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AB - A panel of 5 immunoenzymetric assays (IEMAs) has been developed for quantification of the total and four subclasses of immunoglobulin G (IgG) in human serum using IUIS/WHO documented monoclonal antibodies (MoAb). Human IgG specific MoAb was adsorbed to microtiter plates and used to capture IgG from serum. Peroxidase conjugated forms of polyclonal mouse anti-human IgG Fc or a mixture of 4 MoAb (anti-kappa, anti-lambda, anti-IgG Fc PAN and anti-IgG Fd PAN) were used as detection antibodies. Use of monoclonal antibody in chromatographically purified form was required for acceptable assay sensitivity (S) and working ranges (WR). All 5 IEMAs displayed good precision (intra-assay %CV <5%, inter-assay %CV <12%) and parallelism (inter-dilutional CV <20%). Both HP6069 or HP6070 (anti-IgG1 Fc) worked well alone or together as capture antibodies in the IgGI IEMA: WR - 20–1250 ng/ml, S = 15 ng/ml. HP6002 (anti-gG2 Fc) alone or in combination with HP6014 (anti-IgG2 Fd) produced an IgG2 IEMA with a WR of 5–200 ng/ml and S of 5 ng/ml. HP6047 (anti-IgG3 hinge) alone generated a sensitive IgG3 IEMA with a narrow working range: WR - 2–50 ng/ml, S = 1.6 ng/ml. Both HP6025 and HP6023 (anti-IgG4 Fc) worked equally well alone and together to produce a useful IgG4 IEMA: WR = 8–250 ng/ml, S = 7.8 ng/ml. HP6017 (anti-IgG PAN Fc) was combined in an equal molar ratio with HP6046 (anti-IgG PAN Fd) to produce a total IgG PAN IEMA with a WR of 5–530 ng/ml and a sensitivity of 5 ng/ml. All 5 IEMAs fulfilled requirements for robust clinical immunoassays that permit the quantitation of human IgG and its 4 subclasses.

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