Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Total Murine Immunoglobulin

Zheng Liu

doi:10.21769/BioProtoc.33

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Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Total Murine Immunoglobulin

Zheng Liu

Published: Feb 5, 2011 DOI: 10.21769/BioProtoc.33 Views: 15217

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Abstract

ELISA is an easy and relatively sensitive way to measure protein concentration. This protocol describes how to measure serum levels of murine total immunoglobulin of various isotypes. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

Keywords: ELISA

Mouse

Immunoglobulin

Serum

Background

Materials and Reagents

Coating antibody: goat anti-mouse IgX* unlabeled (Southern Biotech)
Note: IgX*: the isotype of the Ig you wish to detect.
Murine immunoglobulin of the desired isotype (Sigma- Aldrich, catalog number: M5284 )
Horseradish peroxidase (HRP) conjugated goat anti-mouse isotype specific antibodies (Southern Biotech)
2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) Peroxidase Substrate Solution A (Kirkegaard & Perry Laboratories, catalog number: 50-64-00 )
ABTS Peroxidase Substrate Solution B (Kirkegaard & Perry Laboratories, catalog number: 50-65-00 )
ABTS Peroxidase Stop Solution (Kirkegaard & Perry Laboratories, catalog number: 50-85-01 )
10x PBS-Tween 20 (see Recipes)
Blocking solution (see Recipes)

Equipment

Clear 96-well Microtest polystyrene assay plate (BD Biosciences)

Procedure