Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Murine  Anti-dsDNA Antibodies

Zheng Liu

doi:10.21769/BioProtoc.32

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Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Murine Anti-dsDNA Antibodies

Zheng Liu

Published: Feb 20, 2011 DOI: 10.21769/BioProtoc.32 Views: 15318

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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 anti-dsDNA antibodies. Circulating anti-dsDNA antibody is a hallmark of SLE both in human patients and in many SLE mouse models. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

Keywords: ELISA

Materials and Reagents

Salmon sperm DNA (Life Technologies, Invitrogen™, catalog number: AM9680 )
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

45-μm syringe top filter (USA Scientific)
Clear 96-well Microtest polystyrene assay plate (BD Biosciences)

Procedure