Immunology Assays for Clinical Research

Yan Ge, Ph.D.University of Virginia

Email: yg9z@virginia.edu

Common Immunology Assays

ELISA ELISPOT Flow cytometry Immunofluorescent staining

ELISA

Enzyme-linked immunosorbent assay

To detect the level of an antibody or an antigen in a sample

Types of ELISA

Indirect ELISA

Sandwich ELISA

Competitive ELISA

Indirect ELISA – Antibody Detection

Sandwich ELISA– Antigen Detection

Enzymes and Substrates horse radish peroxidase (HRP)

OPD o-phenylenediamine dihydrochloride, maximum absorbance: 492 nm

TMB3,3',5,5'-tetramethylbenzidine, maximum absorbance: 450 nm

ABTS2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt,maximum absorbance: 410 nm

Enzymes and Substrates

alkaline phosphatase (AP) PNPP p-Nitrophenyl Phosphate, Disodium Salt, maxium absorbance: 405 nm

Measurement

Optical density (OD) at a certain wavelength

Standard curve absolute concentration or arbitrary units

ELISPOT

Enzyme Linked Immunospot Principle: sandwich ELISA To detect secreted proteins (e.g.

cytokines, growth factors)

Flow Cytometry (FACS, Flow)

A technique used to measure the physical and chemical properties of cells or cellular components

Cells are measured individually, but in large numbers.

Applications Immunophenotyping

Identification of cells using cell-surface markers

Intra-cellular proteins (e.g. cytokines, chemokines)

Kinetics E.g. Ca2+ mobilization using Indo-1

DNA and cell cycle DAPI, propidium iodide, 7-AAD

Cell sorting

Cell Staining with Fluorochromes

Flow Cytometer

Components: Fluidics Optics Detectors Electronics

Fluidics

FSC and SSC

Side-scattered light (SSC): cell granularity or internal complexity

Forward-scattered light (FSC): cell-surface area or size

Compensation

An example

Stain cells with:

FL1: CD4-FITC FL2: CD19-PE FL3: 7-AAD (live/dead cells) FL4: CD3-Alexa Fluor 647 FL5: CD8-Alexa Fluor 750

Data Analysis

Live cells

(7-AAD negative)lymphocytes

Data Analysis

FL4: CD3-Alexa 750

FL1:

CD

4-

FITC

FL5:

CD

8-A

lexa

750

FL4: CD3-Alexa 750

CD4+ T cells

CD8+ T cells

Data Analysis

B cells

Cou

nts

FL2: CD19-PE

References Introduction to Flow Cytometry: A

learning Guide. Manual Part Number: 11-11032-01, April 2000, BD Sciences.

Introduction to Flow Cytometry. Flow cytometry core facility. Biomedical Instrumentation Center, Unifored Services University.

Introduction to Flow Cytometry. By Misha Rahman, AbD Serotec.