Quantifying lateral flow

tests Reader and label options

Innovation Results Partnership Quality

Steven Lamont

Technical Manager

Quantification

How to?

Reporting

Calibration curves

Label options

Comparison

Readers

Options

Reader technologies

Novarum

Overview

Version 1 Final

To determine or

express the

quantity of

Quantification

Version 2 Draft

Semi- quantitative Semi-quantitative Fully-quantitative

Score card Number of

test lines

Calibration

curve

How to quantify

Version 2 Draft

Requires the use of a score card

Control

3

2

1

High positive

Score card Result

Control

3

2

1

Weak positive

Visual semi-quantification

Version 2 Draft

Reader quantification requires numbers

A calibration curve needs to be generated from which data points can be

read off

Quadratic

Log

Linear

4-PL

mV Grayscale RGB

Concentration

Va

lue

Reader quantification

Version 2 Draft

Quantification of an assay can be presented to the end user as:

Yes / No

Qualitative

Pre-defined

bins

Semi-quantitative

ng/ml readout

Fully-quantitative

In most cases test values require to be read off a calibration curve with the

difference being the post-processing of the data

Reporting quantification

Version 2 Draft

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1 10 100 1000 10000

Test

valu

e

Log of concentration

4PL Fit: y=(A-D) / (1 + (x/C)^B) + D

0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

OD

valu

e

Concentration

4-P

L

Quad

tra

tic

Calibration curve

Version 2 Draft

An appropriate number of standards across the range of interest

Number of replicates of each standard

Curve fit and specifications around the curve parameters

Simplest curve fit should be chosen

Concentration

Va

lue

Calibration curve

Version 2 Draft

Points to consider

Algorithm – Test line only or a Test/Control

ratio?

What is the goal of the assay?

Curve dynamics and your analytical range

% CV requirements for calculated

concentration

Detection Particle Advantages Disadvantages

Gold colloid

Compatible with most

reflectance, absorbance and

camera system readers

Can be visually read

Polystyrene beads (latex)

Range of colours available

Clear visual multiplexing

Range of sizes

Flow up the strip can be more

problematic due to size –

membrane can require

treatment to improve flow

Fluorescent labels

Huge range commercially

available

No visual signal for the end

user to interpret

Particle based or direct

labelling

Need to be careful of

potential auto fluorescence

with materials eg.

Nitrocellulose

Dependent on reader

Filters required

Magnetic latex Perceived sensitivity increase Dependent on reader

Limited choice of readers

Label options

Version 2 Draft

There are a wide range of lateral flow readers on the market now:

Reader options

Version 2 Draft

Utilises the camera on a smart phone to quantify your test strip

Novarum uses intelligent real time image processing which ensures the data is

usable before it is captured

Novarum

Version 2 Draft

Pro’s Con’s

Cheap Tend to have a small

aperture

Tuneable to appropriate

wavelength

Can be fixed or scanning

Pro’s Con’s

Provides an image Expensive

Technology moves on

quickly

LED / Photodiode

CCD / CMOS

Reader technologies

Version 2 Draft

Scanning

Test strips or optics move over a pre-defined distance to scan the length of

the test strip

These readers are more flexible

Fixed point

Disposable in-cassette readers

These readers require no moving parts, but there is then a necessity for

tight tolerances on line position

This has an impact on the equipment used to manufacture the test

Reader technologies

Version 2 Draft

Quantification can be visual or reader based

Particle choice will determine what readers

can be used

Think about the goal of your assay – what

benefit does quantification bring?

Assay precision is key to reliable

quantification

Summary

Version 1 Final

Version 1 Final

Contract Development & Manufacture

Lateral Flow Test Development

Lateral Flow Test Manufacture

Full Regulatory & Post Market Support

Sensitivity Enhancement Technology

Platforms

Readers

Raw Materials

Antibodies

Antigens

Enzymes

Gold Nanoparticles

Serum & Plasma

Cell Culture Reagents

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