While excellent lateral flow dipstick systems have been made for large molecular weight analytes — shown so clearly by the home pregnancy test — the performance of classical immunoassay is often found to be equally clearly lacking when applied to small molecular weight analytes such as toxic synthetic chemicals, drugs, explosives and natural toxins.
The problem is simply one of size. Large analytes can support the simultaneous binding of both capture and detector antibodies allowing typical sandwich immunoassays to be formatted in which increasing analyte concentration provides an increase of observable signal over a zero background. Small molecules are simply not large enough to support such dual binding and so alternative 'competitive-format' systems have been used.
In these, analyte competes with a detectable analyte-analogue for capture antibody sites and so in effect the system measures how much analyte is not present. This introduces major problems not only in terms of precision and sensitivity but also read-out where classically reducing observable signal is indicative of increasing concentration of analyte.
In particular, this makes point-of-need devices difficult to read and assess. On dipsticks, for example, zero analyte provides the maximal line against which other determinations, providing possibly less intense lines, have to be gauged. What is required is a robust generic system in which there is no observable signal in the absence of analyte (i.e. a clear strip) and even low level samples give an obvious observable line over this zero background. Devising an excellent system to overcome this problem and open up many possibilities of providing high performance testing systems has been our goal. This has successfully resulted in the following system.
The Selective Antibody Solution
A dip stick is provided with a visualisation zone that can bind an antibody against the target analyte. The primary antibody is coupled to red gold particles. This coupled antibody is applied to the end of the dip stick with the sample and a blocking substance which binds the primary antibody in the absence of target analyte.
NO TARGET ANALYTE - NO SIGNAL
In the absence of target analyte gold-labelled antibody binds the blocking substance and is prevented from binding in the visualisation zone. No red line is seen indicating absence of target analyte.
ANALYTE PRESENT - STRONG SIGNAL
When present, target analyte binds the gold-labelled antibody, leaving it unblocked to bind as a red line in the visualisation zone. The more analyte present the more intense the line appears over a blank background - a critical advantage over classical competitive systems where the most intense line is seen with no analyte and addition of analyte slowly reduces it - presenting the challenge of distinguishing one line from another.
SAL Technology in Action
The presence of small molecular weight analyte gives rise to a positive line appearing out of a clear white background, as if the analyte were actually being seen on the stick. Intuitively, the more line is seen the more analyte is present.
The cassette dipstick on the right receives anayte-free buffer, whereas the cassette dipstick on the right receives buffer containing the small molecular weight analyte.
During the, real-time run, both cassette dipsticks display a positive control line towards the top of the window to show the devices have been used correctly. Only the cassette receiving a positive sample shows a positive test line, towards the bottom of the window. This occurs very quickly. The fact that the positive sample is indeed positive can be seen by eye within a matter of seconds, allowing immediate action to be taken if necessary. Further development of the sticks over two minutes, allows the control line to stabilise and a quantitative determination of the concentration of the analyte.