FAQs

Quantify your lateral flow test

Lateral flow tests can be run both qualitatively and quantitatively. A qualitative test only examines the sample for the presence or absence of a specific analyte and can be read with the naked eye and the positive/negative result is enough. Pregnancy test is a good example of it.

However, in many day-to-day decisions, it is not enough to just obtain the “yes” or “no” answer.  Oftentime, it comes down to the amount of constituents of the sample. This is where we come to talk about the quantitative testing. We decided to give this field of activity the recognition it deserves and compiled a list of FAQs that often come up in this respect.

In contrast to pure qualitative test, the change in the intensity of the line cannot be detected by naked eye. To determine a concentration on the test strip, a reader is required to convert color change into concentrations using a stored calibration standard curve.  

To quantify a lateral flow test, the intensity of the test line has to be evaluated, using a calibrated camera or a detection system. Typically, the intensity is translated into a 2D peak profile and its area or height is quantified. The peak height value can then be traced against a standard curve, listing known concentration values against previously measured peak heights.  

Following hardware requirements of the reader have to be taken into consideration:

  • Linear camera picture in order get the raw data
  • Adequate and homogeneous illumination
  • Dynamic integration times

A standard curve is an experimentally generated graph in which the concentration of the solution is plotted on the x-axis and the measured variable – gray value, for instance – is plotted on the y-axis. The curve is created by measuring the concentration and gray level of several prepared solutions/probes, called calibration standards. Once the curve has been drawn, the concentration of the unknown solution can be determined by placing it on the curve based on the gray level measurement. 

The following requirements should be fulfilled:  

  • Low variability of the entire system (CVs* ≤20 %, ≤25% @ LoQs) like sampling, assay and reader 
  • Accurate –≤20 % from nominal concentration 
  • Specific 
  • Stable 

The components are: 

  • Assay design and components 
  • Biological reagents 
  • Labels 
  • Sample collection and handling methods 
  • Readers 
  • Manufacturing process 

The sources are: 

  • Assay design and components 
  • Biological reagents 
  • Sample collection and handling methods 
  • Manufacturing process 

Following aspects have to be considered when creating the calibration curve:

  • Generated for each lot
  • Loaded on multiple readers
  • Programmed into reader via RFID or barcode
  • Algorithm determines concentration of test

Following hardware requirements of the reader have to be taken into consideration:

  • Linear camera picture in order get the raw data
  • Adequate and homogeneous illumination
  • Dynamic integration times

We recommend to use at least 7 different concentration levels – from low to high. This should cover the entire quantifiable range.

Yes, to create your own standard curve, you need standard fitting curves. All the immunoassays are following a sigmoidal behavior, but depending on the target concentration range – low, medium or high. Respectively, one can use exponential, linear or logarithmic functions.

No, the test needs to be designed for quantitative read out.

Generally speaking, there are a few parameters which need to be optimized in order to switch from qualitative to quantitative assay formats:

  • The capture molecule (antibody) should have high concentration and high affinity constants. Thereby capacity of the test band for high analyte concentrations is maximal.
  • The gold conjugate should be optimized (coupling pH of the gold colloids, coupling time, concentration of the detector molecule/antibody) and formulation of the conjugate buffer and the material for preparation of the Conjugate Release Pad.
  • The concentration of a given conjugate also may affect the standard curve. Titration of the gold conjugate concentration also is a good tool to guarantee lot-to-lot consistency.

This is really hard to say. The market trend we observe today is that more and more companies develop and offer quantitative lateral flow assays. This is due to the fact that the quality of the lateral tests has significantly improved over time. However, they still don’t get the recognition in the public (and even scientific community) they actually deserve.

Definitely not. The application of the label for conjugation should depend on the analyte concentration which is addressed by a particular lateral flow assay. If the analyte concentration of interest is in a μg-ng/mL range, there is no particular need to use fluorescence detection. For highly sensitive assays, however, you might require fluorescence labels (organic dyes, quantum dots, etc.).

Preparation and application of gold conjugates is rather simple, flexible and robust. If you use fluorescence labels you have to address basic fluorescence of the analytical membrane as well as diffusion problems of conjugate during test run (background fluorescence).

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