I am working through a paper, Lateral flow immunochromatographic assay on a single piece of paper. I'm having trouble understanding one of the points!

Under the Device fabrication question, it says:

The sample and conjugate release regions were treated with a blocking solution to prevent nonspecific binding of AuNP-antibody conjugates.

I didn't understand this phrase, so I did a bit more research and found a question by the author on ResearchGate asking how to prevent nonspecific binding.

However I still don't quite understand. What exactly is nonspecific binding of AuNP-antibody conjugates, and how does a blocking solution prevent this?

Any overview/pointers about where to research further would be very much appreciated.

  • $\begingroup$ Isn't Biology SE more suitable? But it is hard to understand your question. Isn't clear if you don't understand the sentence (which I do understand) or you want to understand details (which I don't know). You could have a title like "How to prevent..... $\endgroup$ – Alchimista May 4 at 9:46
  • $\begingroup$ @Alchimista the former: I don't understand the sentence. Probably the basics: what is a blocking solution? what is nonspecific binding? how does a blocking solution prevent this? This is probably pretty basic stuff, I would love to know how to learn more about these points. $\endgroup$ – Anonymous May 4 at 19:55
  • $\begingroup$ Biology SE more chances to get an answer. $\endgroup$ – Alchimista May 5 at 7:43
  • $\begingroup$ @Alchimista The answer on Chemistry SE posted today is pretty good. And it is pretty chemical (conjugated proteins, surfactant, buffer). $\endgroup$ – Karsten Theis Jun 8 at 1:57

Let's start with defining the terms.

  • The sample region is the portion on a lateral flow immunoassay that receives a sample (of blood, urine, oral fluid, ...). This has often been treated to adjust the sample's pH, viscosity, ionic strength, or protein content.
  • The conjugate release region is the portion on a lateral flow immunoassay that holds immobilised, conjugated antigens. It provides a site for the conjugated antibodies to bind to the target before migrating further.
  • A lateral flow membrane is commonly made of nitrocellulose. In this specific paper, the authors evaluate nitrocellulose membrane and cellulose chromatography paper. They mention, "Nitrocellulose membrane and cellulose chromatography paper both offer excellent protein binding capabilities".
  • Protein binding is where the protein 'sticks' to a surface. This is great for binding to the test and control lines (specific binding), but no good when binding to the membrane (non-specific binding).

The substance tested for is usually a protein. When the sample travels up an untreated nitrocellulose strip, the protein will bind to the membrane instead of the test line.

To prevent this, a nitrocellulose strip is treated with a blocking buffer. This is a mixture of proteins that binds to the membrane, preventing binding from the protein being tested for.

In this paper, the protein tested for was PfHRP2 (Plasmodium falciparum histidine-rich protein 2), a biomarker for malaria detection. Unfortunately, the authors were getting false-positive results (no test line showing up) because the PfHRP2 was binding to the nitrocellulose membrane and not to the test line.

This was prevented by treating the membrane with a blocking buffer of 20% sucrose (stabilises proteins), 0.25% Tween-20 (a surfactant, enhances the flow speed of the sample), and 2% BSA (bovine serum albumin, the protein which binds to the membrane) in PBS (phosphate buffer saline, a buffer solution).


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