A century-old mistake
Scientists from Pushchino criticized the century-old method of amyloid research, which became popular due to incorrect citation
Natalia Bykova, ITEB RAS Press Service
The first mention in the scientific literature of the Congo red dye for the detection of amyloids dates back to 1923. Despite the emergence of new, more modern methods of research, this amyloid detector still remains a working tool in science. A group of scientists from the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences proved that the use of Congo red for the determination of amyloids was initially questionable and began with an incorrect citation of a scientific article. The researchers published a historical review and the results of their own experiment with the dye in the journal BioscienceReports (Yakupova et al., Congo red and amyloids: History and relationship).
The Congo red dye was used in science in the first half of the last century. It was used to detect amyloids in human tissues with amyloidosis. Amyloidosis is a group of diseases in which proteins in the body begin to acquire not their spatial form, but a rigid, so–called cross-beta structural one. This structure provides resistance to degradation and the ability to accumulate in tissues. It was considered proven that during polarization microscopy with Congo red, amyloids emit an "apple-green" glow. The method is not considered reliable enough due to its low specificity, but it continues to be used in modern laboratories, despite the emergence of new methods such as X-ray diffraction, circular dichroism or the use of specific antibodies.
(A) Amorphous aggregates of smooth muscle titin formed in a solution containing 0.2 M KCl have an amyloid structure according to X-ray diffraction data and do not bind to the Congo red dye.
(B) Amorphous aggregates of smooth muscle titin formed in a solution containing 0.15 M glycine also have an amyloid structure according to X-ray diffraction data, but the sample binds to the Congo red dye.
(C) Aggregates of smooth muscle titin in a solution containing 0.2 M KCl after partial disaggregation.
(D) Amorphous aggregates of smooth muscle titin formed in a solution containing 0.15 M glycine after partial disaggregation.
(E) Microscopy in polarized light of a dried drop of a buffer solution containing 0.15 M glycine-KOH, pH 7.2–7.4 and a Congo red dye (presumably glycine is stained).
The interest of scientists from ITEB RAS in the identification of amyloids using Congo red staining arose during experiments with amyloid aggregates of the titin protein conducted by invitro.
"Our study revealed that the glow occurs when the amino acid glycine is used in a buffer solution. In this case, the buffer itself, without any proteins, has both green and yellow, red and other glow. When studying the literature, it also became clear that this method does not allow to determine amyloids in solutions with high accuracy," explains the junior researcher, the first author of the article Elmira Yakupova.
Having delved into the history of scientific publications about Congo red, scientists have found out that the low specificity of the method of coloring amyloids with this dye can be traced at all times of its use. At the same time, the results of the study could be influenced by both the pH values of solutions and the content of various substances, as well as the spread between the results of researchers (different researchers could interpret the same samples differently).
"The following question was logical: if the method does not have a high specificity, then why has it been used all this time and is still used in modern laboratories? The answer to it is quite trivial, but it really struck us. It all started with incorrect or inaccurate citations," says Elmira Yakupova. – In particular, researchers picked up "green" or "apple-green" birefringence in the articles of a well-known scientist at that time (Missmahl, 1957). In addition, Ladewig's research in 1945 did not attract due attention at the time, and the article stated that not only green was found with amyloids. Modern researchers who have closely approached the physics of Congo red coloring (Howie et al., 2008) also note that the green color, which is acutely established in the diagnosis of amyloids in Congo red coloring, is not so necessary and it is important to take into account other colors. But other colors, according to their study of the literature, are mostly ignored by scientists (Howie and Owen-Casey, 2012)."
An article published in Bioscience Reports warns researchers about pitfalls when using Congo Red in their work and suggests more modern, alternative techniques for detecting amyloids. It also describes all the points that should be taken into account when using this coloring, including checking all components of solutions that can give a glow.
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