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Page 6 of 15 Alekseeva et al. Microbiome Res Rep 2023;2:10 https://dx.doi.org/10.20517/mrr.2023.06

Nunc MaxiSorp 96-well polystyrene plates were sensitized with recombinant FN3, 2D FN3 fragment and
CD FN3 fragment at a concentration of 5 µg/mL in 100 µL carbonate-bicarbonate buffer (CBC) (18 mM Na 2
CO , 450 mM NaHCO , pH 9.5) for one h at 37 °C. The plates were subsequently washed four times with
3
3
phosphate-buffered saline with 0.05% Tween 20 (PBST). In order to exclude nonspecific adsorption, 1%
casein was added to the wells and incubated for 1 h at 37 °C. Next, 100 μL of polyclonal rabbit IgG
antibodies was added to the wells in two-fold dilutions starting from a concentration of 1 mg/mL, incubated
at 37 °C for one h and washed four times with PBST. To exclude nonspecific binding between rabbit IgG
and FN3 and its fragments, nonspecific rabbit γ-globulin was added to FN3 instead of polyclonal rabbit IgG
antibodies. Goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate was added to the wells at a
1:20,000 dilution. The plates were incubated for 60 min at 37 °C, washed four times with PBST, and 3,3'5,5'-
tetramethylbenzidine was added to them. The reactions were stopped with 1 M sulfuric acid. Optical density
was measured at a wavelength of 450 nm using a Universal Microplate Reader (Biotek, Winooski, VT,
USA). The results were treated statistically with an unpaired t-test.

After confirming the binding of polyclonal rabbit IgG antibodies to the whole FN3 protein including its 2D
FN3 and CD FN3 fragments, their ability to bind human TNFα was assessed. The whole FN3 protein was
used as a positive control. To do this, we used the previously devised ELISA scheme . Below is a brief
[24]
description of it.

Nunc MaxiSorp 96-well polystyrene plates were sensitized with 100 µl polyclonal rabbit IgG antibodies to
the FN3 protein in concentration 1 µg/mL, incubated in carbonate-bicarbonate coating buffer (CBB) for 1 h
at 37 °C, followed by washing with the phosphate-buffered solution with 0.05% Tween 20 (PBST). In order
to exclude nonspecific adsorption, 1% casein was added to the wells and incubated for 1 h at 37 °C. After
washing, 100 µL of the FN3 protein solution, 2D FN3 or CD FN3 fragments at a concentration of 2 µg/ml
were added to the wells, incubated for 1 h at 37 °C, and washed 4 times with PBS. In the following step, a
cytokine solution was added at a concentration of 250 pg/ml, incubated for 1 h at 37 °C, and washed as
described above. Finally, the HRP conjugate of IgG to TNFα (CYTOKINE, Russia) was added to the wells.
Incubation, washing and color reaction using 3,3'5,5'-tetramethylbenzidine were carried out as described
previously .
[24]

All the obtained data were subjected to statistical analysis using the GraphPad Prism 6 software and the
nonparametric Mann-Whitney test.

Bioinformatics analysis
Bioinformatics analysis of the occurrence of proteins with FN3 domains in the sequenced genomes of
bacteria belonging to the genus Bifidobacterium and the families Lactobacillaceae, Bacteroidaceae,
Clostridioides isolated from the human gastrointestinal tract was carried out using the NCBI database (http:/
/www.ncbi.nlm.nih.gov/). Only proteins with annotated functions were used in the analysis.

The following programs were used in this study: BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for
detection of amino acid polymorphisms; Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) for
multiple amino acid sequence alignments; SMART (http://smart.embl-heidelberg.de/) for identification of
FN3 protein domains; UniProt (http://www.uniprot.org/) for identification of signal peptides and
transmembrane domains; SAS (https://www.ebi.ac.uk/thornton-srv/databases/sas/) to identify homology
with 3D structures of proteins presented in the Protein Data Bank (PDB) database; trRosetta (https://
yanglab.nankai.edu.cn/trRosetta/) for predicting the spatial structures of proteins .
[33]

RESULTS
Study of the binding of fragments of the FN3 protein of the strain B. longum subsp. longum GT15 to