The increased flexibility of CDR loops generated in antibodies by Congo red complexation favors antigen binding.
The dye Congo red and related self-assembling compounds were found to stabilize immune complexes by binding to antibodies currently engaged in complexation to antigen. In our simulations, it was shown that the site that becomes accessible for binding the supramolecular dye ligand is located in the V domain, and is normally occupied by the N-terminal polypeptide chain fragment. The binding of the ligand disrupts the beta-structure in the domain, increasing the plasticity of the antigen-binding site. The higher fluctuation of CDR-bearing loops enhances antigen binding, and allows even low-affinity antibodies to be engaged in immune complexes. Experimental observations of the enhancement effect were supported by theoretical studies using L lambda chain (4BJL-PDB identification) and the L chain from the complex of IgM-rheumatoid factor bound to the CH3 domain of the Fc fragment (1ADQ-PDB identification) as the initial structures for theoretical studies of dye-induced changes. Commercial IgM-type rheumatoid factor (human) and sheep red blood cells with coupled IgG (human) were used for experimental tests aimed to reveal the dye-enhancement effect in this system. The specificity of antigen-antibody interaction enhanced by dye binding was studied using rabbit anti-sheep red cell antibodies to agglutinate red cells of different species. Red blood cells of hoofed mammals (horse, goat) showed weak enhancement of agglutination in the presence of Congo red. Neither agglutination nor enhancement were observed in the case of human red cells. The dye-enhancement capability in the SRBC-antiSRBC system was lost after pepsin-digestion of antibodies producing (Fab)2 fragments still agglutinating red cells. Monoclonal (myeloma) IgG, L lambda chain and ovoalbumin failed to agglutinate red cells, as expected, and showed no enhancement effect. This indicates that the enhancement effect is specific.
Krol
Marcin
M
Dept. of Bioinformatics and Telemedicine, Medical College, Jagiellonian University, Kopernika 17, 31-501 Krakow, Poland. mbkoniec@cyf-kr.edu.pl
Roterman
Irena
I
Drozd
Anna
A
Konieczny
Leszek
L
Piekarska
Barbara
B
Rybarska
Janina
J
Spolnik
Paweł
P
Stopa
Barbara
B
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D004396:Coloring Agents; D022801:Complementarity Determining Regions; D003224:Congo Red; D006386:Hemagglutination Tests; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D000906:Antibodies; D000936:Antigen-Antibody Complex; D004396:Coloring Agents; D022801:Complementarity Determining Regions; D003224:Congo Red