Evgenii A. Lekchnov1, Sergey E. Sedykh1, Pavel S. Dmitrenok2, Valentina N. Buneva1,3, and Georgy A. Nevinsky1,3
- SB RAS, Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia
- Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, Vladivostok 690022, Russia
- Novosibirsk State University, 2 Pirogova Street, Novosibirsk 630090, Russia
The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS–PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5±15.0% kappa-kappa-IgGs and 41.6±17.0% lambda-lambda-IgGs, while 15.0±4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs.
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Using MALDI-TOF mass spectrometry, purified sIgAs were analyzed both before and after treatment with 10 mM of DTT. Before analysis, a saturated solution of sinapic acid in a mixture of 0.1% acetonitrile and trifluoroacetic acid (1:2) was created. 1 μl of 0.2% trifluoroacetic acid and 1 μl of matrix were added to 2 μl of sIgAs (0.5-1mg ml-1). 1 μl of this final mixture was spotted on a plate and air dried before being analyzed using a mass spectrometer modified with a CovalX HM1 detection system. From the data, the researchers were able to compare MMs of human milk sIgAs with those of placenta sIgAs and observe slight differences in the MMs of light and heavy chains.