Andrey N. Glushkov

Generation and Characterization of Human Single-Chain Antibodies Against Polycyclic Aromatic Hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. BP is one of the most toxicologically active PAHs and is often used as a prototype for this entire class of contaminants. In order to select anti-BP antibodies, the conjugate of BP with BSA (BP-BSA) was used to screen naïve combinatorial phage library of human scFvs. Seven unique scFvs against BP-BSA were selected after three rounds of selection. Analysis of the genes encoding the scFvs subdivided them to gene families and subfamilies. Homology with the closest germline ranged from 80.21% to 97.57% for heavy chains and 88.89% to 98.57% for the light chains. Four of the seven scFv amino acid residues sequences without stop codons in frame were selected for proteomic analysis with each other. Four scFvs encoded unique non-related proteins with low-sequence identity among them. All CDRs and the boundaries in the CDR3 formation were carried out. Two of the scFvs (T68 and T72) with the highest binding capabilities to PAHs were expressed in E. coli and purified using a nickel resin. The KDs of T68 to BP-BSA, chrysene, pyrene, and benzo[a]anthracene were almost similar, approximately 10−7 M. The KDs of T72 to benzo[a]anthracene and chrysene were 9.42 × 10−8 M and 2.63 × 10−7 M, respectively. The computational models of T68 and T72 active centers were different.

Immunological disbalance in carcinogenesis.

It is postulated a conception of immunological disbalance between carcinogenesis inhibiting and stimulating antibodies (Ab). Inhibiting Ab prevent the carcinogens and estradiol but increase the progesterone penetration into the target cells. And vise versa do stimulating Ab. Inhibiting Ab could be blocked by corresponding antiidiotypic Ab. The processes of carcinogenesis initiation and promotion are intensified when stimulating Ab prevail over inhibiting ones.

Purification and Characterization of Mouse Single-Chain Antibody against Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyren mainly induce lung cancer in humans. We characterized the mouse single chain antibody against benzo[a]pyren (pSh). pSh was expressed and purified as cellulose binding domain fusion (pSh-CBD). The pSh-CBD bound five different PAH with high affinity. The 18 amino acid linker connected pSh-CBD heavy and light chains provided correct protein folding. The KDs for pSh-CBD and polycyclic aromatic hydrocarbons were similar to KDs for monoclonal antibody, approximately 10−8. Separately heavy and light chains of pSh-CBD did not interact with benzo[a]pyren. Previously defined eleven pSh-CBD aa involved to benzo[a]pyren binding were confirmed by mutagenesis.

Immunological mechanisms of adaptation to the low-weight chemical compounds in ontogenesis

It is postulated that adaptation to the low-weight chemical compounds includes six consistent ontogenetic periods: the reciprocal selection of the marriage partners; the maturation of the gametes; the formation of the zygote; the development of the fetus; the birth and nursing of the child; and the sexual maturation of a human being. The main immunological parts of adaptation are: the antibodies to the low-weight xeno- and endobiotics; the reciprocal immune-like recognition of the maternal and paternal gametes; and the maternal immune reaction on the paternal histocompatibility antigens of the fetus. The main immunological mechanisms of adaptation are: the promotion of the selectivity in the smell recognition of the marriage partners by the antibodies to the xeno- and endobiotics expanding the individual spectrum of their metabolites; the inhibition of the xenobiotic genotoxic action on the gametes, fetus and child by the antibodies; the prevention of the development of gene-damaged gametes and fetus by antibodies to the sexual hormones; and the immune preservation (elimination) of the heterozygote (homozygote) fetus.

New human single chain anti-idiotypic antibody against benzo[a]pyrene

The nal¨ve library from the lymphocytes of healthy humans was screened by murine single-stranded idiotypic antibodies against benzo[a]pyrene (pSh). The phage clone which contained of anti-idiotypic antibody against benzo[a]pyrene, designated as A4, was chosen for further work because of highly specific to pSh. The available protein databases were searched. The A4 amino acid sequence was unique and 76% identical to a sequence in antibody against interferon g. The A4 protein was expressed in bacteria and purified by two different methods: His-tagged A4 and CBD-fusion A4. Both the A4 bound to pSh and also to the human single chain idiotypic antibody against the benzo[a]pyrene (T72) by ELISA. The Kd values of A4 for pSh and T72 were very close: 4.44 × 10-7 M and 5.71 × 10-7M, respectively. A4 was a competitor with benzo[a]pyrene for binding sites of both idiotypic pSh and T72 in competitive ELISA. Thus, A4 was a high affinity anti-idiotypic against benzo[a]pyrene which recognised pSh and T72 active sites.

Isolation, production, and characterization of a new single chain anti-idiotypic antibody against benzo[a]pyrene

ABSTRACT Polycyclic aromatic hydrocarbons are chemical carcinogens which could induce the development of human cancers. Anti-idiotypic antibodies against benzo[a]pyrene (BP) are perspective for human cancer immunoprophylaxis and tumor immunodiagnostic techniques. The purpose of this study was to isolate anti-idiotypic antibodies against BP from human lymphocytes naïve phage library. The anti-idiotypic antibody, named B5, was selected. Analysis of the nucleotide and amino acid sequences B5 showed no similarity to known protein databases antibodies. B5 bound idiotypic antibodies against BP in direct and competitive ELISA. It was suggested that the B5 carried an immunological image of BP and bound the idiotypic antibodies against BP. Abbreviations: scFv: single-chain variable fragment; Ab1: idiotypic antibodies; Ab2: anti-idiotypic antibodies; CBD: cellulose binding domain; BSA: bovine serum albumin; PBS: phosphate buffer; BP-BSA: benzo[a]pyrene-BSA conjugate; Cr-BSA: chrysene-BSA conjugate; Py-BSA: pyrene-BSA conjugate; Ac-BSA: anthracene-BSA conjugate; Ba-BSA: benz[a]anthracene-BSA conjugate; PAH: polycyclic aromatic hydrocarbons; pSh: mouse idiotypic single-chain variable fragment against benzo[a]pyrene; T72: human idiotypic single-chain variable fragment against benzo[a]pyrene.

Genotoxic effects of the combined influence of radon and heavy metals depending on polymorphism of genes of monooxygenase system enzymes

The correlation between the level of chromosomal aberrations and CYP1A genotypes in adolescents of the Tashtagol boarding school (Kemerovo region) exposed to a combined effect of high doses of radon and heavy metals has been studied. It was found that the level of chromosomal aberrations shows a statistically significantly increase in carriers of at least one CYP1A1*2A (A2455G) allele. The frequency of ring chromosomes shows a statistically significant increase in carriers of the CYP1A1 *1A*1A (T3801C) genotype. The frequency of multiple chromosomal aberrations was higher in carriers of the CYP1A2*1A*1A genotype. A conclusion on the important role of polymorphic CYP1A loci in genotoxic effects of radon and heavy metals is made.