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.

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.

Idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbon in human blood serum are new biomarkers of lung cancer.

Evaluation of epidemiologic risk factor in relation to lung cancer invoked by polycyclic aromatic hydrocarbons has been inconsistent. To address this issue, we conducted a prospective evaluation of new biomarkers for lung cancer classified according levels of idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbons in human blood serum. The blood serums of 557 lung cancer patients and 227 healthy donors were analysis of these antibodies by ELISA. Collected data were regrouped and analyzed by gender, smoking, and age as predictors of risk lung cancer factors. Also, the data of lung cancer patients were additionally analyzed by stages and types of lung cancer, surgery, and chemotherapy. It was suggested to use ratio of idiotypic and anti-idiotypic antibodies rather than distinguish level each of them separately. The ratio of levels in healthy people was 3.32 times higher than in lung cancer patients. This approach gave more precisely results and great prognostic value. The logistic regression model (AUC = 0.9) and neural networks (AUC = 0.95) were built to compare lung cancer patients and healthy donors by predictors. The ELISA data of 49 people random sampled from the originally ELISA data and ELISA data of 52 coal miners as a group of lung cancer risk were confirmed logistic regression model. So, suggested idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbons were not only shown difference between healthy donors and lung cancer patients also elicited group of lung cancer risk among healthy people.