New monoclonal antibodies to the Ebola virus glycoprotein: Identification and analysis of the amino acid sequence of the variable domains.

We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained.

[Production of humanized F(ab')2 fragment of rabies blocking antibodies in Pichia pastoris yeast].

The yeast strain Pichia pastoris, a producer of humanized F(ab')2 fragments of rabies-blocking antibodies, has been obtained. Human chaperone BiP coexpression caused a twofold increase of the immunoglobulins secretion level. The use of Fos and Jun zippers in the composition of heavy chains facilitated the dimerization of F(ab')2 fragments of the shared pool of secreted immunoglobulins up to 75%.

[Production of antibodies to aflatoxins].

A panel of ten monoclonal antibodies to aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA. The structures of monoclonal antibody genes were comprehensively studied and the variable and constant domains of the antibody genes were cloned and sequenced. Sequencing analysis confirmed the results of isotyping the light and heavy antibody chains obtained by ELISA. Variable and constant fragments of the antibody genes were cloned into a bicistron expression vector for the recombinant Fab' fragment for one of the antibodies expressed in Escherichia coli and purified. Thus, data were obtained that can be useful for the development of an aflatoxin detection system on the basis of the described monoclonal antibodies and the creation of recombinant antibodies with changed parameters of specificity using protein engineering methods.

Anthracycline antibiotics non-covalently incorporated into the block copolymer micelles: in vivo evaluation of anti-cancer activity.

The chemosensitising effects of poly(ethylene oxide)-poly(propylene oxide)-poly-(ethylene oxide) (PEO-PPO-PEO) block copolymers (Pluronic) in multidrug-resistant cancer cells has been described recently (Alakhov VY, Moskaleva EY, Batrakova EV, Kabanov AV 1996, Biocon. Chem., 7, 209). This paper presents initial studies on in vivo evaluation of Pluronic copolymers in the treatment of cancer. The anti-tumour activity of epirubicin (EPI) and doxorubicin (DOX), solubilised in micelles of Pluronic L61, P85 and F108, was investigated using murine leukaemia P388 and daunorubicin-sensitive Sp2/0 and -resistant Sp2/0(DNR) myeloma cells grown subcutaneously (s.c.). The study revealed that the lifespan of the animals and inhibition of tumour growth were considerably increased in mice treated with drug/copolymer compositions compared with animals treated with the free drugs. The anti-tumour activity of the drug/copolymer compositions depends on the concentration of the copolymer and its hydrophobicity, as determined by the ratio of the lengths of hydrophilic PEO and hydrophobic PPO segments. The data suggest that higher activity is associated with more hydrophobic copolymers. In particular, a significant increase in lifespan (T/C> 150%) and tumour growth inhibition (> 90%) was observed in animals with Sp2/0 tumours with EPI/P85 and DOX/L61 compositions. The effective doses of these compositions caused inhibition of Sp2/0 tumour growth and complete disappearance of tumour in 33-50% of animals. Future studies will focus on the evaluation of the activity of Pluronic-based compositions against human drug-resistant tumours.

[Cloning the Staphylococcus aureus enterotoxin B gene, obtained by polymerase chain reaction, and its expression in Escherichia coli cells]. / Klonirovanie gena énterotoksina B Staphylococcus aureus, poluchennogo metodom polimeraznoi tsepnoi reaktsii, i ego ékspressiia v kletkakh Escherichia coli.

To determine the Staphylococcus aureus enterotoxigenicity, we have developed an approach based on polymerase chain reaction (PCR). Using this method several S. aureus strains have been screened for the presence of the enterotoxin B gene. A DNA fragment of the selected strain (FRI 722H) containing enterotoxin B gene has been obtained by the PCR method and cloned in the pUC19 vector. It is shown that enterotoxin B with the leader peptide forms insoluble complexes in E. coli cells, whereas the mature toxin is present in cytoplasmic fraction in a soluble form. The recombinant toxin made up for 1.7% of the total cellular protein in E. coli JM 109 cells.

Identification of functionally active fragments of staphylococcal enterotoxin B.

It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N-terminal sequences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N-terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin. The toxin's C-terminal fragment possesses an ability to activate calmodulin-dependent enzymes and is probably the toxicogenic part of the enterotoxin.

Antibodies to the regulatory subunit of cAMP-dependent protein kinase type II from bovine brain inhibit the holoenzyme formation.

Mouse monoclonal antibodies to the bovine brain regulatory subunit type II (RII) were produced and antibodies of 11 clones were characterized. We determined their subclass, affinity constants, competivity and influence on two RII functions, namely cAMP binding and inhibition of the catalytic subunit (C) activity. 4 clones produced antibodies that increased RII-cAMP binding 1,5-2-fold. Antibodies of other 3 clones prevented R-C association and inhibition of C phosphotransferase activity. Some antibodies affected neither of these RII functions.