[Human single chain antibodies directed to tumor necrosis factor].

Six unique phage antibodies to human TNF have been selected from a combinatorial library of human single chain fragment variable. ELISA and Western-blotting was used to study selected phage antibodies binding with TNF. The specificity of selected antibodies was determined by binding with interferon alpha and gamma, bovine serum albumin, ovalbumin and ubiquitin. Two antibodies, sA1 and sB3, were converted into a soluble single-chain antibody form and their affinity was 2.5 and 13.7 nM respectively.

Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins.

To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7.0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

[Recombinant full-size human antibody to Ebola virus].

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.

[Construction and characterization of combinatorial naive phage library of human scFvs].

A combinatorial phage display library of human single-chain antibody fragments (scFv) was constructed on the basis of variable domains of heavy (Vh) and light (VI) genes cloned from the lymphocytes of six healthy donors. The size of the library was 2? 10(8) independent clones. Single-chain antibodies against recombinant human TNF?, vaccinia virus and virus-like particles formed by core protein of hepatitis B virus were selected from the library. Unique scFv sequences were identified using the HaeIII fingerprinting. The specificity of the selected clones was proved by the Western-blot analysis.

[Human recombinant antibodies to Ebola virus: preparation and characteristics].

Human recombinant antibodies against a purified Ebola virus (EV) lysate were selected from a combinatorial library of scFv-antibodies using the phage display technique. Nine unique antibodies were identified after sequencing the Vh- and Vl-genes encoding the selected antibodies. Solid-phase enzyme immunoassay (EIA) indicated that these antibodies were able to bind both inactivated and native EV. Immunoblotting showed that 6 antibodies identified nucleoprotein (NP), one antibody did VP24 and another antibody did VP40. One of the selected antibodies reacted with two EP proteins: VP24 and VP40. Solid-phase EIA demonstrated cross-reactivity with Marburg virus (MAR) and defined VP24 MAR as a target protein for the antibody.

[Human recombinant antibodies to variola virus].

Eight specific antibodies to live variola virus (VV), Ind-3a strain, and 7 antibodies to VV, Butler strain, were selected from the synthetic combinatorial phage display library on single-chain (scFv) human antibodies. Indirect solid-phase enzyme immunoassay showed the ability of these antibodies to bind the VV strains Ind-3a, Butler, Brazil-131, Kuw-5, and Congo-2. Moreover, earlier selected human scFv antibodies were also tested in the reaction of binding to the above VV strains. The experiments could reveal the antibodies that bound alastrim strains more effectively that did other VV strains. The nucleotide sequences encoding for the selected scFv antibodies were determined.

[Human mini-antibodies to orthopoxviruses]. / Mini-antitela cheloveka protiv ortopoksvirusov.

A library of human scFv antibodies displayed on the surface of bacteriophages (MRC, Cambridge, England) was panned against the Elstree strain of vaccinia virus (VACV), which resulted in the phage repertoire enriched with clones positive to the strain. Individual clones from the repertoire were screened for binding, independently, to the vaccinia and ectromelia viruses; phage antibodies to the orthopoxviruses were selected. Ten unique antibodies were identified after their Vh- and Vl-genes were sequenced. All selected antibodies were assayed by ELISA for binding to the vaccinia, cowpox and ectromelia viruses. Furthermore, all selected antibodies were assayed for binding with major alastrim strains of the live variola virus. According to the results, the above phage antibodies recognized genus-specific epitopes, some of which differed in their conformation.

Phage antibodies from combinatorial library neutralize vaccinia virus.

The library of human scFv antibodies displayed on the surface of bacteriophages was panned against Vaccinia virus (VACV), strain Elstree. 75% binding with Vaccinia virus. 5 clones were characterized for their binding with VACV and their ability to neutralize VACV in plaque reduction neutralization test (PRNT). Antibodies from the clones were obtained as soluble individual molecules and their binding activities were confirmed in ELISA.