[Effect of conditions of monoclonal antibody adsorption on antigen-binding activity].

The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013-0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.

[Use of pH-sensitive immobilized monoclonal antibodies for optimization of immunoenzyme sandwich technique of detection of HBsAg of hepatitis B virus].

AIM: To develop highly sensitive sandwich technique for identification of surface hepatitis B virus antigen (HBsAg) in serum and analyse of possible improvement of solid phase for immunoenzyme sandwich technique of HBsAg identification through variation of pH-dependent sorption of monoclonal antibodies on the surface of immune plates. MATERIALS AND METHODS: Calibration curves for identification of HBsAg in sandwich techniques using 36 possible binary combinations of monoclonal antibodies of our panel (including high affinity antibodies to HBsAg produced by 6 hybridomas) were compared. Immobilization of antibodies on solid phase (by passive sorption) was performed at different pH values (2.8, 7.5, and 9.5). RESULTS: Analysis of panel of antibodies to HBsAg produced by 6 hybridomas revealed pH-dependent monoclonal antibodies (18C8), which immobilization at low pH values together with detecting antibodies F4F3 allowed to greatly improve sensitivity of the sandwich technique. Minimal credibly detectable concentration of HBsAg in sera of persons infected with hepatitis B virus was 0.013 - 0.017 ng/ml. Validation of sandwich technique was performed on certified panel of serum samples with various concentrations of HBsAg (different serotypes). CONCLUSION: Highly sensitive sandwich technique for detection of HBsAg was developed. It was shown that analysis of panel of monoclonal antibodies on pH-dependence could be used as simple methodical approach for optimization of immunoenzyme sandwich techniques for detection of different antigens.

[The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens]. / Sviazyvanie bispetsificheskikh monoklonal'nykh antitel s antigenami, adsorbirovannymi na tverdoi faze.

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.

[Levels and molecular heterogeneity of serotonin transporter protein in platelets of patients with different mental diseases: a comparative analysis with the use of monoclonal and polyclonal antibodies]. / Soderzhanie i molekuliarnaia geterogennost' belka--perenoschika serotonina v trombotsitakh bol'nykh s razlichnymi psikhicheskimi zabolevaniiami: sravnitel'nyi analiz s ispol'zovaniem monoklonal'nykh i poliklonal'nykh antitel.

Polyclonal (PAb) and monoclonal (MAb) antibodies to CT2-epitope of the C-terminal fragment of serotonin transporter (SERT) protein were used to study the levels and molecular heterogeneity of platelet SERT in healthy donors and patients with affective (AD) and somatoform (SD) disorders, schizoaffective disorder (SAD) and schizophrenia. SERT was found to exist as high molecular wight (HMW) and low molecular weight (LMW) forms separated after electrophoresis. The levels of HMW and LMW forms of SERT were significantly, decreased in mentally ill patients as compared to healthy individuals. Unlike PAb, horse radish peroxidase (HRP)-conjugated MAbs were more sensitive and specific to SERT and could detect the LMW form of SERT as a duplet protein form with MW about 40 and 43 kDa. The MAb to CT2 C-terminal fragment of SERT conjugated with HRP is considered to be a new valuable tool for further investigation of SERT expression, properties, and posttranslation modification in the controls and in patients with different psychopathology.

[Bispecific monoclonal antibodies: the isolation and study of their antigen-binding properties]. / Monoklonal'nye bispetsificheskie antitela: poluchenie i izuchenie antigensviazyvaiushchikh svoistv.

Bifunctional antibodies (bABs) having a double specificity to alpha-endorphin (alpha-END) and horseradish peroxidase (HRP) were produced by hybridoma technology. The antibodies constituted about 28-29% of all immunologically active IgG secreted by hybrid hybridoma (quadroma). The quadroma was isolated by fusion of two murine hybridomas (anti-HRP and anti-alpha-END) with distinct phenotypes: double mutant AMD(R)/NAT(S) and its wild type. To produce the double mutant phenotype, an actinomycin D-resistant (AMD(R)) mouse myeloma was used to initiate one of the parental hybridomas. bABs were purified from quadroma culture medium and ascitic fluids by sequential HRP-sepharose and alpha-END-sepharose affinity chromatography. With radioimmunoassay, the affinity of the individual anti-alpha-END combining sites of bABs was shown to be identical to that of parental monoclonal antibodies. Binding to the second antigen (HRP) did not affect the binding of bABs to alpha-END. bABs proved to be efficient for the determination of endorphins and their precursor proopiomelanocortin in immunohistology and immunoblotting.

[The interaction of bifunctional monoclonal antibodies with antigens studied by a radioimmunological method]. / Izuchenie vzaimodeistviia bifunktsional'nykh monoklonal'nykh antitel s antigenami radioimmunologicheskim metodom.

The quadroma produced bifunctional antibodies (bAbs) with double specificity to alpha-endorphin (alpha-END) and horseradish peroxidase (HRP) were compared with the parental anti-alpha-END monoclonal antibodies (mAbs) in respect to their binding to alpha-END. bAbs were purified from quadroma culture medium by sequential HRP-sepharose and alpha-END-sepharose affinity chromatography. Using radioimmunological method the affinity of the individual anti-alpha-END combining sites of bAbs was shown to be identical to that of parental mAbs. Binding to the second antigen (HRP) didn't affect binding of bAbs to alpha-END.

[A new approach to the design of hybrid hybridomas based on the use of an actinomycin D-resistant line of murine myeloma]. / Novyi podkhod k konstruirovaniiu gibridnykh gibridom, osnovannyi na ispol'zovanii aktinomitsin-D-rezistentnoi linii mielomy myshi.

The hybrid hybridomas (tetradomas) were produced from the fusion of the double mutant actinomycin Dr (ADr)/HATs hybridoma to horseradish peroxidase (HRP) and wild type hybridoma to alpha-endorphin (EP). The double mutant phenotype was constructed using the new strategy, based on the fusion of immune mouse splenocytes with mouse myeloma (X63.Ag8, 653) cell variants, made resistant to 30 ng/ml of AD by stepwise selection. This allowed the direct introduction of the dominant selective marker (ADr) into the hybrid cells. Tetradomas secreted the bispecific monoclonal antibodies (bi Mabs), simultaneously binding to EP and HRP in double antigen ELISA, the ELISA plates covered with EP-bovine serum albumin conjugate. Using rat pituitary the bi Mabs were shown to be effective for immunostaining of EP-producing cells. EP-producing cells.

[Monoclonal antibodies to alpha-endorphin effective in immunohistochemistry and immunoblotting]. / Monoklonal'nye antitela k al'fa-éndorfinu, éffektivnye v immunogistokhimii i immunoblottinge.

Four mouse monoclonal antibodies (E11, A8, F8, H5) to alpha-endorphin have been produced. The antibodies bind 12.5, 20.6, 9.6, 6.6% of 125I-beta-endorphin and 35.5, 15.1, 12.8, 12.2% of 125I-gamma-endorphin; the binding of 125I-alpha-endorphin being taken for 100%. The binding of antibodies E11, A8, F8 and H5 to 125I-alpha-endorphin was 50% inhibited by unlabeled ligand in concentrations 5, 50, 30 and 35 nM respectively. Using tissue sections of rat pituitary it was shown that antibody E11 can be used for the localization of endorphin producing cells by immunofluorescence. The antibodies F8 and H5 effectively detected endorphin precursor proopiomelanocortin by immunoblotting.

[Changes in the genotype and phenotype determining the resistance of Djungarian hamster somatic cells to adriablastin]. / Izmenenie genotipa i fenotipa, opredeliaiushchie ustoichivost' somaticheskikh kletok dzhungarskogo khomiachka k adriablastinu.

The development of adriablastin resistance in Djungarian hamster DM-15 cells is accompanied by the appearance of small chromatin bodies (SCB) and long homogeneously staining regions (HSRs) in the chromosomes--the structures that contained amplified genes. The pattern of karyotypic alterations (the appearance of additional chromosome 4, and emergence of SCB, formation of the HSRs in one of three of chromosome 4, transposition of the HSRs from chromosome 4 to other chromosomes) during the development of adriablastin resistance is identical to that found in these cells before, namely during the development of colchicine resistance. Adriablastin- and colchicine-resistant cells have similar changes in plasma membrane permeability for 3H-colchicine, 3H-actinomycin D, 3H-puromycin, 3H-cytochalasin B, and 3H-vinblastine. Apparently, adriablastin resistance has the same mechanism as colchicine resistance, being connected with gene amplification and decreased plasma membrane permeability for these drugs.

[Characteristics of the karyotype changes in Djungarian hamster cells resistant to methotrexate]. / Osobennosti izmennenii kariotipa v kletkakh dzhumgarskogo khomiachka, ustoichivykh k metotreksatu.

The chromosomes stained by trypsin G-banding technique were studied in five Djungarian hamster cell sublines, resistant to different concentrations of methotrexate. In all cells of two independent sublines, approximately 13 times more resistant to the drug, an additional material on the distal part of the short arm of chromosome 3 was revealed. The size and banding pattern of this new material were different in two sublines and in individual cells of each subline. In cells, which were 25-fold resistant to methotrexate, the additional material was found both in the short arm of chromosome 3 and in the long arm of chromosome 4. In some cells the additional material in chromosome 4 contained the long homogeneously staining regions (HSRs). In a subline which was 100-fold resistant to methotrexate, all cells had the chromosome 4 bearing the long HSR. The further increase in the level of drug resistance (300-fold) was accompanied by the increase in the size of HSRs in chromosome 4, the appearance of the second HSR in the short arm of chromosome 3 and emergence of small chromatin bodies. In cells with trisomy 4 and a low level of colchicine-resistance, methotrexate-resistance arises more frequently than in colchicine-sensitive cells bearing two chromosomes 4, or in cells possessing the high level of colchicine-resistance and trisomy of the short arm of chromosome 4 only. The similarities and differences of karyotypic alterations accompanying the development of colchicine- and methotrexate-resistance in Djungarian hamster cells, are discussed.

[Relation of mammalian cell resistance to actinomycin D with a change in the karyotype and a decrease in cytoplasmic membrane permeability]. / Sviaz' rezistentnosti kletok mlekopitaiushchikh k aktinomitsinu D s izmeneniem kariotipa i umen' sheniem pronitsaemosti tsitoplazmaticheskoi membrany.

Djungarian hamster cell lines, selected for resistance to 2 microgram/ml of actinomycin D (AD) have been studied. These lines are 1000-4000 times more resistant to AD than the parent cells. AD-resistance is an unstable property. It is lost or diminished when the cells are grown in the absence of AD. The resistant cells show markedly reduced uptake of AD and unrelated agent - colchicin, which indicated that resistance to AD is due to the decrease of plasma membrane permeability. The chromosomal analysis of resistant lines revealed a specific abnormality in their kariotypes, namely, chromosomes containing "homogeneously staining regions" (HSR). These data support the suggestion that AD-resistance is associated with gene amplification.

[Actinomycin D and 6-mercaptopurine-resistant Djungarian hamster cell line: karyotype, morphology, malignancy]. / Rezistentnye k aktinomitsinu D i 6-merkaptopurinu linii kletok dzhungarskogo khomiachka: kariotip, morfologiia, zlokachestvennost'.

Djungarian hamster cell lines resistant to actinomycin D (AD) were developed from SV40 transformed HGPRT- cell, line DM-15. Increase in resistance to AD up to 4000 fold was obtained. The acquisition of resistance to AD did not influence the expression of the first mutation--HGPRT-. The cells retained resistance to 6-mercaptopurine and could not grow in HAT medium, as well as the parent cell line DM-15. The acquisition of resistance to AD resulted in production of cell cultures with a less malignant phenotype, than that of the parent cell line DM-15. So, the cells resistant to AD had lower tumorigenicity in vivo, the reduced ability to form colonies in soft agar and were less transformed, as shown by morphological criteria. The obtained cell lines with two genetic markers--resistance to 2 microgram/ml of AD and HGPRT- can be used in somatic cell genetics, especially, for somatic hybridisation, and also to study the role of the cell membrane in malignant transformation.

[Somatic hybrids of normal and tumor Djzungarian hamster cells. I. Preferential elimination of chromosomes of the normal partner]. / Somaticheskie gibridy opukholevykh i normal'nykh kletok dzhungarskogo khomiachka. Soobshchenie I. Preimushchestvennaia utrata khromosom normal'nogo partnera.

Normal Djungarian hamster lymphoid cells were fused with SV40 transformed malignant fibroblasts. The resulting 11 hybrid clones were subjected to the chromosome analysis. The karyotype of hybrids proved to be unstable. In some cases the total tetraploid number of chromosomes in hybrids drastically decreased up to the near-diploid level close to that of the malignant parent cells. The G-band chromosome analysis showed that as a rule morphologically unchanged chromosomes were preferentially lost from the hybrid cells, the markers of the malignant partner being retained. On the basis of these data it is assumed than the hybrids between normal and tumour cells of Djungarian hamster preferentially lose the chromosomes of the normal parent cells during cultivation in vitro.

[Somatic hybrids of normal and tumor Djzungarian hamster cells. II. Manifestation of malignancy and karyotype features of hybrid tumors]. / Somaticheskie gibridy opukholevykh i normal'nykh kletok dzhungarskogo khomiachka. Soobshchenie II. Proiavlenie zlokachestvennosti i osobennosti kariotipa gibridnykh opukholei.

The hybrid clones derived from the fusion of tumour and normal cells of Djungarian hamster were tested for their ability to grow progressively in vivo and to form colonies in semisolid medium. In all cases the hybrids were able to produce tumours in animals, but tumorigenicity of different clones varied. Some clones had high take incidence of tumours comparable to that of malignant partner, others had a very low one. The hybrid clones differed in their ability to form colonies in soft agar. No correlation was found between the malignancy of the hybrid clones in vivo and their ability to grow in semisolid medium. Chromosome analysis of 23 hybrid tumours arising from the injections of the hybrid cells showed that in 18 tumours the drastic reduction of chromosomes from tetraploid to near-diploid level, comparable to that of malignant parent, took place. As a rule, morphologically unchanged chromosomes were preferentially lost from the hybrid tumour cells, the markers of the malignant partner being retained. Some hybrid tumours showed insignificant chromosome elimination of all pairs, except chromosomes of the IV and VIII pairs, their number always being reduced.

[Production and characteristics of a Djzungarian hamster cell line (DX-TK-) resistant to 5-bromodeoxyuridine]. / Poluchenie i kharakteristika linii kletok dzhungarskogo khomiachka (DKh-TK-), rezistentnoi k 5-bromdezoksiuridinu.

New biochemically marked Djungarian hamster cell line (DX-TK-) was established. These cells are resistant to 5-bromodeoxyuridine (25 mkg/ml) and deficient in thymidine kinase activity (TK-). Due to this biochemical defect they have lost the ability to grow in HAT medium. DX-TK- cells are malignant. They grow as tumours after the inoculation to newborn Djungarian hamsters. Tumorigenecity of DX-TK- cells was decreased as compared with the parent TK+ cell line. DX-TK- cell line is a hypodiploid cell culture (26 chromosomes) with 7 chromosome markers easily identified by means of G-band staining. This line is a new model for somatic cell genetic experiments, particularly for somatic cell hybridization.

[Resistance of a line of Djungarian hamster cells to 6-mercaptopurine]. / Rezistentnaia k 6-merkaptopurinu liniia kletok dzhungarskogo khomiachka.

A cell line resistant to 6-mercaptopurine (6-MP) is isolated from Djungarian hamster embryonic fibroblasts transformed with SV-40, 6-MP resistance is due to the absence or complete inhibition of GGPRT activity. Initial and resistant cell cultures are similar in the growth rate and in the inoculation efficiency. Caryological analysis (differential chromosome staining--S-bands) revealed considerable caryotype rearrangements in both resistant and sensitive lines as compared with Djungarian hamster normal chromosome set, and also the appearance of specific chromosome markers.

[Mutagenesis under the influence of simian vacuolating virus 40 (SV40). I. Induction of chromosome aberrations in Chinese hamster cells during the first days following infection]. / Mutagenez pod vozdeistviem obez'ian'ego vakuoliziruiushchego virusa 40 (sv40). Soobshchenie I. Induktsiia khromosomnykh aberratsii v kletkakh kitaiskogo khomiachka v pervye dni posle zarazheniia

Chromosome aberrations were studied in cultured chinese hamster cells on the 1--4th days after infection with Simian Virus 40. In the first and second mitoses after infection a statistically significant increase of the percentage of aberrant metaphases was observed (up to 22% as compared to 8--10% in intact cells). Already after 3 days following treatment the percentage of aberrant metaphases decreased sharply, reaching the control level. The virus induced a significant increase of the frequency of chromosome and chromatid breaks, as well as the appearance of fragments of an unknown origin. Chromosome breaks were distributed randomly among 5 morphologically distinct chromosome groups, according to the comparative length of respective chromosomes. An increase of the frequency of gaps and coiling deficiencies was also observed in virus-infected cultures. An earlier appearance of the first mitosis was observed in virus-treated cultures. It is suggested that the mutagenic effect of SV40 in the cells studied may be related to mechanisms controlling the integration of viral genomes into the cell chromosomes.