[Contemporary molecular-biological methods of cancer diagnosis and monitoring based on the humoral immune response to tumor-associated antigens].

Humoral immune response to tumor-associated antigens in cancer patients can be used as a basis for disease diagnosis and monitoring. Moreover, identification of molecular targets of such response may be used to develop antigen-specific anticancer vaccines. Here, we review the main approaches to identification and study of tumor-associated antigens recognized by serum antibodies. We also focus on the challenges that must be met before serological antigens can be used in clinical cancer diagnostics.

[Expression of human immunodeficiency virus gag antigens and formation of virus-like particles in a cell culture, infected with recombinant vaccinia virus strains (an electron-microscopic study)]. / Ekspressiia gag-antigenov virusa immunodefitsita cheloveka i obrazovanie virusopodobnykh chastits v kul'ture kletok, infitsirovannykh rekombinantnymi shtammami virusa ospovaktsiny (élektronno-mikroskopicheskoe issldovanie).

The synthesis of gag antigens of human immunodeficiency virus (HIV) by recombinant vaccinia virus strains containing the expressed genes gag and gag-pol and the capacity of these proteins to formation of virus-like particles during infection of various cell cultures were studied. The recombinant strain containing the truncated gag gene (p48gag) was shown to effectively synthesize gag polypeptides and to form immature virus-like particles during infection of all the cell cultures tested (CV-1, Hep-2, HT-29). The morphogenesis of mature virus-like particles was detected by electron microscopy only in infection of Hep-2 cells with a strain containing a complete gag-pol sequence of HIV.

[The content of metallothioneins in mouse bone marrow and liver and in human lymphocytes after whole-body gamma irradiation]. / Soderzhanie metallotioneinov v kostnom mozge i pecheni myshei i v limfotsitakh cheloveka pose obshchego gamma-oblucheniia.

The increase in the content of bone marrow and hepatic metallothioneins (MT) in mice with the maximum at 30 hr after whole-body gamma irradiation was shown. The MT level in that tissues at that time correlated with the exposure dose. The MT content in lymphocytes of patient with acute lympholeucosis was increased after fractionated whole-body irradiation, that index also correlated with accumulated exposure dose.

[Lowering the acute toxicity of ethanol with a zinc-metallothionein compound]. / Snizhenie ostroi toksichnosti étanola preparatom tsink-metallotioneina.

The homogeneous drug of zinc-metallothionein (zinc-MT) which characterized according to the molecular weight and UV-absorption spectra was obtained from the liver of the rats preliminary injected with the zinc chloride. It was shown that zinc-MT (0.5-4 mg/kg of protein) injected to the mouse 10 min before the ethanol decrease the acute toxicity of the alcohol. The preliminary injection of the control mixture consisted the albumin, cysteine and zinc chloride in proportions and dose corresponding to the dose 3 mg/kg of zinc-MT did not change LD50 of ethanol. The possible mechanism of the effect is discussed. The most possible is the stabilization of the lipid peroxidation in the liver or the restoration of the thiol groups pool at the alcohol intoxication by the zinc-MT.

[The connection between the content of metallothioneins in the bone marrow and liver and the survivability of irradiated mice after cadmium chloride administration]. / Sviaz' mezhdu soderzhaniem metallotioneinov v kostnom mozge, pecheni i vyzhivaemost'iu obluchennykh myshei posle vvedeniia khloristogo kadmiia.

The survival rate of mice gamma-irradiated at different times after administration of Cd2+ (0.75 mg/kg) was shown to be tightly connected with the amount of metallothioneins induced by this heavy metal in the bone marrow (r = 0.96). The connection between the amount of metallothioneins in the liver and the survival rate was less pronounced.

[Preparation of immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horseradish peroxidase with human antibodies to HIV-1]. / Poluchenie immunofermentnykh kon"iugatov beta-laktamazy iz Bacillus licheniformis 749/c i peroksidazy khrena s chelovecheskimi antitelami k VICh-1.

By using three different linkage methods with carbodiimide, glutaraldehyde and periodite, immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horse radish peroxidase with human antibodies to HIV-1 were prepared. The human antibodies were purified by the affinity procedure on Protein-A-Sepharose 6B. The conjugates were tested in a solid phase immunoenzymatic system for the HIV-1 antigen. It was shown that the conjugates prepared by the carbodiimide linkage method had the highest titer, the beta-lactamase conjugate being superior by its titer to the respective peroxidase conjugate. In the lyophilized state the conjugates prepared with the carbodiimide linkage method were stable.

[Isolation of monoclonal antibodies for the identification of strains of the causative agent of plague that do not contain a capsular antigen]. / Poluchenie monoklonal'nykh antitel dlia identifikatsii shtammov vozbuditelia chumy, ne soderzhashchikh kapsul'nogo antigena.

In most cases the immunological identification of Y. pestis strains is based on the use of capsular antigen as an immunological marker. However, there are Y. pestis strains without capsular antigen. For the immunological identification of such strains, homogeneous antigen with a molecular weight of 43 KD has been isolated and monoclonal antibodies to it have been obtained. The enzyme-linked immunosorbent assay, carried out with the use of these monoclonal antibodies and intended for the detection of antigen with a molecular weight of 43 KD, has been developed. The sensitivity of the assay is about 10 ng/ml.

[Isolation and immunoenzyme determination of human beta 2-microglobulin]. / Poluchenie i immunofermentnoe opredelenie beta 2-mikroglobulina cheloveka.

Two procedures for isolation of homogeneous beta 2-microglobulin from urine of patients with systemic lupus erythematosus were developed: a procedure for isolation of beta 2-microglobulin with two-stage gel chromatography on Sephacryl S-200 and a procedure for isolation of homogeneous beta 2-microglobulin with affinity chromatography on rabbit polyclonal antibodies. The microglobulins isolated with the two procedures showed identical physicochemical properties and were used in development of a competitive immunoenzymatic assay method for determination of beta 2-microglobulin in the blood. The method was approved for determination of beta 2-microglobulin in blood serum of patients.

[Determination of the O antigen of Shigella sonnei by a competitive immunoenzyme method]. / Opredelenie O-antigens Shigella sonnei konkurentnym immunofermentnym metodom.

A technique for immunoenzymatic diagnosis of dysentery by Shigella sonnei O-antigen was developed. For induction of antibodies to O-antigen rabbits were immunized by intravenous administration of a commercial antidysentery vaccine. Specific antibodies to O-antigen belonging to class G immunoglobulins and not binding to O-antigens of Sh. flexneri and Salmonella typhimurium were obtained. beta-Lactamase of Bacillus licheniformis 749/c was used as a marker enzyme in the immunoenzymatic assay. To increase the sensitivity, beta-lactamase molecules were preliminarily linked with glutaric aldehyde into oligomers. Conjugates of Sh. sonnei O-antigen with the oligomers of B. licheniformis 749/c beta-lactamase were prepared with the periodate method by oxidizing O-antigen. The conjugate was used in competing solid phase immunoenzymatic assay for determination of Sh. sonnei O-antigen in blood serum of patients with dysentery. The sensitivity of the assay is 0.5-1 ng per 1 ml of O-antigen.

[Immunoenzyme method of determining Pseudomonas aeruginosa exotoxin A by using a conjugate of beta-lactamase with specific antibodies]. / Immunofermentnyi metod opredeleniia ékzotoksina A Pseudomonas aeruginosa s ispol'zovaniem kon''iugata beta-laktamazy so spetsificheskimi antitelami.

Specific antibodies for immunoenzymatic assay of P. aeruginosa exotoxin A were isolated with affinity chromatography on exotoxin A-Sepharose 4B. A conjugate of the antibodies to exotoxin A with beta-lactamase from Bacillus licheniformis 749/c was prepared by linking glutaric aldehydes. The conjugate showed high immunospecific and enzymatic activity and was used for competitive solid phase immunoenzymatic assay. Sensitivity of the assay provided determination of exotoxin A in concentration of 20 ng/ml. It was used for determining P. aeruginosa exotoxin A in culture fluids.

[Solid-phase immunoenzyme analysis of insulin using Bacillus licheniformis 749/c beta-lactamase and horseradish peroxidase as markers]. / Tverdofaznyi immunofermentnyi analiz insulina s ispol'zovaniem beta-laktamazy iz Bacillus licheniformnis 749/c i peroksidazy khrena v kachestve markerov.

Two modifications of solid phase enzyme immunoassay (EIAA) for insulin with the use of beta-lactamase from B. licheniformis 749/c and horse radish peroxidase as the markers were developed. beta-Lactamase and peroxidase conjugates with insulin were prepared with the glutaric aldehyde and periodate methods respectively. Both the conjugates were stable and preserved high immunospecific and enzymatic activity. Sensitivity of EIAA with the use of the beta-lactamase or peroxidase as the markers was the same and amounted to 15 ng/ml of insulin. However, with the use of the beta-lactamase in EIAA not only spectrophotometric recording but also visual registration of the results was possible.

[Use of beta-lactamase from Bacillus licheniformis 749/C for solid-phase immunoenzyme analysis]. / Ispol'zovanie beta-laktamazy iz Bacillus licheniformis 749/C dlia tverdofaznogo immunofermentnogo analiza.

beta-Lactamase from Bacillus licheniformis 749/c was used as a marker for ELISA. Conjugates of the beta-lactamase with the capsule antigen of the plague causative agent and with the monoclonal antibodies to it were prepared by glutaric aldehyde "linking". The conjugates preserved high immunospecific and beta-lactamase activity. High sensitivity of the modification of ELISA allowed detecting up to 8 ng/ml of the capsule antigen of the plague causative agent. The enzymatic activity of the beta-lactamase was determined microiodometrically which provided visual recording of the results of ELISA.

[Isoelectric focusing of different classes of beta-lactamases in cell-free extracts of Gram-negative bacteria]. / Izoélektrofokusirovanie beta-laktamaz razlichnykh klassov v beskletochnykh ékstraktakh gramotritsatel'nykh bakterii.

A method of electrofocusing of beta-lactamases in cell-free bacterial extracts was developed. It provided determination of the isoelectric points of beta-lactamases without preliminary preparation of the homogeneous enzyme. Isoelectrofocusing of beta-lactamases of 14 poly-resistant strains of gram-negative bacteria of six species was performed. It was found that the isoelectric points of the beta-lactamase belonging to the same class according to Richmond were different. No species specificity of the beta-lactamases of the gram-negative bacteria was observed.

[Isolation and properties of beta-lactamase of the cephalosporinase type from cells of Enterobacter aerogenes 6803]. / Vydelenie i svoistva beta-laktamazy tsefalosporinaznogo tipa iz kletok Enterobacter aerogenes 6803.

beta-Lastamase with the molecular weight of 32500 was isolated from the cells of clinical strain 6803 of Enterobacter aerogenes and purified. By the substrate profile determined microiodometrically beta-lactamase was classified as belonging to the cephalosporinase type. The activity of the electrophoretically homogenous enzyme was equal to 430 microM a minute per mg protein with respect to benzylpenicillin. The Km for benzylpenicillin, dicloxacillin, cephaloridin and cephalothin was 6.5410(-5), 3 X 10(-4), 2.1 X 10(-5) and 5.7 X 10(-5) M, respectively. The isoelectric point of the enzyme equal to 5.45 was estimated with the method of preparative isoelectrofocusing. The presence of the serine residue or residues was shown with the use of selective reagents applied to the functionally important groups. With the method of circular dichroism the ratio of alpha- and beta-structures in the enzyme molecule was determined, the slow hydrolysis of cephazolin was demonstrated and the values of Km and Kcat for this process were estimated.

[Substrate specificity of the beta-lactamases found in a number of clinical strains of gram-negative bacteria]. / Izuchenie substratnoi spetsifichnosti beta-laktamaz, obnaruzhennykh u riada klinicheskikh shtammov gramotritsatel'nykh bakterii.

The substrate profiles of beta-lactamases defected in 46 clinical polyresistant strains of gram-negative bacteria were determined. By the substrate profile and sensitivity to inhibitors (dicloxacillin and p-CMB) beta-lactamases were considered to belong to classes I, II, III, IV and V of the Richmond classification. The molecular weights of the enzymes were measured. Enterobacter aerogenes 6803, Enterobacter aerogenes 11030 and Klebsiella pneumoniae 970 produced simultaneously two beta-lactamases belonging to different classes. beta-Lactamases of classes I and III were detected in the cells of Enterobacter aerogenes 6803. The cells of Enterobacter aerogenes 11030 contained beta-lactamases of classes V and III and the cells of Klebsiella pneumonia 970 beta-lactamases of classes II and III. Therefore, in all the cases one of beta-lactamases belonged to the class III enzyme close to TEM beta-lactamases by its substrate profile, molecular weight and sensitivity to the inhibitors. Cephalexin and dicloxacillin were most frequently stable to the effect of the above beta-lactamases. The enzymes from 26 strains did not hydrolyse or hydrolysed slightly cephalexin and the enzymes from 19 strains did not hydrolyse of hydrolysed slightly dicloxacillin.

[Determination of beta-lactamase types of clinical strains of bacteria using a modified microiodometric method]. / Opredelenie tipov beta-laktamaz u klinicheskikh shtammov bakterii s pomoshch'iu modifitsirovannogo mikroiodometricheskogo metoda.

The substrate profiles and sensitivity to dicloxacillin inhibition were studied in the enzymes of the clinical strains of Pseudomonas aeruginosa and the transconjugants of E. coli carrying the plasmids discovered earlier in P. aeruginosa. The study was performed with a modified microiodometric method for determination of the activity of beta-lactamases. According to the M. Richmond classification of beta-lactamases the enzymes detected in P. aeruginosa strains 4529, 5290 and 9902 may correspond to the 5th class, the enzymes of P. aeruginosa strain 8208 to the 2nd class and the beta-lactamases of the E. coli transconjugants to the 3rd class. Two different beta-lactamases were detected in P. aeruginosa strain 10294.

[TEM-type beta-lactamase coded by the plasmid from Citrobacter sp]. / beta-laktamaza TEM-tipa, kodiruemaia plazmidoi iz Citrobacter sp.

beta-Lactamase was isolated from the cells of E. coli, strain 1039, a transconjugant carrying the plasmid first detected in Citrobacter sp. beta-Lactamase was purified to obtain a homogeneous preparation. The activity of the enzyme was estimated by a modification of the potentiometric method providing determination of up to 0.1 mumol of penicilloinic acid. The activity of the pure enzyme was 206 mumol per 1 mg of protein per minute. The molecular weight determined by gel filtration was 21500. The substrate profile of beta-lactamase corresponded to the TEM type. Preliminary incubation with methicillin resulted in a significant decrease in the enzyme activity. Carbenicillin, dicloxacillin, oxacillin and cephalothin induced no decrease in the activity of the enzyme when subjected to preliminary incubation with it. The use of the functional group reagents allowed detecting residues of serine and lysine in the enzyme active centre or close to it. Km and Kcat for some antibiotics were evaluated. The ratio of alpha- and beta-structures in the molecule of beta-lactamase was determined with the method of circular dichroism (CD). The fraction of the alpha-spiral areas amounted to 30 +/- 5 per cent and the fraction of beta-structures amounted to 20 +/- 5 per cent. On attachment of methicillin to the molecule of the enzyme the ratio of alpha- and beta-structures did not change which may be considered as a preliminary indication of the centre "immobility" in the process of catalysis. Slow hydroysis of methicillin by beta-lactamase was shown with the method of CD.