Effect of the chemical structure of N-(2-hydroxypropyl)methacrylamide copolymers on their ability to induce antibody formation in inbred strains of mice.

The homopolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) and copolymers of HPMA differing in oligopeptide side chains (-Gly-Gly-OH; -Acap-Phe-OH; -Acap-Leu-HMDA and -Gly-Phe-Tyr-OH) or in their content (1%, 3.5% and 8.4% mole of -Gly-Gly-OH side chains) were investigated with respect to their ability to induce antibody formation and mitogenic reaction in inbred strains of mice. The dependence on the antigen dose, on composition of the side chain and on the genetic background of the immunized organism was defined. It was demonstrated that the specificity of the antibody formed is predominantly directed against oligopeptide side chains, though some part of the antibody is also produced against hydroxypropyl chains. Neither the homopolymer nor the copolymers behave in the tissue culture as mitogens.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

The characteristics of monoclonal antibodies against human albumin.

Three hybridoma lines secreting antibodies against human albumin have been characterized. Monoclonal antibody (mAb) A1-01 reacted only with human albumin, whereas antibodies A1-02 and A1-03 reacted with both albumin and transferrin but differed in their affinity for these proteins. The mAbs did not react with albumins or transferrins of other mammalian species. Their specificities were tested by isolating the proteins that bind specifically to the purified immobilized antibodies. Only albumin was isolated on immobilized antibodies A1-01 and A1-02, whereas albumin and transferrin were isolated on antibody A1-03. The minor bands specifically detected by antibody A1-01 were fragments or aggregates of albumin, as shown by two-dimensional electrophoresis and transfer to nitrocellulose sheets. It was found by ELISA that as much as 3 ng/ml human albumin is detectable by mAb A1-01.

Immunogenicity of N-(2-hydroxypropyl)-methacrylamide copolymers--potential hapten or drug carriers.

After repeated i.p. immunizations of mice with 10 micrograms of homopolymer poly (HPMA) no antibodies were detected by the ELISA test. Immunization with copolymer P-Acap-Leu-HMDA leads to a weak antibody response, while immunization with a copolymer with some side chains modified with ARS or FITC groups (P-Acap-Leu-HMDA-ARS or P-Acap-Leu-HMDA-FITC) leads to a significant antibody response detectable by PFC, ELISA and haemagglutination tests. Most of these antibodies are aimed against the modifying haptenic group, a smaller amount against side oligopeptide sequences of the carrier. Intensity of the antibody response depends on: 1) the antigen dose--the optimal dose was 10 micrograms: both the higher (100 micrograms) and the lower doses (1 and 0.1 micrograms) induced considerably lower antibody responses; 2) molar mass of the immunizing fractions--fractions of high molar mass induced up to five times higher responses than those of a low molar mass; 3) the bound haptenic group--the ARS-copolymers induced ten times lower response than the FITC-copolymers. We detected no difference between capacities of the H-2a, H-2b and H-2d haplotypes to react with anti-ARS antibodies after immunization with P-Acap-Leu-HMDA-ARS.

Stimulation of human blood lymphocyte by different polyclonal B cell activators of bacterial and plant origin: production of IgM, IgG and IgA estimated by the ELISA method.

Lymphocytes isolated from peripheral blood of healthy donors were stimulated in vitro with pokeweed mitogen, concanavalin A, flagellin, Nocardia delipidated cell mitogen (NDCM) and heat-killed bacteria Escherichia coli and Actinomyces viscosus. A simple and sensitive technique, enzyme-linked immunosorbent assay (ELISA) was used for the detection of nanogram levels of IgM, IgA and IgC in media from lymphocyte cultures after polyclonal stimulation, Pokeweed mitogen, NDCM and E. coli were shown to stimulate a high production of IgM; after stimulation with A. viscosus a higher production of IgA was detected. No immunoglobulin production was observed after stimulation with polymerized flagellin.

[The importance of screening for hepatitis B surface antigens in pregnancy]. / Význam skríninku povrchového antigenu viru hepatitidy B u tehotných.

Transmission of hepatitis B virus (HBV) from mother to infant during the perinatal period represents very efficient mode of HBV infection and often leads to severe long-term sequelae. Perinatal transmission can occur when an infant is born to mother positive for hepatitis B surface antigen (HBsAg), frequency of transmission is high if the mother is also HBeAg positive. Prevention of perinatal transmission of HBV is important since the majority of these infants who are infected at birth become chronic carriers and can subsequently develop chronic hepatitis, cirrhosis or primary hepatocellular carcinoma. Routine screening for HBsAg of all pregnant women attending prenatal clinic at the Institute for the Care of Mother and Child, Prague in the third trimester of pregnancy by Sevatest ELISA HBsAg Micro I. method (Sevac, Prague) began in June 1986. HBsAg positive patients were subsequently tested for HBeAg and anti-HBe hepatitis markers. Of the 2744 women examined 22 were found HBsAg positive, of these mostly asymptomatic carrier mothers 2 were positive for HBeAg, 14 for anti HBe and 6 were negative for both HBeAg and anti-HBe. All infants were given passive HBIG prophylaxis in conjunction with HB vaccine after birth regardless of HBeAg status of mother. At present no infant were found HBsAg positive at 6 month of age, observation of this risk group infants continues.

[Detection of specific antibodies to all classes, including IgE, against pollen allergens using the ELISA test]. / Prukaz specifických protilátek vsech tríd, vcetne IgE, proti pylovým alergenum testem ELISA.

The authors describe a modification of the immunoenzymatic test suitable for estimation (sensitive dedection) of specific antibodies against pollen allergens of all classes of immunoglobulins, incl. specific IgE. When introducing the method, sera of experimental rabbits immunized with selected allergens were used. After elaboration of suitable conditions, the method was used to assess the presence of specific antibodies in sera of patients with the diagnosis of pollinosis. It was found that the most suitable concentration of pollen allergen combined with the solid phase for detection of specific IgE is 100 micrograms/ml, the optimal concentration of glutaraldehyde used to increase the amount of allergen combined with the solid phase by its polymerization and fixation is 0.25%. The use of the method does not call for special equipment and uses locally produced products. The method is useful for supplementing the diagnosis and its more accurate assessment under clinical conditions when biological tests cannot be used for diagnosis. The test makes monitoring during immunotherapy possible.

Detection and quantification of anti-HBs antibodies by the Czechoslovak ELISA SEVAC Kit (Sevatest ELISA anti-HBs).

We invented and verified the possibilities of clinical use of the ELISA Kit for quantification of human serum anti-HBs antibodies. The kit does not employ any labelled antigen but is based on the principle of neutralization. The kit was tested on the panel of sera of the normal population, blood donors, workers at risk workplaces and persons vaccinated with Heptavax-B vaccine. The sensitivity of the kit is 80 mIU/ml, its capture is only slightly worse (in the convalescents) than the capture of the AUSAB (Abbott, USA). Statistical values and frequency distribution at various levels of anti-HBs antibodies in the followed-up group, have been indicated. The kit can be used very well for the follow-up of patients afflicted with virus hepatitis B, for the observation of workers at risk workplaces, for selection of persons eligible for vaccination and for checking the vaccination. It is suitable for the selection of sera for the preparation of specific globulin against hepatitis B and for standardization of this preparation. It is less suited for the compilation of epidemiological surveys where it is important to detect even as low levels of antibodies as 10-80 mIU/ml.

Comparability of some serum protein determinations by radial immunodiffusion, laser nephelometric and turbidimetric assays employing Q-antisera SEVAC.

Quantitation of IgG, IgA and IgM immunoglobulins and C3, C4 components in human serum samples by the radial immunodiffusion technique and by the nephelometric and turbidimetric assays was compared using the linear regression analysis method. Comparisons of the two methods run in polyethylene glycol showed close agreement between methods and a relatively high degree of correlation between the parameters studied. Compared to the radial immunodiffusion technique, nephelometry and turbidimetry gave good correlation between parameters, but the agreement between tests was worse, especially in the case of C3 component determinations in fresh samples of patients' sera. All tests were carried out using the Q-antisera and Control human serum preparations SEVAC.

The first clinical trial for determination of alpha 1 fetoprotein by means of Sevatest-ELISA AFP Kit (micro I).

At the Institute of Sera and Vaccines, Praha, was invented and tested on clinical samples a kit for detection and quantification of alpha 1 fetoprotein in human serum. It is a heterogeneous EIA on the "sandwich" principle. Rabbit antibody to alpha 1 fetoprotein (further AFP) was used for coating the solid surface and goat horse-radish peroxidase labelled antibody to AFP was used as the tracer. Microtitration plate of Czechoslovak manufacture (KOH-I-NOOR, Dalecín) type P with 96 wells was used as the solid phase. The range of an approximately linear part of the calibration curve was intentionally chosen between 10 and 400 ng/ml, since in this way it fills the detection gap in AFP determination between 10 and 200 ng/ml, which is, on the one hand, a physiological value of AFP in human serum and, on the other hand, the bottom limit of sensitivity of counter immunoelectrophoresis (CIEP). Attention was devoted both to reproducibility of the method, i.e. results of intra- and interassays, and comparability with other foreign ELISA Kits. According to the correlation analysis, the kit was ascertained to be very well comparable with kits of foreign provenance. The coefficient of variation (CV) for the interassays varied between 11 and 16% and for intraassays it equalled 15%.

The development and evaluation of Sevatest ELISA hCG Micro I. kit as a test for pregnancy.

The enzyme-linked immunosorbent assay (ELISA) method of sandwich type for determination of human chorionic gonadotropin (hCG) in serum or urine using horseradish peroxidase as an enzyme label and microtiter ELISA plates (or polystyrene microtubes respectively) as a solid phase support for antibody was developed. Test sensitivity of 200 mIU hCG per milliliter is approximately sixfold greater than the available hemo- or latex agglutination tests; quantitative hCG ELISA method has sensitivity of 6 mIU hCG per milliliter. In order to evaluate the usefulness of the method for early pregnancy detection 5,000 urine samples were prospectively collected and results correlated with outcome of pregnancy. Reliability of the test performed on routine basis at the Institute for the Care of Mother and Child in Prague proved to be 97.2% for intrauterine pregnancy detection, in 2.52% the test result was "+ -", and only in 0.28% the results were erroneous. For samples sent with the diagnosis of suspected extrauterine pregnancy 93.5% of correct results, 4.35% of "+ -" and 2.17% of erroneous results was found.

PIP: The enzyme-linked immunosorbent assay (ELISA) method of sandwich type for determination of human chorionic gonadotropin (hCG) in serum or urine using horseradish peroxidase as an enzyme label and microtiter ELISA plates (or polystyrene microtubes respectively) as a solid phase support for antibody was developed. Test sensitivity of 200 mIU hCG/milliliter is approximately 6 times greater than the available hemo- or latex agglutination tests; quantitative hCG ELISA method has sensitivity of 6 mIU hCG/milliliter. In order to evaluate the usefulness of the method for early pregnancy detection, 5000 urine samples were prospectively collected and results correlated with outcome of pregnancy. Reliability of the test performed on a routine basis at the Institute for the Care of Mother and Child in Prague proved to be 97.2% for intrauterine pregnancy detection, in 2.52% of the test result was "+ -", and only in 0.28% were the test results in error. For samples sent with the diagnosis of suspected extrauterine pregnancy, 93.5% were correct results, 4.35% were "+ -", and 2.17% were results in error.

[Routine screening for serological markers of viral hepatitis B in pregnancy]. / Rutinní screening sérologických markeru viru hepatitidy B u tehotných.

Transmission of hepatitis B virus (HBV) from mother to infant during the perinatal period represents very efficient mode of HBV infection and often leads to severe longterm sequelae. Perinatal transmission can occur when an infant is born to mother positive for hepatitis B surface antigen (HBsAg), frequency of transmission is high if the mother is also HBeAg positive. Prevention of perinatal transmission of HBV is important since the majority of these infants who are infected at birth become chronic carriers and can subsequently develop chronic hepatitis, cirrhosis or primary hepatocellular carcinoma. Routine screening for HBsAg of all pregnant women attending prenatal clinic at the Institute for the Care of Mother and Child, Prague in the third trimester of pregnancy by Sevatest ELISA HBsAg Micro I. method (Sevac, Prague) began in June 1986. HBsAg positive patients were subsequently tested for HBeAg and anti-HBe hepatitis markers. Of the 2744 women examined 22 were found HBsAg positive, of these mostly asymptomatic carrier mothers 2 were positive for HBeAg, 14 for anti-HBe and 6 were negative for both HbeAg and anti-HBe. All infants were given passive HBIg prophylaxis in conjunction with HB vaccine after birth regardless of HBeAg status of mother. At present no infant were found HBsAg positive at 6 month of age, observation of this risk group infants continues.

The localization of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and fallopian tube.

The localization of Thy-1, MRC OX 2 and Ia antigens as defined by monoclonal antibodies MRC OX 7, MRC OX 2 and MRC OX 6 was determined by an indirect immunoperoxidase technique on cryostat sections of rat ovaries. Thy-1 antigen was present significantly in the theca interna of growing antral follicles. Developing corpora lutea exhibited an increasing presence of Thy-1 antigen and it was still present in degenerating ones. Thy-1 antigen was constantly present in fallopian tube tunica propria. The MRC OX 2 antigen was expressed most on ovarian structures that do not develop further, i.e. granulosa of degenerating antral follicles and third generation of corpora lutea. MRC OX 2 antibody stained the capillaries of the fallopian tube; the most heavily MRC OX 2+ were the cells of ovarian germinal epithelium. The Ia+ cells were occasionally found within the growing ovarian structures but they were more frequent in degenerating ones. Rare or no la+ cells within the ovary and heavily Ia-depleted thymus medulla and Ia areas in the spleen were, however, observed in some rats. The role of these antigens with respect to the structures they label is discussed.

Enzyme immunoassay to determine serum myoglobin in patients with acute myocardial infarction.

A method of "sandwich" enzyme immunoassay was developed for determination of human serum myoglobin with the use of myoglobin isolated from human myocardium and gammaglobulin fraction of a specific sheep antiserum labelled with horseradish peroxidase. The linear part of the calibration curve within the range of 0.08-2.2 nmol/l is suitable for accurate quantitative reading of myoglobin concentration. Intra- and interassay variation coefficients are 7% and 11.2%, respectively. A comparison of 100 serum samples assessed by means of commercially available RIA kit and by the given method revealed a correlation coefficient of 0.86.

Sensitivity and specificity of Czechoslovak ELISA HBsAg Sevac tests: comparison with other third-generation tests and counter-immunoelectrophoresis.

Two variants of sandwich-type ELISA (Enzyme Linked Immunosorbent Assay) kits for HBsAg detection (Sevatest ELISA HBsAg Macro I and Sevatest ELISA HBsAg Micro I) in human sera and plasmas were developed. As the solid phase, the ELISA Macro kit and ELISA Micro kit make use of polystyrene microtubes, and polystyrene microtitration plates, respectively, of Czechoslovak production (Koh-i-noor, Dalecín). Capture anti HBs antibody for adsorption to solid phase and rabbit anti HBs antibody for labelling with horse-radish peroxidase were prepared for both tests. The sensitivity of both ELISA kits for HBsAg, equal to approx. 2 ng/ml, was determined by titrating six selected HBsAg-positive sera and the WHO Agk 76 panel of HBsAg-positive sera and the results were compared with those obtained by ELISA, RIA (Radioimmunoassay) and RPHA (Reverse passive hemagglutination) kits of different producers and by counter-immunoelectrophoresis (CIEP). The sensitivity of the new ELISA kits was comparable to that of other producers' ELISA kits, higher than that of RPHA kits and only a little lower than that of RIA kits. A set of sera of patients hospitalised with different diagnoses was tested for HBsAg. The detection rate by ELISA Macro kit 2.8 and 1.5 times higher than by CIEP and RPHA (Raphadex B), respectively, and 1.1 time lower than by RIA (Austria II).

Rapid diagnosis of tick - borne encephalitis by immunofluorescence assay of specific serum IgM antibodies.

The indirect IF technique, using suspensions of TBE virus infected and uninfected PS cells as antigen-containing substrate, furnishes a rapid and practical test making possible the detection of specific IgM class serum antibodies in the initial stage of clinically manifest TBE. It enables early confirmation of diagnosis already in the acute phase of the disease and thus it can be instrumental in differential diagnosis and rational therapy, e.g., the administration of specific hyperimmune gamma-globulin.

Opsonic, cytotoxic, precipitating, blocking of bacterial adherence, and other activities of monoclonal IgE antibody compared with IgA and IgM.

Monoclonal IgE anti-TNP antibodies were compared with monoclonal anti-TNP, IgM and IgA isotypes in different biological reactions. The reaginic activity of IgE antibodies demonstrated in passive cutaneous anaphylaxis reactions and degranulation of mast cells was accompanied by a number of activities known to be associated with other isotypes. The occurrence of relatively high numbers of lymphoid cells and macrophages bearing Fc epsilon receptors suggests a possible role of IgE antibodies in host defence mechanisms acting systematically and/or locally on mucosal surfaces.

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization.

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.