Detection of Legionella pneumophila antibodies in the blood serum of pneumonia patients by an immunoenzyme method

The conditions of making the enzyme immunoassay (EIA) for the detection of antibodies to L. pneumophila have been optimized. The use of L. pneumophila purified serotypic antigen at a concentration of 0.25 micrograms/ml for the sensitization of polystyrene plates has been shown to increase the sensitivity and specificity of the assay. 220 patients with severe pneumonia have been examined. As revealed in this investigation, antibodies to L. pneumophila can be detected in 12.2% of cases. A high degree of correlation (94.4%) between the results of EIA and the indirect immunofluorescence test has been shown.     

Determination of amyloid P component in blood by an immunoenzyme method

An ELISA procedure was developed for measuring serum amyloid P-component (SAP). The assay is based on our finding of binding AP to polystyrene microtiter plates. The amount of SAP is determined by concurrent ELISA, wells being sequentially incubated with rabbit anti-AP antiserum and goat anti-rabbit antiserum, conjugated with peroxidase. The limit of sensitivity of the assay is 0.5 micrograms/ml. When applied to the screening of patients plasma, elevation of SAP concentration in patients with rheumatoid arthritis and amyloidosis were found.     

Determination of Mycoplasma pneumoniae antigens and antibodies by the immunoenzyme method in patients with respiratory tract diseases

The optimum conditions for carrying out the enzyme immunoassay (EIA) with a view to determine M. pneumoniae antigen and antibodies to them in the sera of patients with different respiratory diseases were established. The use of the specially modified EIA technique made it possible to reveal that patients with tuberculosis and chronic pneumonia showed similar occurrence of M. pneumoniae (35.7% and 35.0% of cases, respectively), while in patients with pulmonary sarcoidosis M. pneumoniae occurred in 27.2% of cases. At the same time the occurrence of antibodies in patients with chronic pneumonia and sarcoidosis was more than three times greater than in tuberculosis patients.     

Determination of antibodies to Streptococcus group A polysaccharide in human sera by an immunoenzyme method

Use was made of the ELISA to develop a highly sensitive quantitative method for detection of antibodies against Streptococcus group A polysaccharide (polysaccharide A) in human sera. The main advantage is that one can use only one optimal dilution of the sera together with the reference serum. Sera of 53 healthy volunteers and 77 patients with a history of Streptococcus group A infections were screened for the presence of polysaccharide A antibodies. Highly reproducible results were obtained in 97% of cases. The specificity of the method was shown with the polysaccharide A-induced inhibition of the reaction. Positive reactions obtained with the tested sera in gel immunodiffusion correlated with the data derived by the ELISA. Using the latter high level of specific antibodies was found in some of the sera that yielded negative reactions when tested by gel immunodiffusion. This may be associated with the presence of non-precipitating antibodies.     

Stress platelets in normal individuals and patients with idiopathic thrombocytopenic purpura.

To test the hypothesis that stress platelets (SPs) described by Tong et al. in rats may be a parameter of young platelets in humans, we examined and characterized SPs in normal individuals and in patients with idiopathic thrombocytopenic purpura (ITP). Our results indicated that SPs comprise about 1.2% of the circulating platelets in normal individuals and 2.6% in ITP patients. The configuration of SPs as well as of various irregular forms of circulating platelets was found to be supported by synergism of both the platelet microfilaments and microtubules. SPs showed some segmentation, the degree of which was similar in normal individuals and ITP patients, and they underwent further segmentation during in vitro incubation, mainly promoted by microtubules, so that they sometimes appeared like discoid platelets in a chain. These observations suggest a new mode of production of discoid platelets in the circulation. Thus, identification and enumeration of SPs may be useful for evaluating thrombocytopoiesis in humans.     

Determination of tularemia antibodies by an immunoenzyme method on a solid-phase carrier (ELISA)

ELISA "sandwich" techniques have been developed and the optimum assay conditions for detecting specific antibodies in human serum samples have been determined. The possibility of using these techniques for the determination of the level of antibodies to tularemia antigens in the sera of persons immunized with live tularemia vaccine has been shown. Statistically significant differences in the level of antibodies to tularemia antigen in the sera of immunized and nonimmunized persons have been established. The comparative study of five serological methods - ELISA, the agglutination test, the passive hemagglutination test, the immunofluorescence test and the defined antigen substrate sera ( DASS ) techniques - has revealed the advantage of ELISA, whose sensitivity has proved to be considerably higher than that of all other methods used in our work.     

Detection of Mycoplasma arthritidis and Mycoplasma fermentans antibodies in rheumatoid arthritis patients by an immunoenzyme method

The optimum parameters of the immunoenzyme assay system for the identification of antibodies to M. arthritidis and M. fermentans in the sera of patients with rheumatoid arthritis have been established. The investigation has shown that the products obtained by the ultrasonic disintegration of the biomass of M. arthritidis and M. fermentans can be used as soluble antigens for adsorption on the polystyrene surface of plates. The use of the immunonenzyme assay, specially modified, has made it possible to establish that antibodies to M. arthritidis can be detected in 6.5% of cases, antibodies to M. fermentans, in 41.9% of cases and the association of antibodies to M. arthritidis and M. fermentans, in 41.9% of cases. At the same time antibodies to M. arthritidis have been found to belong mainly to IgM and antibodies to M. fermentans, to IgG or to IgG and IgM simultaneously.     

Prognostic significance of splenic follicle size in splenectomized idiopathic thrombocytopenic purpura patients

The prognostic significance of splenic follicle (B-lymphocyte compartment) size was studied in 62 patients splenectomized for idiopathic thrombocytopenic purpura (ITP). Patients with hyperplasia of splenic follicles (mean follicle diameter greater than 500 micron) were more likely to relapse or to develop additional autoimmune disorders than patients without hyperplastic splenic follicles (mean follicle diameter less than 500 micron) (p less than 0.01). The enlargement of splenic follicles had a positive predictive value of 27% and a negative predictive value of 100% for a poor outcome of splenectomy. Thus, the histological examination of spleens surgically removed for ITP seems to be an appropriate method to obtain the first indication of the possible long-term effect of splenectomy almost immediately after the operation.     

Detection of pneumococcal antibodies in children with acute pneumonia and pleurisy by an immunoenzyme method

The samples of sera and pleural fluid from sick children have been analyzed by means of EIA techniques. To detect the time course of antibody production, the antigenic preparations of pneumococci (monovalent capsular polysaccharides, polyvalent polysaccharide vaccine and complex pneumococcal antigen) have been used. Antibody response observed in the forms of pneumococcal infection, studied in this investigation, has proved to be highly variable. It is expedient to determine antibodies to polysaccharide antigens not earlier than on days 10-12 from the beginning of the disease. But, besides the positive dynamics of antibodies, their unchanged level is sometimes observed in the patients at the beginning of the disease. As a rule, there is a coincidence between the dynamics of antibody formation in response to polysaccharide antigens and to complex pneumococcal antigen.     

Complement-dependent cytotoxic factor to megakaryocyte progenitors in sera from patients with idiopathic thrombocytopenic purpura

The influence of sera from patients with idiopathic thrombocytopenic purpura (ITP) was examined on colony formation from megakaryocyte (M) progenitors. Though incubation of marrow cells in Iscove's modified Dulbecco's medium (IMDM) containing 50% sera from several ITP patients stimulated M-colony formation in 8 of 13 cases, incubation in the sera from the patients and in baby rabbit serum as a source of complement significantly suppressed the colony formation. Experiments showed that sera of immunoglobulin G from ITP patients had significant complement-dependent cytotoxicity to M-progenitors in normal marrow cells or in the marrow cells from corresponding patients, but not to CFU-e, BFU-e or CFU-gm. Cytospin preparations of individually collected M-colonies from marrow cells treated with ITP patients' sera and complement revealed a reduction of megakaryocyte colonies containing cells of multilineages. These results indicate that autoantibodies detected in ITP patients can bind not only to platelets and megakaryocytes, but may also bind to M-progenitors.     

Quantitative evaluation of serum IgG subclass levels by an immunoenzyme method

We developed a enzyme linked immunosorbent assay (ELISA) for measuring IgG subclasses concentration in serum. For this we used monoclonal antibodies. The specificity of these antibodies was evaluated with a panel of myeloma proteins belonging to the 4 IgG subclasses. The ELISA was sensitive (allowing the detection of subclasses at ng level) and accurate (inter-assay coefficient of variation of 14%). Using the WHO serum 67/97 as reference, we determined the concentration of IgG subclasses in a pool of sera. In addition concentrations were measured in 69 healthy adults to study the distribution of each IgG subclass. A good correlation (r = 0.78) was obtained between the sum of the subclasses measured by ELISA and total IgG measured by immunonephelometry.     

A case of silent thyroiditis associated with idiopathic thrombocytopenic purpura

A 51-year-old woman had symptoms of thyrotoxicosis which disappeared spontaneously within two months. She was diagnosed as a case of silent thyroiditis on the basis of both the clinical course and the laboratory data such as low uptake of radioactive iodine and technesium. She also had petechiae in her arms which were diagnosed as an idiopathic thrombocytopenic purpura (I.T.P.). This case would seem to expand the spectrum of the coexistence of autoimmune thyroid diseases and I.T.P. which is believed to be an autoimmune disease.     

Planned splenectomy in treatment of idiopathic thrombocytopenic purpura.

The results of a policy of treatment in idiopathic thrombocytopenic purpura based on previous observations on the natural history of the disease and its response to corticosteroids are described. The results of splenectomy were better when the history was less than 100 days. Three patterns of response to splenectomy were observed: complete remission, symptomatic remission, and relapse. The prognosis can be determined by the level of the platelet count six weeks after splenectomy. Corticosteroid treatment for more than three weeks before splenectomy noticeably increased the incidence of complications after operation. Splenectomy can safely be performed in pregnancy. The decision to operate should be made on the maternal condition and its response to corticosteroids.     

Kinetics of megakaryocyte progenitor cells in idiopathic thrombocytopenic purpura

The number and proliferative state of megakaryocyte progenitor cells (CFU-Meg) were compared between 13 patients with idiopathic thrombocytopenic purpura (ITP) and hematologically normal controls. The mean frequency of CFU-Meg assayed by the plasma clot method was 27.8 +/- 12.2 (+/- SD)/2 x 10(5) bone marrow light-density cells for the ITP patients, which did not differ significantly from the control value of 31.9 +/- 16.1. The percentage of CFU-Meg in DNA synthesis estimated by the 3H-thymidine suicide technique was 41.3% +/- 9.2% in ITP, which was significantly greater than the control value of 27.1% +/- 7.4% (P less than 0.01). The megakaryocyte counts for histological sections prepared from bone marrow aspirates from the ITP patients and controls were 34.5 +/- 8.5/mm2 and 11.2 +/- 5.8/mm2, respectively, with the difference being highly significant (P less than 0.001). These results suggest that increased cycling activity in a quantitatively unchanged CFU-Meg pool may lead to increased megakaryocytes in the bone marrow of ITP patients.     

Long-term prognosis of the results of splenectomy in idiopathic thrombocytopenic purpura

The value of selected clinical and histological parameters for the prognosis of long-term results of splenectomies was investigated retrospectively in 50 patients with idiopathic thrombocytopenic purpura 4-14 years after surgery. Two parameters showed an association to the results of splenectomy, viz. spleen follicle size as well as site of thrombocytopenia degradation. Non-responders to splenectomy (n = 7) only turned up in spleen follicle hyperplasia, never in follicle normoplasia, however (p less than 0.01), four times more frequently in mainly extralienal thrombocyte degradation, also in predominantly lienal ones (p greater than 0.05).