Emil Slavov

Comparative study of circulating immune complexes quantity detection by three assays : CIF-ELISA, C1Q-ELISA and anti-C3 ELISA

The assessment of the soluble immune complexes (IC) in human sera is traditionally performed by the C1q binding assay. In the present study, a novel method for the quantity of immune complexes was reported. The methodology was based on measuring their deposition on solid-phase C3 binding glycoprotein (CIF), using an enzyme-linked immunosorbent assay. We also used ELISA that employed anti-C3 antibodies to determined the quantity of immune complexes. The three assays were evaluated for their performance characteristics on the same specially prepared samples: 55 normal sera, 99 sera from RA, 88 sera from SLE, and 27 sera from PSS. The results were compared by reference to a common standard-heat aggregated IgG that possesses many activities of immune complexes. Three of the tests used displayed almost the same specificity (over 95%), while their relative sensitivity varied depending on the disease sera tested. The sensitivity of the assays used was recorded highest for C1q ELISA-28.97% of positive sera, followed by CIF-ELISA-19.63% and lowest for anti-C3 ELISA-17.29%. A well-expressed correlation was found between CIF-ELISA and anti-C3 ELISA data (r=0.42), and a week correlation was noted when comparing CIF-ELISA and C1q ELISA IC levels detected (r=0.28). When the correlation coefficients were calculated individually for each disease category, they were clearly different, and that reflected indirectly in different sensitivities of the test for various disease categories. We also found that the results from the simultaneous performance of the tests demonstrated low percentage positive results when three or two assays were used. This is most probably due to the different assay abilities to detect IC with different sizes and composition, which shows that a small part of IC in the tested sera can be detected simultaneously by more than one assay. On the basis of the results obtained, we concluded that optimal screening for IC could be achieved by parallel application of several different methods.

Correlation between TNF-alpha and IL-12p40-containing cytokines in silicosis

The aim of the present study was to investigate the correlation between tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-12p40-containing cytokines, in silicosis patients and healthy donors exposed to silica dust, in an attempt to clarify the reason for variety of clinical outcomes between humans with similar exposure history. Serum levels of TNF-α, IL-12p40, IL-12p70 and IL-23 in total group of 62 silicosis patients, 24 healthy donors with similar exposure history like patients and 19 healthy donors without exposure were determined by enzyme-linked-immunosorbent assay (ELISA) method. The serum level of TNF-α was significantly higher in healthy donors exposed to SiO2 (22.4 ± 11.1 pg/mL) in comparison with non-exposed healthy donors (14.8 ± 8.8 pg/mL; p = 0.022) and similar to that in silicosis patients. In total, group of silicosis patients significantly elevated levels of TNF-α (20.9 ± 12.9 vs. 14.8 ± 8.8 pg/mL; p = 0.047) and IL-12p40 (94.5 ± 51.6 pg/mL vs. 68.7 ± 26.2 pg/mL; p = 0.029) compared to non-exposed healthy donors were observed. In addition, a strong positive correlation between TNF-α and IL-23 levels (r = 0.678; p = 0.022) and between TNF-α and IL-12p70 levels (r = 0.75; p = 0.0003) was detected in the group of exposed healthy donors, while in the group of silicosis patients, a significant positive correlation was observed only between TNF-α and IL-12p40 (r = 0.434; p = 0.00048), in contrast to other IL-12p40 containing cytokines. In conclusion, we could assume that the elevated serum levels of TNF-α are associated with exposition to silica particles, while the elevation of both TNF-α and IL-12p40 is associated with silicosis development and severity. Additionally, the balance between IL-12p40-containing cytokines may also contribute to the silicosis progression.

Serum neopterin in workers exposed to inorganic dust containing free crystalline silicon dioxide

The study was conducted among 92 male workers, divided into two groups depending on workplace and level of inorganic dust containing free crystalline SiO2 in the work environment, and 43 healthy workers without exposure to dust aerosols. The measured inhalable and respirable dust concentrations, as well as the concentration of free crystalline silica (FCS) in the respirable fraction were different for the two groups, but the percentage of free crystalline silica in the respirable fraction was almost identical. Significantly higher neopterin levels were found in workers exposed to dust, compared to the control group: 12.72 nmol/L and 6.32 nmol/L respectively (p<0.05). No significant difference was found between serum neopterin levels in both groups of the exposed workers. Among the groups with different length of service, a statistically higher neopterin level was evident only in the workers with length of service less than 10 years (p<0.05). The correlation analysis did not find a significant dependence of neopterin levels on the age of the studied workers or on the duration of smoking in packet years. The difference between neopterin levels in smokers and non-smokers was nonsignificant. The results obtained show that increased neopterin levels in the exposed workers are not influenced by individual features, duration and level of exposure to inorganic dust, but mostly by the presence of FCS in the respirable fraction.

New ELISA Kits using C3 Binding Glycoprotein from Cuscuta europea Detect Mainly IgM CIC in Rheumatoid Arthritis and Progressive Systemic Sclerosis, but not in Systemic Lupus Erythematosus

Elevated levels of circulating immune complexes (CIC), containing IgG, IgM or IgA antibodies were detected in the sera of patients with autoimmune diseases. This might indicate a different biological meaning of the three isotypes of immunoglobulin (Ig) in the CIC. Each CIC assay detected only certain classes and subclasses of Ig in CIC material or fixed complement protein. In this study, a new method based on C3binding glycoprotein named CIF-ELISA and a well-known method ANTI-C3 ELISA, were used for quantitative assessment of IgM-CIC, IgG-CIC and IgA-CIC levels in human sera. A modified CIF-ELISA and ANTI-C3 ELISA for simultaneous detection of CIC, containing IgG, IgM and IgA, (stCIC), were also performed. The assays were evaluated on the same specially prepared samples: 55 normal sera, 99 sera from rheumatoid arthritis (RA), 88 sera from systemic lupus erythematosus (SLE), and 27 sera from progressive systemic sclerosis (PSS). We found that the sensitivity of the tests used varied depending on the diseases studied. CIF-ELISA displayed higher sensitivity of IgM-CIC when compared to ANTI-C3 ELISA in RA patients (40.0 and 20.95%, respectively) and PSS (44.43 and 37.04%, respectively). Results for the sensitivity of IgA-CIC were in adverse direction in the RA group (14.28 and 19.05%) and PSS (14.81 and 25.93%) by both methods. It was also established that the concordance of IgM-CIC positives by both methods was 48.84% in RA and 46.67% in PSS, while in SLE it was 18.78%. These results are most probably due to the different assay abilities to detect antibody isotype of the CIC material and help to explain what specific role each Ig isotype in CIC has in the course of the disease.