Pekka Palomäki’s research while affiliated with Research Institute of the Finnish Economy, Finland, Helsinki and other places

New enzyme immunoassays (EIAs) for determination of specific IgG, IgA, and IgM antibody titers to Chlamydia pneumoniae were evaluated independently in three research laboratories. Specificity of the EIAs was enhanced by removing LPS from the chlamydial antigen. The performance of these EIAs was evaluated in comparison with the microimmunofluorescence (MIF) test using specimens from: (i) a group of adult patients with community-acquired pneumonia (CAP) previously diagnosed as having an acute chlamydial infection by the complement fixation test or the whole inclusion fluorescence test; (ii) from a group of adult patients with acute respiratory tract infections; and (iii) from a group of young children consecutively presenting with acute respiratory tract infections. The MIF test and the EIAs detected acute infections in paired serum specimens from 12 of 14 patients from the first group. Eleven of these 12 patients were positive in both tests. The MIF test detected seven acute infections in single convalescence serum specimens from eight patients. Two of these were also positive in the EIAs. Paired serum specimens from the second group of adult patients (n=12) were collected during an epidemic of C. pneumoniae. The EIAs detected six acute infections. The MIF test detected two additional patients with acute infections. From the group of young children (n=30), the EIAs detected two patients with acute infections. Our conclusion from this preliminary evaluation is that these EIAs could be useful for laboratory diagnosis of acute C. pneumoniae infections. Comprehensive prospective studies should provide suitable data to calculate the sensitivity, specificity, and predictive values.

Serology is commonly used for the diagnosis of acute Chlamydia pneumoniae infections and also for the diagnosis of complicated Chlamydia trachomatis infections. Furthermore, recent sero-epidemiological studies have linked C. pneumoniae infection with several diseases traditionally considered non-infectious. The objectives of this mini-review are to critically review and discuss some selected analytical and methodological aspects, controversies and current problems in chlamydial serodiagnosis. To illustrate our views we present some original data of the comparison of current technologies. The review of the literature revealed high variability in methodologies applied to different studies. This observation was supported by our own data, which explains occasional conflicting clinical interpretation. Although the microimmunofluorescence (MIF) technique is generally considered as the gold standard for serodiagnosis of chlamydial infections, assay conditions are highly variable and hence pose a major problem in the interpretation of the results. For instance, many recent studies linking C. pneumoniae and atherosclerosis have utilized MIF techniques with variable threshold criteria for the positivity, in combination with selection bias of cases and controls possibly leading to conflicting results. Variability of assay conditions is also a common problem with Western blots, and interpretation is problematic when both anti-C. pneumoniae and anti-C. trachomatis antibodies are present. Furthermore, there is a lot of disagreement in serological criteria applied to recently emerged enzyme immunoassay (EIA) techniques when these assays are used for acute and non-acute clinical conditions and their association with Chlamydiae. In conclusion, standardization of serological techniques and the development of uniform criteria for interpretation of serologic findings is necessary to increase our knowledge of the biology of Chlamydiae, pathogenesis of any chlamydial infection and chronic infections in particular.

In the Toxoplasma gondii immunoglobulin M (IgM) capture fluorometric enzyme immunoassay used as a model, nonspecific responses due to the binding of human IgM to horseradish peroxidase (HRP) conjugates were observed despite the removal of the Fc portion of the immunoglobulin. This interaction may be mediated through the binding of human IgM to the HRP moiety of the conjugate. Addition of polymerized HRP into the reaction mixture reduced nonspecific signals in the majority of low false-positive serum reactions. Other plausible sites of interaction are conserved epitopes of mouse immunoglobulins presenting antigenic similarities with the allotopes of other species. Fragmentation of mouse antimicrobial IgG to Fab' and selection of proper conjugation procedure improved assay specificity.

A one-step third generation enzyme immunoassay (EIA) was developed for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma using a polyclonal (Pab-HBsAg) and two monoclonal antibodies to HBsAg (Mab1-HBsAg and Mab2-HBsAg). In this assay, the solid phase is coated with Mab1-HBsAg and the specimen is incubated simultaneously with peroxidase labelled Pab-HBsAg and Mab2-HBsAg. If HBsAg is present in a specimen it will form sandwich complexes with capture and tracer antibodies. The hook effect, observed in some HBsAg detection tests when a high concentration of HBsAg is present, was minimized in this assay by increasing the concentration of the peroxidase-labelled Mab2-HBsAg. The sensitivity of this assay for HBsAg/ay and HBsAg/ad subtypes in a standard (2 h incubation) procedure was 0.6 and 0.3 ng/ml and in an overnight (16-22 h incubation) procedure 0.2 and 0.15 ng/ml, respectively. Strong elimination of the hook effect was observed with specimens containing high levels of HBsAg compared with test results using peroxidase-labelled Pab-HBsAg alone as enzyme tracer. This EIA offers a procedure, with a high specificity and wide range of sensitivity for the detection of HBsAg in human sera or plasma.

The application of 3-p-hydroxyphenylpropionic acid (HPPA), a fluorogenic substrate of horseradish peroxidase (HRP) to an automated microplate fluorometric enzyme immunoassay is described. Fluorescence intensity of the end product was highly dependent on the pH of the buffer and on the concentrations of the substrate mixture ingredients. The determination of human thyrotropin (TSH) and recombinant hepatitis B surface antigen (rHBsAg) were performed using a fluorometric enzyme immunoassay (FEIA) with HPPA as the substrate, and a colorimetric one with tetramethylbenzidine (TMB) as the chromogenic substrate. The sensitivity of both types of assays proved comparable. The distinct advantage of a fluorometric assay is the possibility to perform a quantitative detection of analyte over a very wide dynamic range. Clinical evaluation of both assays showed good correlation between the FEIA and conventional methods.