JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: , Volume 2, Issue 2, March 2014

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1 PRELIMINARY EVALUATION OF CHORUS SYSTEM IN COMPARISON WITH COBAS 6000 SYSTEM FOR DETECTION OF ANTI-TOXOPLASMA-IGM ANTIBODIES BLERTA LAZE* ANILA MITRE** *Dept. of Biology, University Ismail Qemali, Vlora, Albania **Dept. of Biology, Faculty of Natural Sciences, Tirana, Medical Clinic Intermedica, Tirana, Albania ABSTRACT Toxoplasma gondii is a parasitic protozoa which can be transmitted by eating infected meat or from mother to fetus during the first trimester of pregnancy. This microscopic parasit can cause fetal infection with unpredictable consequences in later life. Medical diagnostic is working to determine the most sensitive techniques for the detection of T. gondii antibodies, in the framework of which is developed this scientific work. An enzyme-linked immunosorbent assay (ELISA, applied in CHORUS instrument) and a new Electrochemiluminescence technique (ECL, applied in Cobas 6000 instrument) have been compared with each other for the detection of Toxoplasma IgM antibodies. 200 patients were analyzed for detection of Toxoplasma IgM antibodies. 185 out of 200 samples (92.5%), gave compatible results with both techniques. In particular, 149 samples gave negative results, 35 samples gave positive results and 1 samples gave doubtful result with both techniques. It was observed that 10 samples were positive in Cobas instrument and doubtful in Chorus instrument. While 5 samples were negative in Chorus instrument and positive in Cobas instrument. The ECL technique was considered more specific and technically more advantageous than ELISA technique in Chorus instrument. KEYWORDS: ELISA, ECL, Toxoplasma IgM, Sensitivity, Specificity INTRODUCTION Toxoplasma gondii is a well-known obligate intracellular protozoa pathogen of virtually all warm-blooded animals and commonly infects human worldwide. The infection is mainly acquired by ingestion of food or water that is contaminated by mature oocysts shed by cats or by undercooked meat containing tissue cysts (4). Acute infection of toxoplasmosis in early pregnancy of women carries the peril of transmitting the infection to the fetus with serious and unpredictable consequences in later life (9, 11, 12). Medical diagnostic is working to determine the most sensitive techniques for the detection of T. gondii antibodies, 105

2 in the framework of which is developed this scientific work. The detection of Toxo IgM antibodies is presumptive of an acute, recent or reactivated Toxoplasma infection. MATERIAL AND METHODS An enzyme-linked immunosorbent assay (ELISA, applied in CHORUS instrument) and a new Electrochemiluminescence technique (ECL, applied in Cobas 6000 instrument) have been compared with each other for the detection of anti-toxoplasma IgM antibodies. There have been analyzed 200 patients with each technique. Principle of ELISA technique: This technique is applied on CHORUS instrument, which is a new device in medical diagnostics. The partially purified Toxoplasma antigen is bound to the solid phase. Through incubation with human serum diluted in diluents which blocks the IgG, the specific IgM are bound to the antigen. After washing to eliminate the proteins which have not reacted, the sample is incubated with the conjugate composed of monoclonal anti-human IgM antibodies labelled with peroxidase. The unbound conjugate is eliminated and the peroxidase substrate is added. The colour which develops is proportional to the concentration of specific antibodies present in the serum. The disposable devices contain all the reagents to perform the test when applied on the CHORUS instrument. Description of Toxo IgM strip: The strip consist of 7 wells covered with a labelled, foil seal. The label comprises a bar code which mainly indicates the assay code, kit lot number and expiration date. The foil of the first well is perforated to facilitate the introduction of the undiluted sample. The wells in the center section of the strip contain the various reagents required for the assay. Position 1: Empty well in which the operator must place the undiluted serum. Position 2: Conjugate 0,35 ml. Position 3: Diluent for the samples 0,35ml. Position 4: TMB substrate 0,35 ml. Position 5: Uncoated microplate well. Position 6: Microplate well coated with purified Toxoplasma antigens. Position 7: Empty 106

3 Specimen type and collection: Human serum collected in separating tube gel in the normal manner from the vein and handled with all precautions. Samples can be stored at 2-8 C for 4 days, or frozen for longer periods at -20 C and can be thawed a maximum of 3 times. Principle of Electrochemiluminescence technique: This technique is applied on Cobas 6000 instrument. The test principle is µ-capture with a total duration of 18 minutes. The first incubation: 10 µl of sample are automatically prediluted 1:20 with Elecsys Diluent Universal. T. gondii-specific recombination antigen labeled with a ruthenium complex is added. Anti-Toxo IgM antibodies present in the sample react with the ruthenium-labeled T. gondii -specific recombination antigen. The second incubation: Biotinylated monoclonal anti-h-igm-specific antibodies and streptavidin-coated microparticles are added. The complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. Results are determined automatically by the software by comparing the electrochemiluminescence signal obtained from the reaction product of the sample with the signal of the cutoff value previously obtained by Toxo IgM calibration. Description of the reagent: M: Streptavidin-coated-microparticles (transparent cap), 1 bottle, 6.5mL. 2+ R1: Toxoplasma-Ag-Ru(bpy) 3 (gray cap), 1 bottle, 9 ml. R2: Anti-h-IgM-Ab-biotin (black cap), 1 bottle, 9 ml. Specimen type and collection: Human serum collected in separating tube gel. Samples can be stored at 2-8 C for up to 5 days; if longer storage is required, freeze at -25 ± 6 C. Calculation and interpretation of the results The Cobas 6000 analyzer automatically calculates the cutoff based on the measurement of Cal1 and Cal2. The result of a sample is given either as reactive or non-reactive as well as in the form of a cutoff index (signal sample/cutoff). The CHORUS instrument expresses the 107

4 result as an index ( ratio between the OD value of the test sample and that of the cutoff) which can be used as a quantitative measure, as it is proportional to the amount of specific IgM present. The test serum can be interpreted as follows: Table 1. Interpretation of the results Toxoplasma IgM ELISA (CHORUS) (index) COBAS 6000 (index) Negative <0.9 < 0.8 COI Positive >1.1 1 COI Doubtful index < 1 COI RESULTS 200 patients were analyzed for detection of Toxoplasma IgM antibodies. 185 out of 200 samples (92.5%), gave compatible results with both techniques. In particular, 149 samples gave negative results, 35 samples gave positive results and 1 samples gave doubtful result with both techniques (tab 2). It was observed that 10 samples were positive in Cobas instrument and doubtful in Chorus instrument (tab 3). While 5 samples were negative in Chorus instrument and positive in Cobas instrument. Table 2. The results for ECL and ELISA techniques. Toxoplasma IgM ECL (COBAS 6000 ELISA (CHORUS) Negative Pozitive E dyshimtë 1 11 Table 3. Details for inconsistent results. Positive (ECL) Doubtful (ELISA) Positive (ECL) Doubtful (ELISA) Positive (ECL) Negative (ELISA) Based on these results, we can calculate sensitivity and specificity for each technique. Table 4. Sensitivity and specificity for ELISA and ECL techniques. Toxoplasma IgM Sensitivity Specificity ELISA (CHORUS) 87.5% 100% ECL (COBAS 6000) 100% 100% 108

5 CONCLUSIONS The CHORUS instrument expresses the result as an index ( ratio between the OD value of the test sample and that of the cutoff) which can be used as a quantitative measure, as it is proportional to the amount of specific IgM present in the sample. The results of the assay must be interpreted with caution and in conjuction with information available from the clinical evaluation and other diagnostic data. Sera from patients in an early or late stage of the disease could give a repeatedly negative result close to the cut-off value. In such cases, a confirmation of the result is recommended (1, 2, 3). Also, all positive test results require careful interpretation since false positive reactions or heterotypic IgM responses may occur with sera from patients with heterophile positive mononucleosis, or Varicella Zoster. ECL technique showed a better ability to detect anti-toxoplasma IgM antibodies during the early stage of acute infection. The principle of both tests are based on the detection of antigen-antibody reactions and both require their own expensive equipment for the reading of the results. The ECL technique was considered more specific and technically more advantageous than ELISA technique in Chorus instrument. But, the detection of IgM antibodies against T. gondii in a single sample is not sufficient to prove an acute Toxoplasma infection since elevated IgM antibody levels may persist even for years after initial infection (7, 8, 10). Further tests or a combination of test methods should be done for clarification. A significant increase of the Toxo IgG antibody titer from a first to a second sample taken e.g. within 2 weeks may support the diagnosis of acute Toxoplasma infection. In rare cases, interference due to extremely high titers of antibodies to immunological components, streptavidine or ruthenium can occur. These effects are minimized by suitable test design. Low sensitivity is the disadvantage of ELISA in CHORUS instrument. This happens because there is a non specific glycolipid antigen for Toxoplasma gondii, which operates in a crossreaction with antigens of different origins. Advantage of ELISA is measuring samples one by one ( even a single analyse) and a short procedure time. The principle of both tests are based on the detection of antigen-antibody reactions and both require their own expensive equipment for the reading of the results. Positive and negative samples produced a large difference in signal strength. ECL technique showed a better ability to detect anti- Toxoplasma IgM antibodies during the early stage of acute infection. Analysis of the results revealed a good level of concordance between the two assays and confirmed the usefulness of ECL technique to diagnose acute acute toxoplasmosis. 109

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