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PLATELIA™ TOXO Igg AVIDITY 72842 PLATELIA™ TOXO IgG AVIDITY 48 72842 DETERMINATION OF ANTI-TOXOPLASMA GONDII IgG AVIDITY IN HUMAN SERUM BY ENZYME IMMUNOASSAY 881130 - 2013/11 Table of Content 1- INTENDED USE ...............................................................................3 2- CLINICAL VALUE ............................................................................3 3- PRINCIPLE ......................................................................................4 4- PRODUCT INFORMATION ..............................................................5 5- WARNINGS AND PRECAUTIONS ..................................................5 6- SAMPLES ........................................................................................7 7- ASSAY PROCEDURE ......................................................................7 8- INTERPRETATION OF RESULTS .................................................10 9- PERFORMANCES .........................................................................11 10- LIMITATIONS OF THE PROCEDURE ...........................................13 11- QUALITY CONTROL OF THE MANUFACTURER .........................13 12- REFERENCES ...............................................................................14 2 [EN] 1. INTENDED USE Platelia™ TOXO IgG AVIDITY is an immuno-enzyme assay for determination of avidity of anti-T. gondii IgG antibodies in human serum. The Platelia™ TOXO IgG AVIDITY assay should be used in association with the Platelia™ TOXO IgG kit (Ref. 72840). 2. CLINICAL VALUE T. gondii is a protozoan causing infection in numerous species of mammals and birds. Toxoplasmosis, frequent in humans and animals, are more typically silent. The prevalence of this infection in the population, established using serological tests, may differ depending upon the country of origin and the age. Toxoplasmosis during pregnancy has been implicated in serious congenital abnormalities (in particular, impaired brain functions) and sometimes stillbirth. Demonstration of Toxo IgG antibody in women prior to conception provides assurance of fetal protection from possible toxoplasmosis during pregnancy. Predisposition to severe toxoplasmosis infection is common in persons known to have Acquired Immune Deficiency Syndrome (AIDS), or who are otherwise immunocompromised. These infections are mainly due to reactivation of T. gondii cysts present prior to the HIV infection. Specific diagnosis of T. gondii infection can be complicated and isolation of the parasite is rare. Serologic confirmation ofT. gondii antibody is indicative of exposure to the parasite and has become widely accepted as a means to determine immune status and susceptibility to infection. Screening of several isotypes allows either the dating of the T. gondii and the implementation of appropriate therapy in case of recent infection or the proposal of prophylactic recommendations: hygiene-diet guidelines in pregnant women, chemo- prophylaxy in immunocompromised population. However, differential diagnosis between recent and past-infection is sometimes difficult due to persistence of anti-T. gondii IgM antibodies or re-apparition of anti-T. gondii IgM in case of re-infection, reactivation of a latent infection or non specific stimulation of the immune system. In such situations, IgG avidity measurement by an immuno-enzyme method can help to differentiate between acute or past-infection. This determination is based on evolution of the immune response maturity, which translates in synthesis of low avidity IgG antibodies during primo-infections and of high avidity IgG antibodies during past-infections. 3 [EN] 3. PRINCIPLE This test is carried out using Platelia™ TOXO IgG AVIDITY (Ref. 72842) in association with Platelia™ TOXO IgG (Ref. 72840). The principle of this method relies on the measurement of the avidity of the IgG antibodies to T. gondii. The use of an agent (as urea) dissociating the link antigen/antibody in parallel with the usual technique of IgG antibodies measurement allows comparison of the optical density (OD) obtained after dissociating agent action and OD obtained without dissociating agent action. The avidity is considered low when the antigen/antibody link is easily dissociated. The avidity is considered high when the antigen/antibody link is not easily dissociated. This test has to be used only on positive samples that demonstrate an anti-T. gondii IgG concentration higher or equal to 9 IU/ml with Platelia™ TOXO IgG (Ref. 72840). • Step 1 Avidity controls and patient sera with a concentration of anti-T. gondii IgG between 9 and 80 IU/ml are diluted to 1/101. Sera from patients with a concentration of anti-T. gondii IgG equal or higher than 80 IU/ml are diluted to 1/808. Diluted samples are then distributed twice into T. gondii antigen coated wells of the microplate. During this incubation of one hour at 37°C, IgG antibodies to T. gondii present in the sample bind to the T. gondii antigen coated on microplate wells. After incubation, unbound non specific antibodies and other serum proteins are removed by washings. • Step 2 Each avidity control and each patient serum is treated in double: control solution is added in one well and dissociating solution in the other well. During this incubation of 15 minutes at room temperature (+18-30°C), the dissociating solution tries to dissociate the antigen/antibody complexes. After incubation, both solutions and dissociated IgG are removed by washings. • Step 3 The conjugate (peroxydase labeled polyclonal antibody specific for human gamma chains) is added to the microplate wells. During this incubation of one hour at 37°C, the labeled antibody binds to the serum IgG captured by the T. gondii antigen. The unbound conjugate is removed by washings at the end of the incubation. • Step 4 The presence of immune-complexes (T. gondii antigen, IgG antibodies to T. gondii, anti-IgG conjugate) is demonstrated by the addition in each well of an enzymatic development solution. 4 [EN] • Step 5 After incubation of 30 minutes at room temperature (+18-30°C), the enzymatic reaction is stopped by addition of 1N sulfuric acid solution. The optical density reading obtained with a spectrophotometer set at 450/620 nm is proportional to the amount of IgG antibodies to T. gondii present in the well. 4. PRODUCT INFORMATION Supplied quantities of reagents have been calculated to allow 48 tests with the Platelia™ TOXO IgG kit (Ref. 72840). All reagents are exclusively for in vitro diagnostic use. Label Nature of reagents Presentation R5a Low Avidity Low Avidity Control: 1 x 0.75 mL Control Human serum reactive for IgG antibodies to T. gondii, and negative for HBs antigen, anti- HIV1, anti-HIV2 and anti-HCV Preservative : 0.15% ProClin™ 300 R5b High Avidity High Avidity Control: 1 x 0.75 mL Control Human serum reactive for IgG antibodies to T. gondii, and negative for HBs antigen, anti- HIV1, anti-HIV2 and anti-HCV Preservative : 0.15% ProClin™ 300 R12 Control Control Solution: 1 x 28 mL Solution TRIS-NaCl buffer (pH 7.6 ± 0.2), 0.1% Tween® 20 and green dye Preservative : 0.15% ProClin™ 300 R13 Dissociating Dissociating Solution: 1 x 13 mL Solution TRIS-NaCl buffer (pH 7.6 ± 0.2), urea, 0.1% Tween® 20 et yellow dye Preservative : 0.001% ProClin™ 300 For storage conditions and expiration date, please refer to the indications stated on the box. 5. WARNINGS AND PRECAUTIONS The reliability of the results depends on correct implementation of the following Good Laboratory Practices: • Do not use expired reagents. • Do not mix or associate within a given run reagents from different lots. • Before use, wait for 1H30 minutes to allow reagents to reach room temperature (+18-30°C). 5 [EN] • Use glassware thoroughly washed and rinsed with deionized water or, preferably disposable material. • Use a new pipette tip for each sample. • Check the pipettes and other equipments for accuracy and correct operations. • Strictly follow the assay procedure. • Refer also to the Platelia™ TOXO IgG product insert (Ref. 72840). Note: Platelia™ TOXO IgG AVIDITY kit can be used with different lots of Platelia™ TOXO IgG (Ref. 72840). HEALTH AND SAFETY INSTRUCTIONS Human origin material used in the preparation of reagents has been tested and found non-reactive for hepatitis B surface antigen (HBs Ag), antibodies for hepatitis C virus (anti-HCV), and to human immunodeficiency virus (anti-HIV1 and anti-HIV2). Because no method can absolutely guarantee the absence of infectious agents, handle reagents of human origin and patient samples as potentially capable of transmitting infectious diseases: • Any material, including washings solutions, that comes directly in contact with samples and reagents containing materials of human origin should be considered capable of transmitting infectious diseases. • Wear disposable gloves when handling samples and reagents. • Do not pipette by mouth. • Avoid spilling samples or solutions containing samples. Spills must be rinsed with bleach diluted to 10 %. In the event of a spill with an acid, it must be first neutralized with sodium bicarbonate, and then cleaned with bleach diluted to 10% and dried with adsorbent paper. The material used for cleaning must be discarded in a contaminated residue container. • Patient samples, reagents containing human origin material, as well as contaminated material and products should be discarded after deconta- mination only. • Chemical and biological residues must be handled and disposed off in accordance with Good Laboratories Practices. • All reagents in the kit are exclusively for in vitro diagnostic use. •
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