Enzyme Immunoassays for the Diagnostics of Toxoplasmosis
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INFECTIOUS SEROLOGY – PARASITOLOGY – Toxoplasma gondii FOLLOW US FOLLOW US Toxoplasma gondii Enzyme immunoassays for the diagnostics of toxoplasmosis ELISA and IMMUNOBLOT kits are optimized and validated for detection of IgA, IgE, IgG and IgM antibodies in human serum and plasma BIOVENDOR.GROUP INFECTIOUS SEROLOGY – PARASITOLOGY – Toxoplasma gondii Introduction Diagnosis of infection Toxoplasmosis is a widespread parasitic disease cau- Diagnosis of the disease is based on epidemiological sed by protozoan Toxoplasma gondii – a parasite with anamnesis, clinical manifestation and laboratory tests. a complicated life cycle consisting of several morpho- Direct detection of the parasite is not available for rou- logically different stadia. Primary hosts are members of tine diagnostics. Serology is the most important tool for the feline family. Humans and most warm-blooded ani- laboratory diagnostics of toxoplasmosis. mals can be infected by either primarily infected food – Screening – determination of total antibodies by (insufficiently heat-treated meat) or by ingestion of oo- complement fixation test (CFT) cysts (secondary contaminated food or contaminated – Determination of specific IgA, IgE, IgM, IgG fingers, objects, etc.). antibodies and IgG avidity by ELISA and confirmation Acquired toxoplasmosis in immunocompetent indi- of results by Immunoblot viduals is usually asymptomatic or can manifest itself with flu-like symptoms (subfebrility, fatigue, lymphade- nopathy, muscle aches) and has no lasting ill effects. Severe life-threatening infections (encephalitis, hepati- tis, chorioretinitis, myocarditis, generalized form of the disease) may develop in immunocompromised patients usually because of a reactivation of a latent infection. Congenital toxoplasmosis is caused by transmission of infection from mother to foetus and it might result in severe damages of the foetus (brain calcification, hydrocepha- lus, vision disorders, mental affections), still birth or abortion. Antibody response IgG IgG Avidity Antibody titer KFR IgM IgE IgA 012345678910 11 12 13 Months ELISA IgM antibodies: Clinical application – Highly sensitive marker of acute infection – Diagnostics and differentiation of toxoplasmosis – Disadvantage: long-time persistence after stage by detection of IgA, IgE, IgG a IgM specific the beginning of infection (more than 1 year) antibodies in human serum or plasma and determination of IgG avidity IgA antibodies: – Sensitive and specific marker of acute infection Summary protocol – Persistence for 6-9 months from the beginning of infection Step Test steps Dilute samples IgE antibodies: 1. – serum/plasma 1:101 (10 μl + 1 ml) – Highly specific marker of acute infection Pipette Controls and diluted samples – Persistence up to 6 months from the beginning 2. 100 μl of infection – Blank = empty well IgG antibodies: 3. Incubate 60 min. at 37 °C – Anamnestic antibodies 4. Aspirate and wash the wells 5 times – Persist for years and ensure protection against new infection Add 100 μl Conjugate or Tracer 5. – IgG avidity reflects stage of infection – Blank = empty well 6. Incubate 60 min. at 37 °C Test Principle Sandwich ELISA Capture ELISA 7. Aspirate and wash the wells 5 times Add 100 μl Substrate (TMB-Complete) 8. – Including blank HRP HRP Anti-Hu IgG Tracer 9. Incubate 30 min. at 37 °C AG Add 100 μl Stopping solution IgG 10. – Including blank IgMI Microtiter AG 11. Read colour intensity at 450 nm plate Anti-HuAAn IgM Antigens Purified and inactivated native Toxoplasma gondii antigen (RH strain). INFECTIOUS SEROLOGY – PARASITOLOGY – Toxoplasma gondii User comfort Advantages – Ready-to-use components (except Tracer) – Identical assay procedure – Colour-coded components – High diagnostic specificity and sensitivity – Interchangeable components – High reproducibility – Breakable colour-coded microplate strips – High dynamics of antibody response – CUT-OFF included – Avidity test available (EIA Toxoplasma IgG) – Semiquantitative evaluation of results – Ready for automation (Index of Positivity) – Customer support – Calibrators (EIA Toxoplasma IgG) – Independent verification (CKS) – Quantitative evaluation of IgG antibodies (IU/ml) – Short total assay time 1.5 hour Test characteristics Diagnostic Diagnostic ELISA Sensitivity Specificity EIA Toxoplasma IgA 96.9% 99.0% EIA Toxoplasma IgE 96.9% 99.0% EIA Toxoplasma IgG 98.9% 99.2% EIA Toxoplasma IgM 96.4% 97.9% Avidity Determination of avidity of IgG antibodies Antibody avidity reflects binding strength of the bin- fection. During secondary infection or reactivation the ding between antigen and antibody. Antibodies with memory B-Cells immediately produces IgG antibody low avidity are created firstly in the process of primary with high avidity. infection . During the progress of infection the immune Determination of avidity of IgG antibody allows more response matures and the avidity increases. Highly avid accurate indentification of the infection stage. antibodies are being observed in the latent phase of in- 100 6 90 9 80 8 70 10 11 5 6 60 3 6 ) 6 3 5 50 (% IA 4 3 0 40 2 0 30 0 0 20 0 0 10 0 0 0 ABCDEFGH Samples of patients A-HTime interval in month (2-11) from the first testing (0) 1. sample 2. sample 3. sample 4. sample Clinical data Seroprevalence and dynamics of IgG antibody classes at blood donors (CZ) Blood donors (n = 483) Positive (n = 119) Negative (seroprevalence) (n = 364) 25% 75% 6 5 4 3 tibody titer (IP) An 2 Borderline 1 0 Samples Types of kits SmartEIA kits are designed for automated processing using the Agility® analyser. EIA SmartEIA INFECTIOUS SEROLOGY – PARASITOLOGY – Toxoplasma gondii IMMUNOBLOT Test principle Summary protocol Recombinant antigens are transferred to a nitrocellulo- Step Test steps se membrane using a micro-dispensing method. 1. Pipette Universal solution 2 ml P Reaction of substrate and enzyme resulting in coloured insoluble Strips soaking 10 min. at room E product 2. temperature Enzyme-conjugated secondary antibody – Shaker binding to primary antibody Specific primary antibody binding to protein 3. Aspirate Blocking Specific antigen protein AG Dilute samples agent 4. – serum/plasma 1:51 (30 μl + 1,5 ml) Membrane Pipette Controls and diluted samples 5. Antigens 1.5 ml SAG1 p30; a highly immunogenic Incubate 60 min. at room temperature 6. and antigenic surface antigen invol- – Shaker ved in the activation of strong immu- ne response of tachyzoites during Aspirate samples and wash strips with the acute phase of toxoplasmosis. 1.5 ml of Universal solution 3-times for Control line 7. IgA 5 min. IgG A good serological marker for anti- IgM – Shaker Start bodies against T. gondii in the acute SAG1, p30 and chronic phases of infection; high 8. Pipette Conjugate 1.5 ml MIC3 titres of IgG, IgM and IgA. GRA7 MIC3 p90; strong adhesin and is Incubate 60 min. at room temperature GRA1 9. one of the major vaccine candidates. – Shaker GRA8 It is a dimeric 90 kDa micronemal SAG2 cysteine-rich protein. It is expressed Aspirate Conjugate and wash strips in tachyzoite, bradyzoite and spo- with 1.5 ml of Universal solution 10. rozoite and has excellent immune 3-times for 5 min. properties. – Shaker GRA1 p24; a highly immunogenic Pipette Substrate solution (BCIP/NBT) 11. protein whose reactivity correlates 1.5 ml with the chronic phase of the disease to a large extent. Incubate 10 min. at room temperature 12. GRA7 p29; expressed in all infectious forms of toxo- – Shaker plasma. It elicits a strong antibody response in the acute Aspirate Substrate solution and wash phase of infection. It is considered an important dia- strips with 2 ml of distilled water gnostic tool, suitable for the chronic phase of infection. 13. 2-times for 5 min. GRA8 p35; highly immunogenic protein, more suitable – Shaker for the diagnosis of acute toxoplasmosis than chronic infections 14. Sticking and evaluation of strips SAG2 p22; a major surface protein known as a binding ligand, characterised by good antigenicity and immuno- genicity. Effective for the detection of IgG antibodies in patients with acute toxoplasmosis. Clinical application Test characteristics Diagnostic Diagnostic – Detailed determination for the presence of Immunoblot Sensitivity Specificity anti-Toxoplasma gondii specific antibodies BLOT-LINE – Diagnostic of Toxoplasma gondii infection, confirma- 95.2% 99.1% Toxoplasma IgA tive test for ELISA – Useful method to trace immune profile of mothers’ BLOT-LINE 95.6% 98.0% and new-borns’ sera – determination of congenital Toxoplasma IgG toxoplasmosis BLOT-LINE 95.6% 98.0% Toxoplasma IgM User comfort – Ready-to-use components – Colour-coded strips Advantages – Positive and Negative controls – Easy interpretation and reproducibility of results – CUT-OFF control is present on the strip – High diagnostic specificity and sensitivity – Interchangeable components – Compatibility with all commercial immunoblot – Easy assay procedure processing systems – Customer support Interpretation results IgE IgA IgM IgG Interpretation Acute infection – – + – (occurs rarely) + + + low + Acute infection + + + + Acute infection Acute or post-acute – + + + infection – low + + + Post-acute infection Post-acute or latent – – + + infection – – – + Latent infection Specific antibodies – – – – were not proven INFECTIOUS SEROLOGY – PARASITOLOGY – Toxoplasma gondii Ordering information ELISA Cat. No. Product Units FOLLOW US TgA096 EIA Toxoplasma IgA 96 wells TgE096 EIA Toxoplasma IgE 96 wells TgG096 EIA Toxoplasma IgG 96 wells TgM096 EIA Toxoplasma IgM 96 wells SK-TgA096 SmartEIA Toxoplasma IgA 96 wells SK-TgE096