Elisa Sandwich Toxoplasma Gondii Test Kit Detect IgM Antibody 37°C Humid Chamber

96wells Toxoplasma gondii (Toxo) IgM ELISA test kit, high accuracy, Elisa Sandwich method
 
 
Product Name:  Toxoplasma gondii (Toxo) IgM ELISA test
 
 
Intended Use:
 
Toxoplasma gondii (Toxo) IgM Enzyme-Linked -Immunosorbent Assay (ELISA) is intended for the presumptive qualitative detection of IgM antibody to Toxoplasma gondii in human serum for the presumptive diagnosis of acute, recent, or reactive Toxoplasma gondii infection. Testing of patient sera must be performed in conjunction with an anti-Toxoplasma gondii IgG antibody assay. This product is not FDA cleared (approved) for use in testing (i.e., screening) blood or plasma donors. The assay’s performance has not been established for screening of prenatal women or newborns. High complexity test.
 
Summary:
 
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 of T. 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, chemoprophylaxy in immunocompromised population.
 
Principle of Test:
 
Enzyme-Linked Immunosorbent Assays (ELISA) rely on the ability of biological materials (e.g., antigens) to adsorb to plastic surfaces such as polystyrene (solid phase). When antigens bound to the solid phase are brought into contact with a patient's serum, antigen specific antibody, if present, will bind to the antigen on the solid phase forming antigen-antibody complexes. Excess antibody is removed by washing. This is followed by the addition of goat anti -human IgM globulin conjugated with horseradish peroxidase which will bind to the antibody-antigen complexes. The excess conjugate is removed by washing, followed by the addition of Chromogen/Substrate tetramethylbenzidine (TMB). If specific antibody to the antigen is present in the patient's serum, a blue color develops. When the enzymatic reaction is stopped with 1N H2SO4, The contents of the wells turn yellow. The color, which is indicative of the concentration of antibody in the serum, can be read on a suitable spectrophotometer or ELISA microwell plate reader.
 
Kit Components
 
 

 
 
 
 
ASSAY PROCEDURE
 

1.  Allow the vacuum-closed aluminium bag with strips to reach room temperature. Withdraw an adequate number of strips and put the unused strips into the provided bag and seal it carefully with the desiccant kept inside.
2.  Pipette 100 µL of Sample diluent, Controls and serum samples to the wells according to the pipetting scheme in Plate Layout: fill the first well with Dilution buffer (DIL) to determine the reaction background. Fill the next two wells with Calibrator 1 (CAL 1). The next wells fill with Calibrator 2-4 (CAL 2-4) and Negative control (CONTROL 1). The remaining wells fill with diluted serum samples (S1...). It is satisfactory to apply one serum into one well (S1, S2, S3...). However, if you want to minimize a laboratory error, apply controls and samples as doublets. Cover the strips with the Cover membrane or cover.
3.  Incubate 60 minutes (+/- 5 min.) at 37°C in a humid chamber.
4.  Aspirate the liquid from wells into a collecting bottle containing appropriate disinfectant (see Safety Precautions). Wash and aspirate the wells four times with 300 µl/well of Wash buffer. Avoid cross-contamination between wells! If some liquid remains in the wells, invert the plate and tap it on an adsorbent paper to remove the last remaining drops.
5.  Add 100 µL Peroxidase conjugate r.t.u. into each well. Incubate 30 minutes (+/- 2 min) at 37°C in a humid chamber.
6.  Aspirate and wash four times with 300 µl/well of Wash buffer.
7.   Dispense 100 µl of TMB into each well. Incubate 15 minutes (+/- 30 seconds) at room temperature. The time measurement must be started at the beginning of TMB dispensing. Cover the strips with opaque cover or keep them in the dark during the incubation with TMB.
8.  Stop the reaction by adding 100 µL of Stop solution. Use the same pipetting rhythm as with the TMB to ensure the same reaction time in all wells. Tap gently the microplate for a few times to ensure complete mixing of the reagents.
9.  Read the absorbance at 450 nm with a microplate reader within 10 minutes. It is recommended to use a reference reading at 630 (620) nm.