Indirect ELISA test - (Apr/14/2015 )

In the indirect ELISA test the enzyme-linked antibody will attach to ?

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The indirect detection method adds a labeled secondary antibody for detection on the basis of direct ELISA and it is the most popular ELISA format. Antigen is passively attached to wells by incubation. After washing, antibodies specific for the antigen are incubated with the antigen. Wells are washed and all bound antibodies are detected by the addition of anti-species antibodies covalently linked to an enzyme. 

Whole Toxoplasma Antibodies Research (WTAR)

Coated plate : reconstitute lyophilized Toxoplasma gondii antigen RH strain from peritoneal exudate from inoculated mice (Diagnostic Pasteur cod. 52721) with 2 ml of dH2O obtaining a concentration of 10⁹ tachyzoites in 2.0 ml. Mix in order to obtain homogeneous suspension and aliquote in tubes with 0.4 ml of suspension (2.0x10⁸ tachyzoites/0.4 ml) that is stored at -20 °C. Mix 0.4 ml of suspension with 0.6 ml of carbonate/bicarbonate buffer (35 mM sodium hydrogen carbonate and 15 mM sodium bicarbonate) at pH 9.6 obtained a tachyzoites concentration of 8x10⁷ per ml. Dispense 100 µl of the final suspension in microplate well having antigen optimum concentration of 0.8x10⁷  tachyzoites per well. Seal plate with adhesive plastic, and incubate overnight at 4 °C. After washing the wells with PBS containing 0.05% Twee-20 for 3 times, invert microplate and shake out the wash solution from the wells and dry. Use immediately or place the coated plate in plastic bag containing a drying agent (silica gel) and store at 4 °C resulting stable for 8 months.

Procedure   

Dispense in wells 100 µl of 1:100 diluted sera (1% BSA in washing solution) and incubate for 1 h at 37 °C. After another wash, dispense 100 µl of 1:1000 diluted peroxidase-conjugated rabbit or goat anti-human IgG and incubate for 1 h at 37 °C. Wash again and dispense 100 µl of TMB substrate.

This is the first immunoenzymatic assay realized by myself in 1993. Use this basis for elaborate a personal ELISA test.