Elisa Blood Test Technique For Toxoplasma Gondii Disease
Toxoplasmosis is an infectious disease caused by the intracellular toxoplasmosis gondii and is absorbed from meat contaminated with the parasite or by contact with cat litter containing pathogens.
If a pregnant woman acquires Toxoplasmosis, the pathogen will follow the placenta to the fetus, resulting in congenital Toxoplasmosis in the child, causing death or deformity. Children without symptoms may develop abnormally later.
Toxoplasma IgG ELISA is an accurate serum method for the detection of Toxoplasma IgG antibody in clinically determined Toxoplasmosis disease.
Purified Toxoplasma antigen is covered on the surface of the micro-well. The diluted serum is added to a well and IgG Toxoplasma antibody, if present, will bind to the antigen.
Unaffected components will be washed away. HRP conjugate is added, binding to the antibody-antigen complex
The leftover HRP conjugate will be washed off and then added to the TMB reagent solution
Enzymatic catalytic enzyme reaction is stopped at the right time. The color intensity is proportional to the amount of IgG specific antibody in the sample.
Results are read with a micro-well reader accompanied with Calibration and Calibration.
- Put the number of covered wells to use in the holding frame.
- Prepare a 1:40 dilution for test samples, Negatives, Positive controls and calibrators by adding 5 μL of the sample to 200 μL of the sample dilution. Mix thoroughly.
- Draw 100 μL of diluted serum, calibrator and control into the appropriate wells
- Incubate at 37 ℃ for 30 minutes.
- At the end of the incubation step, discard the solution from the wells. Wash and tap the well 5 times with diluted Wash Buffer Solution
- Suck 100 μL of Enzyme Conjugate into each well.
. Mix gently for 10 seconds.
- Incubate at 37 ° C for 30 minutes.
-Remove Enzyme Conjugate from wells. Wash and tap the microwell well 5 times with Wash Buffer Solution
-Send 100 μL TMB reagent into each well. Mix gently for 10 seconds.
-CAP at 37 ℃ for 15 minutes.
-Add 100 μL Reaction Stop Solution (1N HCM) to stop the reaction.
-Smoothly mixing for 30 seconds. Make sure that the solution from blue turns completely yellow.
Note: Make sure there are no air bubbles in each well before reading the results.
- Read the OD value at 450 nm for 15 minutes using the micro-well tray reader.
CALCULATION OF RESULTS
- Calculate the average of the Threshold Calibration values (32 IU / mL) xc.
- Calculate the average of the positive (xp), negative (xn) and sample (xs) values.
- Calculate the Toxoplasma IgG index for each determination by dividing the average value of each sample by the calibrator mean value, xc.
Testing is considered standard when meeting the following issues:
O.D. of the White reagent according to the air from the reader should be lower than 0.250.
If the value O.D. of Calibration Threshold lower than 0.250, the test is not satisfactory and needs to be repeated.
Toxo IgG for Positive and Negative Control should be within the range shown in the Analytical Certificate.
Negative: The Toxoplasma gondii IgG index is lower than 0.90 indicating no Toxoplasma presence (<32 IU / mL). Unknown: Toxoplasma gondii IgG index between 091 - 0.99 is not determined.
. The sample needs to be retested.
Positive: Toxoplasma gondii IgG index 1.00 or higher, or a WHO IU / mL value higher than 32 IU / mL are serum positive. Shows exposure to Toxoplasma.
If suspected, receive a second sample for 8 to 14 days later for simultaneous IgM antibody testing.
The ratio of Toxo G index between the two samples is higher than 15, indicating a significant increase in antibody. Can be considered as a sign of acute Toxoplasma infection
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