Testing Procedure Using Elisa Technique To Find Toxoplasma Gondii Cat Parasite

Toxoplasma gondii is an original parasite isolated in 1908 from a North African rodent, the most common of which is in a cat. Since then, creatures have been found in many species of birds, reptiles and mammals. Here folk often called cat parasites.
Humans are infected with Toxoplasma gondii from various sources of doubt: ingesting contaminated meat, especially lamb and pork, or eating soil-contaminated vegetables contaminated with cat parasites. Congenital toxoplasmosis is a disease with a variety of different manifestations

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When Toxoplasmosis is present, antibody IgM levels are usually detected early in the infection and peak within a month or two after the onset. They are usually still detectable within a few weeks but can survive for up to 2 years.
PRINCIPLES FOR TESTING
The enzyme-linked immunosorbent assay (ELISA) is based on the ability of a biological substance (Antigen) to absorb onto plastic surfaces such as polystyrene (solid phase) When the interwoven antigen on the solid phase is exposed to the patient's serum, the antigen specific antibody, if any, binds to the antigen on the solid phase to form an antigen-antibody complex Next, the wells must be washed off the rest of the sample, and the goat-anti-IgG conjugated antibody conjugated horseradish peroxidase will be added to attach to the antibody antigen complex. After a second wash, a colorant / substrate, tetramethylbenzidine (TMB) is added.



. If antigen specific antibodies are present in the patient's serum, the color mixture becomes green. The green color turns yellow after the solution stops enzyme reaction. The color result indicates antibody concentration, which is read visually or using an ELISA reader.
TESTING PROCESS
Put the desired number into a micro-well bracket. Leave 4 Control (one Negative Control, two Calibrators and one Positive Control) each run A reagent well (RB) should be run on each test Check the software and reader required for the correct control layout. Return the unused micro-well into a sealed bag with desiccant, close tightly and immediately refrigerate.
Dilution test serum, control ratio 1:81 (eg 10 μl + 800 μl) in Serum Diluent Plus. (For manual dilution, apply Serum Diluent to the test tube first and then add patient serums).



. Mix.
For each well, add 100 μl control and appropriate diluted patient serum. Add 100 μl Serum Diluent Plus to the reagent well. Check the software and ask the reader to correctly position the reagent drum well
Incubate the wells at room temperature (21-25 ° C) for 30 minutes.
Suck or pour liquid out of all wells. If using a semi-automatic or automatic washing device add 250-300 μl Wash Buffer diluted into each well. Suck or empty all liquids. Repeat the washing process twice (a total of three washings) for manual or semi-automatic washing or four times (a total of five washing) for automatic equipment. After the final wash, smash the tray upside down on paper towels to remove all liquid from the well.



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Add 100 μl Conjugate to each well, including the reagent blank well. Avoid the formation of foaming as they may yield false results
Incubate the wells at room temperature (21-25 ° C) for 30 minutes.
Repeat washing step.
Add 100 µL Substrate Solution (TMB) to each well, including reagent wells, maintaining the same small velocity between the wells.
Incubate the wells at room temperature (21-25 ° C) for 15 minutes.
Stop the reaction by adding 100 µL Stop Solutio in small Substrate order, including reagent wells. Tap along the tray gently, to mix ingredients of the well. The tray may be left to stand for 1 hour after the addition of the Stop Solution and before reading.
The resulting color should be read on the ELISA micro-tray reader, which is equipped with a 450 nm filter.



If dual wavelength is used, set the reference filter to 600-650 nm. Equipment should be referenced with air.
The reagent well has an absorbance at 450 nm which must be less than 0.150. If the well is reagent ≥ 0.150 the test must be rerun. Refer to the reader on the reagent drum well and then continue to read the entire tray. Remove the trays used after reading
RESULTS OF RESULTS
- ≤ 0.90: negative
- 0.



.91-1.09: Doubt
- ≥1.10: Positive
A positive or negative conclusion on serum does not mean that a person is not infected with parasites or is infected with parasites. The conclusion of whether or not to be infected will be determined by the Doctor who, at the time of examination, the patient's expression combined with all the tests, then comes to the conclusion of an accurate diagnosis of Toxoplasma gondii infection and treatment decisions.
KTV. HUYNH HUONG

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