GB/T 18448.2-2008Laboratory animal - Method for examination of toxoplasma gondii (English PDF)

3.1 Reagents

3.1.1 ELISA antigen

3.1.1.1 Specific Antigen

Toxoplasma gondii (RH strain) tachyzoites inoculated intraperitoneally cleaning stage above mice (KM, ICR, BALB/c, etc. can be), after the 3d ~ 5d, to

Sterile saline lavage of the abdominal cavity of mice vaccinated, mouse peritoneal fluid was collected containing the worm, 3000r/min centrifugal 10min, the precipitate,

PBS three times. Add appropriate amount of distilled water to precipitate five times repeated freezing and thawing, or ultrasonic treatment, 10000r/min centrifugal 30min, take on

Serum. Supernatant Sephadex G200 gel (column diameter × column height. 1.5cm × 60cm), a flow rate of 0.2mL/min. each

Collection tube about 2mL. We collected a total of 60 or more. Each collection tube protein absorbance values were measured at 280nm under. Isolation of pure

After technology, the emergence of two protein peaks; the first peak of the tubes were combined, namely Toxoplasma-specific antigen (also known as Toxoplasma gondii soluble antigen).

3.1.1.2 normal antigen

Sterile saline injection cleaning stage above mice (mice with an antigen preparation of the same species or strain) intraperitoneal, after 3d ~ 5d, to

Sterile saline is injected in murine peritoneal lavage collected mouse peritoneal fluid, 3000r/min centrifugal 10min, the precipitate, PBS washed 3 times.

Add appropriate amount of distilled water to precipitate five times repeated freezing and thawing, or ultrasonic treatment, 10000r/min centrifugal 30min, the supernatant is normal antigen.

3.1.2 antigen sheet

Toxoplasma gondii RH tachyzoites inoculated intraperitoneally cleaning stage above mice (KM, ICR, BALB/c, etc. can be), 3d ~ 5d at the rear

Dead, collect worms, trypsin, to a certain concentration smear, fully dry after cold acetone fixed, -20 °C preservation.

3.1.3 Toxoplasma antigen-sensitized sheep red blood cells

The tannic acid was reacted with a certain concentration of sheep red blood cells, red blood cells formed tanning, then, with Toxoplasma gondii soluble antigen in a suitable strip

Member under tanning sensitized red blood cells, made of Toxoplasma antigen-sensitized sheep red blood cells.

3.1.4

normal sheep red blood cells.

3.1.5 positive control serum

Naturally infected with Toxoplasma corresponding animal antibody positive serum or serum Toxoplasma gondii.

3.1.6 negative control serum

No confirmed toxoplasmosis animal serum.

3.1.7 conjugate

Horseradish peroxidase-labeled anti-mouse, rat, hamster, guinea pigs, rabbits, dogs and monkeys IgG antibody; or horseradish peroxidase-labeled Portugal

Aureus protein A (SPA).

3.1.8 fluorescein conjugates

Isothiocyanate hydrogen acid fluorescein-labeled anti-mouse, rat, hamster, guinea pigs, rabbits, dogs and monkeys IgG antibodies.