Cryptosporidium Parvum ELISA 试剂盒

Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.

(1) Label a test tube (12x75 mm) or a 1.5 mLplastic vial.
(2) Add 1 mL of assay buffer to each tube or vial.
(3) Add 100 μL of liquid stool sample to the above tube.(4) With solid stool sample, take an equivalent amount (about 50 100 mg) with a spatula or a disposable inoculation loop. Suspend the solid stool sample with 1 mL patient sample diluent and mix well on a vortex mixer.(5) Centrifuge the diluted fecal sample at 3000 rpm (1500 g) for 10 15 minutes. The supernatant can be directly used in the assay. As an alternative to centrifuging, let the diluted samples sit and sediment for 15 minutes and take the clear supernatant for testing.Note: If the test procedure is performed on an automated ELISA system, the supernatant must be particle-free by centrifuging the sample.

(1) Place a sufficient number of Anti-Cryptosporidium antibody coated microwell strips in a frame to run Cryptosporidium controls and unknown samples in duplicate.
(2) Test Configuration
(3) Add 100 μL of controls (Cat. 30470-30471) and diluted patient stool samples into each designated microwell.
(4) Cover the plate with a plate sealer and also with aluminum foil to avoid exposure to light.
(5) Incubate plate at room temperature for 1 hour.
(6) Prepare working Anti-Cryptosporidium tracer antibody working solution by 1:21 fold dilution of the Anti-Cryptosporidium Tracer Antibody with the Tracer Antibody Diluent. For each strip, it is required to mix 1 mL of Tracer Antibody Diluent with 50 μL of Tracer Antibody in a clean test tube.
(7) Remove the plate sealer. Decant the contents of each well. Wash each well 5 times by dispensing 350 μL to 400 μL of diluted wash buffer into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(8) Add 100 μL of above diluted tracer antibody working solution to each of the wells.
(9) Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
(10) Incubate plate at room temperature for 40 minutes.
(11) Remove the plate sealer. Decant the contents of each well. Wash each well 5 times by dispensing 350 μL to 400 μL of diluted wash buffer into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(12) Add 100 μL of ELISA HRP Substrate into each of the wells.
(13) Cover the plate with aluminum foil to avoid exposure to light.
(14) Incubate plate at room temperature for 15 minutes
(15) Remove the aluminum foil. Add 100 μL of ELISA Stop Solution into each of the wells. Mix gently.
(16) Read the absorbance at 450 nm within 10 minutes in a microplate reader.

1. Calculate the average absorbance for each pair of duplicate test results
   2. Calculate the cut-off:The positive cut-off and the negative cut-off are established by using following formula.Positive Cut-Off = 1.1 x (mean extinction of negative control + 0.10)Negative Cut-Off = 0.9 x (mean extinction of negative control + 0.10)3. Interpret test result? Positive: patient sample extinction is greater than the Positive Cut-Off.? Negative: patient sample extinction is less than the Negative Cut-Off.? Equivocal: patient sample extinction is between the Positive Cut-Off and the Negative Cut-Off.4. Assay quality control? Positive control must show an average OD reading greater than 0.500.? Negative control should show an average OD reading less than 0.200.