Purpose

Our Equine Babesiosis Antibody ELISA Kit is to for the qualitative determination of Babesiosis-Ab in Equine serum, plasma, culture media or any biological fluid.

Principle

The ELISA is based on the the qualitative enzyme immunoassay technique. The Microplate provided in this kit has been pre-coated with an antigen specific to Babesiosis-Ab, make it to solid-phase antigen. Samples are added to the Microplate wells and combined to the specific antigen. Then a Horseradish Peroxidase (HRP)-conjugated antigen specific for Babesiosis-Ab is added to each Microplate well and incubated, so the antigen-antibody-Enzyme labeled antigen complex is formed. Following a wash to remove any unbound reagent, then the TMB substrate solution is added to each well. Only those wells that contain Babesiosis-Ab and HRP conjugated Babesiosis antigen will appear blue in color and then turn yellow after the addition of the stop solution. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The qualitative determination of Babesiosis-Ab is determined by comparing with the CUT OFF value.

Sample Collection and Storages

Serum – Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

Plasma – Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture super nates and other biological fluids – Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Materials Required but not Supplied

• Standard microplate reader(450nm)
• Precision pipettes and Disposable pipette tips
• 37 ℃ incubator

Materials Supplied

Reagent Preparation

20×wash solution: Dilute with Distilled or deionized water 1:20.

Assay Procedure

• Prepare all reagen t s before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Micro Elisa  Strip plate
• Separately add Positive control and Negative control 50μl to the Positive and Negative well; Add testing sample 10μl then add Sample Diluent 40μl to testing sample well.
• Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
• Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or auto washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
• Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
• Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
• Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

Determine the Result

• Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤0.15.
• Calculate Critical (CUT OFF): Critical= the average of Negative control well + 0.15.
• Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.
• Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.