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 Porcine apolipoprotein E (Apo-E) ELISA kit

Catalog No. CSB-E17889p

(96T)

This immunoassay kit allows for the in vitro quantitative determination of porcine Apo-E concentrations in serum and plasma.

Expiration date six months from the date of manufacture

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

PRINCIPLE OF THE ASSAY

The microtiter plate provided in this kit has been pre-coated with Apo-E. Standards or samples are then added to the appropriate microtiter plate wells with Horseradish Peroxidase (HRP) -conjugated antibody preparation specific for Apo-E, mix well and incubated. The more the amount of Apo-E in samples, the less HRP-conjugated antibody preparation specific for Apo-E bound by pre-coated Apo-E. Then a TMB (3,3’,5,5’ tetramethyl-benzidine) substrate solution is added to each well. And the color develops in opposite to the amount of Apo-E in the sample. The color development is stopped and the intensity of the color is measured.

DETECTION RANGE

3.12 ug/ml-200 ug/ml. The standard curve concentrations used for the ELISA’s were 200 ug/ml, 100 ug/ml, 50 ug/ml, 25 ug/ml,

12.5 ug/ml, 6.25 ug/ml, 3.12 ug/ml.

SPECIFICITY

This assay recognizes porcine Apo-E. No significant cross-reactivity or interference was observed.

SENSITIVITY

The minimum detectable dose of porcine Apo-E is typically less than 0.8 ug/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

MATERIALS PROVIDED STORAGE

1.    Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit, provided it is stored as prescribed above. Refer to the package label for the expiration date.

2.    Opened test plate should be stored at 2-8°C in the aluminum foil bag with desiccants to minimize exposure to damp air. The kits will remain stable until the expiring date shown, provided it is stored as prescribed above.

3.    A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

REAGENT PREPARATION

Bring all reagents to room temperature before use.

1         Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer.

2         Standard Centrifuge the standard vial at 6000-10000rpm for 30s. Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 200 ug/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (200 ug/ml). The Sample Diluent serves as the zero standard (0 ug/ml). Prepare fresh for each assay. Use within 4 hours and discard after use.

3         HRP-conjugate Centrifuge the vial before opening. Dilute to the working concentration using HRP-conjugate Diluent (1:100), respectively.

Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

OTHER SUPPLIES REQUIRED

1          Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.

2          Pipettes and pipette tips.

3          Deionized or distilled water.

4          Squirt bottle, manifold dispenser, or automated microplate washer.

5          An incubator which can provide stable incubation conditions up to 37°C±0.5°C.

SAMPLE COLLECTION AND STORAGE

Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

ASSAY PROCEDURE

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well.

1         Add 50µl of Standard or Sample per well. Add 50µl HRP-conjugate working solution to each well immediately. Mix well with the pipette or shake the plate gently for 60 seconds.

2         Then incubate for 30 minutes at 37°C.

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