Porcine apolipoprotein E (Apo-E)ELISA kit

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

Reagent Quantity

Assay plate 1

Standard 2

Sample Diluent 2 x 20 ml

HRP- conjugate Diluent 1 x 10 ml

HRP- conjugate 1 x 60 μl

Wash Buffer

1 x 20 ml

(25×concentrate)

TMB Substrate 1 x 10 ml

Stop Solution 1 x 10 ml

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

Microplate reader capable of measuring absorbance at 450

nm, with the correction wavelength set at 540 nm or 570 nm.

Pipettes and pipette tips.

Deionized or distilled water.

Squirt bottle, manifold dispenser, or automated microplate

washer.

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 HRPconjugate

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.

3. Aspirate each well and wash, repeating the process five

times for a total of five washes. Wash: Fill each well with

Wash Buffer (200μl) and let it stand for 2 minutes, then

remove the liquid by flicking the plate over a sink. The

remaining drops are removed by patting the plate on a paper

towel. Complete removal of liquid at each step is essential to

good performance.

4. Add 90μl of TMB Substrate to each well. Incubate for 20

minutes at 37°C. Keeping the plate away from drafts and

other temperature fluctuations in the dark.

5. Add 50μl of Stop Solution to each well when the last four

wells containing the lowest concentration of standards

develop obvious blue color. If color change does not appear

uniform, gently tap the plate to ensure thorough mixing.

6. Determine the optical density of each well within 15 minutes,

using a microplate reader set to 450 nm.

CALCULATION OF RESULTS

Using the professional soft "Curve Exert 1.3" to make a standard curve is

recommended, which can be downloaded from our web.

Average the duplicate readings for each standard, control, and

sample and divide the average zero standard optical density.

Create a standard curve by reducing the data using computer

software. As an alternative, construct a standard curve by

plotting the absorbance ratio for each standard on the x-axis

against the concentration on the y-axis and draw a best fit curve

through the points on the graph. The data may be linearized by

plotting the Apo-E concentrations versus the ratio and the best fit

line can be determined by regression analysis. This procedure

will produce an adequate but less precise fit of the data. If

samples have been diluted, the concentration read from the

standard curve must be multiplied by the dilution factor.

LIMITATIONS OF THE PROCEDURE

The kit should not be used beyond the expiration date on the

kit label.

Do not mix or substitute reagents with those from other lots or

sources.

If samples generate values higher than the highest standard,

dilute the samples with the appropriate Diluent and repeat the

assay.

Any variation in operator, pipetting technique, washing

technique, incubation time or temperature, and kit age can

cause variation in binding.

This assay is designed to eliminate interference by soluble

receptors, binding proteins, and other factors present in

biological samples. Until all factors have been tested in the

Immunoassay, the possibility of interference cannot be

excluded.

TECHNICAL HINTS

Centrifuge vials before opening to collect contents.

When mixing or reconstituting protein solutions, always avoid

foaming.

To avoid cross-contamination, change pipette tips between

additions of each standard level, between sample additions,

and between reagent additions. Also, use separate reservoirs

for each reagent.

When using an automated plate washer, adding a 30 second

soak period following the addition of wash buffer, and/or

rotating the plate 180 degrees between wash steps may

improve assay precision.

To ensure accurate results, proper adhesion of plate sealers

during incubation steps is necessary.

Substrate Solution should remain colorless or light blue until

added to the plate. Keep Substrate Solution protected from

light. Substrate Solution should change from colorless or light

blue to gradations of blue.

Stop Solution should be added to the plate in the same order

as the Substrate Solution. The color developed in the wells

will turn from blue to yellow upon addition of the Stop Solution.

Wells that are green in color indicate that the Stop Solution

has not mixed thoroughly with the Substrate Solution.