大鼠不对称二甲基精氨酸(ADMA)ELISA Kit说明书

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Rat Asymmetrical

Dimethylarginine(ADMA)

ELISA Kit

(96T)

This immunoassay kit allows for the in vitro quantitative determination of rat

ADMA concentrations in serum, 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

a goat-anti-rabbit antibody. Standards or samples are then added

to the appropriate microtiter plate wells with a HRP-conjugated

ADMA and antibody preparation specific for ADMA and

incubated. Then substrate solutions are added to each well. The

enzyme-substrate reaction is terminated by the addition of a

sulphuric acid solution and the color change is measured

spectrophotometrically at a wavelength of 450 nm ± 2 nm. The

concentration of ADMA in the samples is then determined by

comparing the O.D. of the samples to the standard curve.

DETECTION RANGE

1.2 ng/ml-300 ng/ml. The standard curve concentrations used for

the ELISA’s were 300 ng/ml, 75 ng/ml, 18.75 ng/ml, 4.69 ng/ml,

1.2 ng/ml.

SPECIFICITY

This assay recognizes recombinant and natural rat ADMA. No

significant cross-reactivity or interference was observed.

SENSITIVITY

The minimum detectable dose of rat ADMA is typically less than

0.75 ng/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

Standards (S1-S5) 5 x 0.5 ml

HRP-conjugate 1 x 6 ml

Antibody 1 x 6 ml

Wash Buffer

1 x 15 ml

(20×concentrate)

Substrate A 1 x 7 ml

Substrate B 1 x 7 ml

Stop Solution 1 x 7 ml

Standard S1 S 2 S3 S4 S5

Concentration

(ng/ml) 1.2 4.69 18.75 75 300

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

1. Bring all reagents and plate to room temperature for at least

30 minutes before use. Unused wells need store at 2-8℃and

avoid sunlight.

2. Wash Buffer If crystals have formed in the concentrate,

warm to room temperature and mix gently until the crystals

have completely dissolved. Dilute 15 ml of Wash Buffer

Concentrate into deionized or distilled water to prepare 300 ml

of Wash Buffer.

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.

Note: Grossly hemolyzed samples are not suitable for use in this assay.

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. Set a Blank well without any solution. Add 50μl of Standard or

Sample per well. Standard need test in duplicate.

2. Add 50μl of HRP-conjugate to each well (not to Blank well),

then 50μl Antibody to each well. Mix well and then incubate

for 1 hour at 37°C.

3. Fill each well with Wash Buffer (about 200μl), stay for 10

seconds and Spinning. Repeat the process for a total of three

washes. Complete removal of liquid at each step is essential

to good performance. After the last wash, remove any

remaining Wash Buffer by aspirating or decanting. Invert the

plate and blot it against clean paper towels.

4. Add 50μl of Substrate A and 50μl Substrate B to each well,

mix well. Incubate for 15 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. If color change does

not appear uniform, gently tap the plate to ensure thorough

mixing.

6. Determine the optical density of each well within 10 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, Blank, and

sample and subtract the optical density of Blank. Create a

standard curve by reducing the data using computer software

capable of generating a four parameter logistic (4-PL) curve-fit.

As an alternative, construct a standard curve by plotting the

mean absorbance for each standard on the y-axis against the

concentration on the x-axis and draw a best fit curve through the

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

log of the ADMA concentrations versus the log of the O.D. 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

Quantikine Immunoassay, the possibility of interference

cannot be excluded.

TECHNICAL HINTS

Centrifuge vials before opening to collect contents.

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. Sealers can not be

reused.

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.

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