鳥褪黑素（MT）ELISA試劑盒說明書

【資料簡介】

Birds MT ELISA Kit

Catalogue Number: BG37883

(96Tests)

Store all reagents at 2-8°C

Collect sample: serum or blood plasma

Assay range ： 0.8ng/L -280ng/L

FOR LABORATORY RESEARCH USE ONLY.

NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!

PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE

BEGINNING!

INTENDED USE

This MT ELISA kit is intended Laboratory for Research use only and is

not for use in diagnostic or therapeutic procedures.The Stop Solution changes

the color from blue to yellow and the intensity of the color is measured at 450

nm using a spectrophotometer. In order to measure the concentration of MT in

the sample, this MT ELISA Kit includes a set of calibration standards. The

calibration standards are assayed at the same time as the samples and allow the

operator to produce a standard curve of Optical Density versus MT

concentration. The concentration of MT in the samples is then determined by

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

PRINCIPLE OF THE ASSAY

The kit assay MT level in the sample，use Purified antibody to coat microtiter

plate wells, make solid-phase antibody, Samples which including standards of

known concentrations and unknowns are pipetted into coated microtiter wells,

after Incubating ,add Biotinylated anti-IgG,and Combined Streptavidin-HRP,

become antibody – antigen - enzyme- antibody complex, after washing

Compley, Add TMB substrate solution, TMB Chromogen Solution Becomes

blue color At HRP enzyme-catalyzed, And at the effect of acid the color finally

become yellow, The intensity of this coloured product is directly proportional to

the concentration of MT present in the samples. measure the optical densit (OD)

at 450 nm with microtiter plate reader, calculate MT concentration by standard

curve.

REAGENTS PROVIDED

All reagents provided are stored at 2-8° C. Refer to the expiration date on

the label.

1. MICROTITER PLATE 96 wells

2. Biotinylated anti-IgG 6.0 mL 1 tube

3. STANDARD(120ng/L) 0.5ml 1 tube

4. STREPTAVIDIN-HRP 6.0 mL 1 tube

5. STANDARD DILUENT 1.5ml 1 tube

6. Chromogen Solution A 6.0 mL 1 vial

7. Chromogen Solution B 6.0 mL 1 vial

8. STOP SOLUTION 6.0 mL 1 vial

9. WASH SOLUTION x30 20 mL 1 vial

10. Instruction 1

SAMPLE COLLECTION AND STORAGE

Serum- Use a serum separator tube(SST) and allow samples to clot for

30minutes before centrifugation for 15minutes at approximay 1000 x

g.Remove serum and assay immediay or aliquot and store samples at -20 °C

or -80°C.

Plasma - Collect plasma using EDTA or heparin as an

anticoagulant.Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within

30minutes of collection.Store samples at -20°C or -80°C.Avoid repeated

freeze-thaw cycles.

Cell culture fluid and other biological fluids - Remove particulates by

centrifugation and assay immediay or aliquot and store samples at -20°C or

-80°C.Avoid repeated freeze-thaw cycles.

NOTE: Serum, plasma, and cell culture fluid samples to be used within 7

days may be stored at 2-8°C, otherwise samples must stored at -20°C(≤2months)

or -80°C(≤6months) to avoid loss of bioactivity and contamination. Avoid

freeze-thaw cycles .When performing the assay slowly bring samples to room

temperature.

DO NOT USE HEAT-TREATED SPECIMENS.

MATERIALS REQUIRED BUT NOT SUPPLIED

1. Microplate reader capable of measuring absorbance at 450 nm.

2. Precision pipettes to deliver 2 ml to 1 ml volumes. .

3. Adjustable 10ml -100ml pipettes for reagent preparation.

4. 100 ml and 1 liter graduated cylinders.

5. Calibrated adjustable precision pipettes, preferably with disposable plastic

tips. (A manifold multi-channel pipette is desirable for large assays.)

6. Absorbent paper.

7. 37°C incubator.

8. Distilled or deionized water.

9. Data analysis and graphing software..

10. Tubes to prepare standard or sample dilutions.

PRECAUTIONS

1. Do not substitute reagents from one kit lot to another. Standard, conjugate

and microtiter plates are matched for optimal performance. Use only the

reagents supplied by manufacturer.

2. Allow kit reagents and materials to reach room temperature (20-25°C) before

use. Do not use water baths to thaw samples or reagents.

3. Do not use kit components beyond their expiration date.

4. Use only deionized or distilled water to dilute reagents.

5. Do not remove microtiter plate from the storage bag until needed. Unused

strips should be stored at 2-8°C in their pouch with the desiccant provided.

6. Use fresh disposable pipette tips for each transfer to avoid contamination.

7. Do not mix acid and sodium hypochlorite solutions.

8. Serum and plasma should be handled as potentially hazardous and capable of

transmitting disease. Disposable gloves must be worn during the assay

procedure, since no known test method can offer complete assurance that

products derived from human blood will not transmit infectious agents.

Therefore, all blood derivatives should be considered potentially infectious

and good laboratory practices should be followed.

9. All samples should be disposed of in a manner that will inactivate viruses.

10. Solid Waste: Autoclave 60 min. at 121°C.

11. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%.

The waste should be allowed to stand for a minimum of 30 minutes to

inactivate the viruses before disposal.

12. Substrate Solution is easily contaminated. If bluish prior to use, do not use.

13. Chromogen Solution B contains 20% acetone, keep this reagent away from

sources of heat or flame.

14. Remove all kit reagents from refrigerator and allow them to reach room

temperature ( 20-25°C).

REAGENT PREPARATION

Standard -The Kit provides a stock standard（120ng/L）. Allow the standard to

sit for a minimum of 5 minutes with gentle mixing prior to making

dilutions.Pipette 150μL of Standard Dilution into each tube.

(total 5 tubes) Use the 150μL of stock solution to produce a 2-fold dilution

series (including 60ng/L,30ng/L,15ng/L,7.5ng/L , 3.75ng/L). Mix each tube

thoroughly before the next transfer.

Wash Buffer - If crystals have formed in the concentrate, warm to room

temperature and mix gently until the crystals have compley dissolved. To

prepare enough Wash Buffer for one plate, add 20mL Wash Buffer Concentrate

into deionized or distilled water to prepare 600mL of Wash Buffer.

ASSAY PROCEDURE

Prepare all Standards before starting assay procedure (see Preparation

Reagents). It is recommended that all Standards and Samples be added in

duplicate to the Microtiter Plate.

1. Determine the number of microwell strips required to test the desired

number of samples, Each sample, standard and blank should be assayed in

duplicate.

2. add sample: Set blank wells separay (blank comparison wells don’t add

sample and ELISA reagent, other each step operation is same). Add 50 μL of

Standards or Samples to the appropriate well of the antibody pre-coated

Microtiter Plate, and Gently mix. Incubate for 45 min at 37℃

3. Configurate liquid: 30 times of wash solution diluted 30 times with distilled

water and reserve.

4. washing: remove Liquid, dry by swing, add washing buffer to every well,

still for 30 second then remove, repeat 4 times.

5. add Biotinylated anti-IgG: Add diluted Biotinylated anti-IgG 50ul to all

wells, Incubate for 30 min at 37℃

6. washing: Operation with 4.

7. add streptavidin-HRP: Add streptavidin-HRP 50ul to all wells, Gently mix

Incubate for 15 min at 37℃

8. washing：Operation with 4.

9. color: Add Chromogen Solution A 50ul and Chromogen Solution B to

each well,Incubate for 15 min at 37℃

10. Stop the reaction: Add Stop Solution50μl to each well, Stop the

reaction(the blue color change to yellow color Immediay).

11. assay: take blank well as zero , measure the optical densit (OD) at 450 nm

after Adding Stop Solution and within 15min.

TYPICAL DATA

CALCULATION OF RESULTS

Average the duplicate readings for each

standard,control, and sample and subtract

the average zero standard optical density.

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. 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.