الوصف
INTRODUCTION
� Operation In struction of Kit for Quantitative Determination by Enzyme Linked Immunosorbent Assay
� Detection of species: Human
� Detection medium:Serum&Plasma&Tissue homogenate&Cell culture medium &Tissue
PRINCIPLE OF TEST
The kit assay Human EPCA level in the sample ， use Purified Human EPCA antibody to coat microtiter plate wells, make solid-phase antibody, then add EPCA to wells, Combined antibody which With HRP labeled EPCA, become antibody -antigen – enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of EPCA in the samples is then determined by comparing the O.D. of the samples to the standard curve.
COMPOSITION OF THE KIT
Microelisa Stripplate 12well × 8strips
Standar d : 450 p g/ml 0.5ml One bottle
Standard diluent 1.5 ml One bottle
HRP- Conjugate Reagent 6mL O ne bottle
Sample diluent 6mL One bottle
Chromogen Solution A 6mL One bottle
Chromogen Solution B 6mL One bottle
Stop Solution 6mL One bottle
Wash Solution 20ml × 30 fold One bottle
Microelisa Stripplate 1 1
Sealed bags 1 1
User manual 1 1
Closure plate membrane 2 2
STORAGE CONDITIONS
� The kit shall be stored at [2-8 ℃ ] while the coated microwell plate shall be stored at a dry place.
� The reagents shall be kept stable in the period of validity, t he Substrate shall be colorless and changed in time in case of development or blueing.
WASHING METHOD
� Manually Manually Manually Manually washing washing washing washing method: method: method: method: shake away the remain liquid in the enzyme plates; place some bibulous papers on the test-bed, and flap the plates on the upside down strongly. Inject at least 0.35ml after-dilution washing solution into the well, and marinate 1~2 minutes. Repeat this process according to your requirements.
� Automatic Automatic Automatic Automatic washing washing washing washing method: method: method: method: if there is automatic washing machine, it should only be used in the test when you are quite familiar with its function and performance.
SPECIMEN REQUIREMENTS
1. S erum-coagulation at room temperature 10-20 mins ， centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
2. P lasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.
4. C ell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS ( PH7.2-7.4 ) , Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
5. Tissue samples- After cutting samples, check the weight,add PBS ( PH7.2-7.4 ) , Rapidly frozen with liquid nitrogen, maintain samples at 2-8 ℃ after melting,add PBS ( PH7.4 ) , Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant.
6. E xtract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can ‘ t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.
7. Can ‘ t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
ASSAY PROCEDURE
Step 1 :Dilute Standard with Standard dilution and add Standard to wells: set 10 Standard wells on the ELISA plates coated, add Standard 100 μ l to the first and the second well, then add Standard dilution 50 μ l to the first and the second well, mix; take out 100 μ l form the first and the second well then add it to the third and the forth well separately. then add Standard dilution 50 μ l to the third and the forth well ,mix ; then take out 50 μ l from the third and the forth well discard, add 50 μ l to the fifth and the sixth well ,then add Standard dilution 50 μ l to the fifth and the sixth well, mix ; take out 50 μ l from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50 μ l to the seventh and the eighth well ,mix ; take out 50 μ l from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50 μ l to the ninth and the tenth well, mix , take out 50 μ l from the ninth and the tenth well discard(add Sample 50 μ l to each well after Diluting , (concentration: 300 p g/ml ， 200 p g/ml ， 100 p g/ml ， 50 p g/ml ， 25p g/ml )
Step 2: Add sample: Set blank wells separately (blank comparison wells don’ t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40 μ l to testing sample well, then add testing sample 10 μ l (sample final dilution is 5-fold), add sample to wells , don’ t touch the well wall as far as possible, and Gentlymix.
Step 3: Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37 ℃ .
Step 4: Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.
Step 5: Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.
Step 6: Add enzyme: Add HRP-Conjugate reagent 50 μ l to each well, except blank well.
Step 7: Incubate: Operation with 3.
Step 8: Washing: Operation with 5.
Step 9: Color: Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37 ℃
Step 10: Stop the reaction: Add Stop Solution50 μ l to each well, Stop the reaction(the blue color change to yellow color).
Step 11: Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.
JUDGMENT OF ASSAY RESULT
� Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper,Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.
DESCRIPTION
1. The standard curve is drawn under ideal conditions, and is just for reference rather than the actual standard curve diagram of the kit.
2. It is advisable to establish proper assay data and standard curve according to respective laboratory conditions.
ASSAY RANGE
The range of the kit is : 5p g/ml – 400 p g/ml
EXPIRATION DATE OF THE KIT
Twelve months [see label on the outer box for the specific date].
ATTENTION
� The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature,ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.
� W ashing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.
� A dd Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 mins, if the number of sample is much , recommend to use Volley .
� I f the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor. ( × n × 5 ) .
� Closure plate membrane only limits the disposable use, to avoid cross-contamination.
� The substrate evade the light preservation.
� Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.
� All samples, washing buffer and each kind of reject should according to infective material process.
� Do not mix reagents with those from other lots.
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