Human Cyclosporine A (CsA) ELISA Kit

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Human Cyclosporine A (CsA) ELISA Kit FOR RESEARCH USE ONLY. Not for clinical diagnosis use CATALOG #:96648 INTRODUCTION  For the quantitative determination of human CsA concentrations.  Detection of species: Human  Detection medium: serum, plasma, tissue homogenates, cell culture supernates. PRINCIPLE OF TEST The kit is for the quantitative level of CsA in the sample, adopt purified human CsA to coat microtiter plate, make solid-phase antibody, then add samples or standards to wells with a labeled antibody specific to CsA, then add labeled HRP to the well. After washing completely, add TMB substrate solution, TMB substrate becomes blue color in wells that contains antibody - antigen - enzyme-antibody complex, reaction is terminated by the addition of a stop solution and the color change is measured at a wavelength of 450 nm. The concentration of CsA in the samples is then determined by comparing the O.D. of the samples to the standard curve. COMPOSITION OF THE KIT Reagent Quantity Microelisa Stripplate 12well×8strips Standard: 900ng/mL 1×0.5ml Standard Diluent 1×1.5ml HRP-Conjugate Reagent 1×6ml Sample Diluent 1×6ml Chromogen Solution A 1×6ml Chromogen Solution B 1×6ml Stop Solution 1×6ml Wash Solution 1×20ml×30 fold User manual 1 Adhesive Strip 2 Sealed bag 1 STORAGE CONDITIONS  The unopened kit shall be stored at [2-8 ℃] .  For opened kit can be stored at [2-8 ℃] for up to 1 month. If not be used recently, the standard should be kept in -20 ℃. WASHING METHOD  Manually washing method: shake away the remained 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 washing 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. SAMPLE PREPARATION 1. Serum-coagulation at room temperature for 10-20 min, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation appeared, centrifuge again. 2. Plasma-use suited EDTA or citrate plasma as an anticoagulant, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation appeared, centrifuge again. 3. Cell culture supernatant-Detect secretory components, remove particulates by centrifugation for 20-min at the speed of 2000-3000 rpm. Remove supernatant detect the composition of cells, dilute cell suspension with PBS (PH7.2-7.4) to make cell concentration 1 million / ml, repeated freeze-thaw cycles, damage cells and release intracellular components, centrifugation 20-min at the speed of 2000-3000 rpm. Remove supernatant, if precipitation appeared, centrifugal again. 4. Tissue homogenates-after cutting samples, check the weight, pipette PBS (PH7.2-7.4), frozen with liquid nitrogen, maintain samples at 2-8℃ after melting. Pipette PBS (PH7.4), homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 rpm. Remove supernatant. 5. Extract as soon as possible after samples collection, and should be tested as soon as possible after the extraction. If not, samples can be kept in -20 ℃.Avoid repeated freeze-thaw cycles. 6. Don’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity. ASSAY PROCEDURE Step 1: Standard: Bring all reagents to room temperature. Dilute the standard：Pipette 50μl standard dilution in each tube. Pipette 100μl standard (900ng/mL) in the first tube. And take out 100μl from the first tube into the second. Pipette 50μl from the second tube to the third tube and produce dilution series as below. Repeat each of the concentration to get the mean value of each well. Tube 0 1 2 3 4 5 ng/mL 900 600 400 200 100 50 Step 2: Prepare sample: Set blank wells separately (blank comparison wells don't add sample and HRP-Conjugate reagent, other each step operation is same). Pipette Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), Pipette sample to wells, don’t touch the well wall as far as possible, and mix gently. Step 3: Incubate: Cover with the adhesive strip provided, incubate for 30 min at 37℃. Step 4: Configurate liquid: Dilute wash solution 30-fold (or 20-fold) with distilled water. Step 5: Washing: Uncover the adhesive strip, discard liquid, Pipette washing buffer to every well, still for 30s then drain, repeat 5 times. Step 6: Add enzyme: Pipette HRP-Conjugate reagent 50μl to each well, except blank well. Step 7: Incubate: Operation with 3. Step 8: Washing: Operation with 5. Step 9: Color: Pipette Chromogen Solution A 50ul and Chromogen Solution B to each well, avoid the light preservation for 15 min at 37℃ Step 10: Stop the reaction: Pipette Stop Solution 50μl to each well, Stop the reaction (the blue change to yellow). Step 11: Calculate: take blank well as zero, Read absorbance at 450nm after Pipetteing Stop Solution within 15min. CALCULATION OF RESULT Take the standard concentration as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding concentration 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 concentration and the OD value, with the sample OD value in the equation, calculate the sample concentration, multiplied by the dilution factor, the result is the sample actual concentration. Graphical Representation as following: 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. DETECTION RANGE Detection range of the kit is: 30ng/mL -700ng/mL EXPIRATION Twelve months [see label on the outer box for the specific date]. ATTENTION  The kit takes out from the refrigeration should be balanced 15-30 minutes in the room temperature, if the coated ELISA plates have not been used up after opening, the plate should be stored in sealed bag.  Washing buffer will Crystallization separation, it can be heated in water to dissolve.  Pipette sample with pipettors each step, and proofread its accuracy frequently to avoid the experimental error. Pipette sample within 5 min, if the number of sample is big, recommend using multichannel pipettor.  If the testing material concentration is excessively high (The sample OD is higher than the first standard well)，please dilute the sample (n-fold).  Adhesive Strip only limits the disposable use to avoid cross-contamination.  The substrate should evade the light to be preserved.  Please refer to the user instruction strictly, the test result determination must take the microtiter plate reader as a standard.  The preparation of samples and all the reagents should refer to infective material process.  Do not mix reagents with those from other lots.

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