Activated Protein C (APC) Pig ELISA Kit. For the quantitative measurement determination of APC in porcine samples.

Transcription

1 ab Activated Protein C (APC) Pig ELISA Kit Instructions for Use For the quantitative measurement determination of APC in porcine samples. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 25 July 2013

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 5 6. MATERIALS REQUIRED, NOT SUPPLIED 6 7. LIMITATIONS 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION SAMPLE COLLECTION AND STORAGE SAMPLE PREPARATION PLATE PREPARATION 13 ASSAY PROCEDURE 13. ASSAY PROCEDURE 14 DATA ANALYSIS 14. CALCULATIONS TYPICAL DATA TYPICAL SAMPLE VALUES ASSAY SPECIFICITY 19 RESOURCES 18. TROUBLESHOOTING NOTES 21 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Abcam s Activated Protein C (APC) Pig ELISA Kit is an in vitro Enzyme-linked immunosorbent assay (ELISA) for the quantitative measurement of APC in serum, plasmas, tissue homogenates and cell extracts. The microtiter plate has been pre-coated with a monoclonal antibody specific for APC. Standards or samples are then added to the microtiter plate wells and APC if present, will bind to the antibody precoated wells. In order to quantitatively determine the amount of APC present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody, specific for APC are added to each well to sandwich the APC immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, substrate solutions are added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain APC and enzyme-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The APC concentration in each sample is interpolated from this standard curve. Discover more at 2

4 INTRODUCTION 2. ASSAY SUMMARY Remove appropriate number of antibody coated well strips. Equilibrate all reagents to room temperature. Prepare all the reagents, samples, and standards as instructed. Add standard or sample to each well used and incubate. Aspirate and wash each well. Add HRP conjugated secondary antibody to each well and incubate Add Development Solution to each well for specified period of time, and then add the stop solution. Read wells at specified O.D. Discover more at 3

5 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at +2-8ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 9 - Reagent Preparation. Discover more at 4

6 GENERAL INFORMATION 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) MICROTITER PLATE 96 Wells 2-8ºC ENZYME CONJUGATE 10 ml 2-8ºC STANDARD A 0 ng/ml 2-8ºC STANDARD B 0.5 ng/ml 2-8ºC STANDARD C 1.0 ng/ml 2-8ºC STANDARD D 2.5 ng/ml 2-8ºC STANDARD E 5.0 ng/ml 2-8ºC STANDARD F 10 ng/ml 2-8ºC SUBSTRATE A 6 ml 2-8ºC SUBSTRATE B 6 ml 2-8ºC STOP SOLUTION 6 ml 2-8ºC WASH SOLUTION (100X) 10 ml 2-8ºC BALANCE SOLUTION 3 ml 2-8ºC NOTE: The BALANCE SOLUTION is used only when the sample is cell culture supernatants, body fluid and tissue homogenate; if the sample is serum or plasma, then the BALANCE SOLUTION is not required. Discover more at 5

7 GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Microplate reader capable of measuring absorbance at 600 nm (or 450 nm after addition of Stop solution - not supplied). 100 ml and 1 liter graduated cylinders. Distilled or deionized water. Tubes to prepare sample dilutions. Microplate washer or washing bottle. Incubator (37 C). Data analysis and graphing software. PBS (ph ). 7. LIMITATIONS ELISA kit intended for research use only. Not for use in diagnostic procedures Do not use kit or components if it has exceeded the expiration date on the kit labels Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. Discover more at 6

8 GENERAL INFORMATION 8. TECHNICAL HINTS It is recommended that all standards, controls and samples be run in duplicate. Standards and samples must be assayed at the same time. The coefficient of determination of the standard curve should be 0.95 and the highest O.D. should be more than 1.0. Cover or cap all kit components and store at 2-8 C when not in use. Microtiter plates should be allowed to come to room temperature before opening the foil bags. Once the desired number of strips has been removed, immediately reseal the bag with desiccants and store at 2-8 C to maintain plate integrity. Samples should be collected in pyrogen/endotoxin-free tubes. When possible, avoid use of badly hemolyzed or lipemic serum. If large amounts of particulate matter are present, centrifuge or filter prior to analysis. When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal incubation times for all wells. Do not mix or interchange different reagent lots from various kit lots. Read absorbance immediately after adding the stop solution. Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Solution provided. All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells. Because TMB is light sensitive, avoid prolonged exposure to light. Also avoid contact between TMB and metal, otherwise color may develop. Discover more at 7

9 GENERAL INFORMATION This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Discover more at 8

10 ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents and samples to room temperature (18-25 C) prior to use. Wash Solution Dilute 10 ml of Wash Solution concentrate (100X) with 990 ml of deionized or distilled water to prepare 1,000 ml of Wash Solution (1X). If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. The 1X wash solution is stable for 2 weeks at 2-8 C. All other components are ready to use and require no further dilutions. Discover more at 9

11 ASSAY PREPARATION 10. SAMPLE COLLECTION AND STORAGE 10.1 Cell Culture Supernatants & other body fluids Centrifuge cell culture media at 1,000 x g (or 3,000 rpm) for 15 minutes Remove supernatant Tissue Homogenates (Preparation of tissue homogenates will vary depending upon tissue type) Rinse tissues in ice-cold PBS (0.02 mol/l, ph ) to remove excess blood Mince the tissues to small pieces and homogenize in PBS with a glass homogenizer on ice Either use ultrasonication or two freeze-thaw cycles to further break the cell membranes Centrifuge homogenates for 15 minutes at 1,500 x g (or 5,000 rpm) Collect the supernatant Serum Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 2-8 C Centrifuge at 1,000 x g (or 3,000 rpm) for 15 minutes Collect the serum Plasma Collect plasma using EDTA or heparin as an anticoagulant Centrifuge samples for 15 minutes at 1,000 x g (or 3,000 rpm) at 2-8 C within 30 minutes of collection. Discover more at 10

12 ASSAY PREPARATION 10.5 Cell Lysates - Cells should be lysed according to the following directions Adherent cells should be detached with trypsin and then collected by centrifugation. Suspension cells can be collected by centrifugation directly Wash cells three times in PBS Cells were resuspended in PBS and subjected to ultrasonication for 3 times. Alternatively, freeze cells at -20 C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times. Centrifuge at 1,000 x g (or 3,000 rpm) for 15 minutes at 2-8 C to remove cellular debris. All samples should be assayed immediately or store samples at -20 C or -80 C. If samples are to be run within 24 hours, they may be stored at 2-8 C. Avoid repeated freeze-thaw cycles. Discover more at 11

13 ASSAY PREPARATION 11. SAMPLE PREPARATION General Sample information: Measure the concentration of samples before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. PBS (ph ) or 0.9% physiological saline can be used as dilution buffer. Owing to the possibility of mismatching between antigen from other resource and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products. Influenced by the factors including cell viability, cell number and also sampling time, samples from cell culture supernatant may not be detected by the kit. This kit can be used with previously stored material. However, note that storage conditions and duration could affect results. Fresh samples that have not been subject to long term storage are recommended for this assay. Prior to assay, frozen samples should be brought to room temperature slowly and mixed gently. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals. Samples containing a visible precipitate must be clarified prior to use in the assay. Care should be taken to minimize hemolysis. Do not use grossly hemolyzed or lipemic specimens. Do not use heat-treated specimens. Discover more at 12

14 ASSAY PREPARATION 12. PLATE PREPARATION The 96 well plate strips included with this kit is supplied ready to use. It is not necessary to rinse the plate prior to adding reagents. Unused well plate strips should be returned to the plate packet and stored at 4 C. For each assay performed, a minimum of 2 wells must be used as blanks, omitting primary antibody from well additions. For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates). Well effects have not been observed with this assay. Contents of each well can be recorded on the template sheet included in the Resources section. Discover more at 13

15 ASSAY PROCEDURE 13. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate Prepare all reagents, working standards, and samples as directed in the previous sections Remove unused microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal Add 50 µl of each of Standards or Samples to per well. Add 50 µl of PBS in the blank control well 13.4 Add 5 µl of Balance Solution into 50 µl specimens, mix well. (NOTE: This step is required when the sample is cell culture supernatants, body fluid and tissue homogenate; if the sample is serum or plasma, then this step should be skipped.) Cover/seal the plate and incubate for 1 hour at 37 C. If available use a plate shaker for all incubation steps at 300 rpm Add 100 µl of Conjugate to each well (NOT blank control well). Mix well. Mixing well in this step is important. Cover and incubate the plate for 1 hour at 37 C Wash the microtiter plate by removing the incubation mixture by aspirating contents of the plate into a sink or proper waste container. Fill in each well completely with 1X wash solution, and then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure five times for a total of FIVE washes. After washing, invert plate, and blot dry by hitting the plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain Discover more at 14

16 ASSAY PROCEDURE securely in frame. Complete removal of liquid at each step is essential to good performance Add 50 µl Substrate A and 50 µl Substrate B to each well including blank control well, subsequently. Cover and incubate for minutes at 37 C. (Avoid sunlight) Add 50 µl of Stop Solution to each well including blank control well. Mix well Immediately determine the Optical Density (O.D.) at 450 nm using a microplate reader. Discover more at 15

17 DATA ANALYSIS 14. CALCULATIONS The standard curve is used to determine the concentration of the samples Average the duplicate readings for each standard and sample. All O.D. values are subtracted by the mean value of blank control before result interpretation Construct a standard curve by plotting the average O.D. for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis, and draw a best fit curve using graph paper or statistical software to generate a linear regression, four parameter logistic (4-PL) curve-fit, or curvilinear regression of second degree. An x-axis for the optical density and a y-axis for the concentration is also a choice. The data may be linearized by plotting the log of the concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis Calculate the concentration of samples corresponding to the mean absorbance from the standard curve. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Discover more at 16

18 DATA ANALYSIS 15. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Standard Curve Measurements Conc. (ng/ml) Mean O.D Discover more at 17

19 DATA ANALYSIS 16. TYPICAL SAMPLE VALUES SENSITIVITY - The sensitivity in this assay is 0.1 ng/ml. RECOVERY Serum Spike Recovery: % LINEARITY OF DILUTION PRECISION Serum % Range 1: : : : Intra- Inter- Assay Assay %CV Discover more at 18

20 DATA ANALYSIS 17. ASSAY SPECIFICITY This assay has high sensitivity and excellent specificity for detection of APC. No significant cross-reactivity or interference between APC and analogues was observed. NOTE: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between APC and all the analogues, therefore, cross reaction may still exist in some cases. Discover more at 19

21 RESOURCES 18. TROUBLESHOOTING Problem Cause Solution Poor standard curve Low Signal Samples give higher value than the highest standard Large CV Inaccurate pipetting Improper standards dilution Incubation times too brief Inadequate reagent volumes or improper dilution Starting sample concentration is too high. Plate is insufficiently washed Contaminated wash buffer Check pipettes Prior to opening, briefly spin the stock standard tube and dissolve the powder thoroughly by gentle mixing Ensure sufficient incubation times; change to overnight standard/sample incubation Check pipettes and ensure correct preparation Dilute the specimens and repeat the assay Review manual for proper wash technique. If using a plate washer, check all ports for obstructions Prepare fresh wash buffer Low sensitivity Improper storage of the ELISA kit Store the all components as directed. Keep substrate solution protected from light. Discover more at 20

22 RESOURCES 19. NOTES Discover more at 21

23 RESOURCES Discover more at 22

24 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. RESOURCES 23