Dkk-1 Rat ELISA Kit. For the quantitative measurement of Rat DKK-1 concentrations in plasma, serum and culture fluids.

Transcription

1 ab Dkk-1 Rat ELISA Kit Instructions for Use For the quantitative measurement of Rat DKK-1 concentrations in plasma, serum and culture fluids. This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Introduction 3 2. Principle of the Assay 5 3. Assay Summary 6 4. Kit Contents 7 5. Storage and Handling 8 6. Additional Materials Required 8 7. Protocol 9 8. Calculation of Results Performance Characteristics Troubleshooting 23 2

4 1. Introduction ab is a complete kit for the quantitative determination of Dkk- 1 in plasma, serum, and culture fluids. Please read the complete kit insert before performing this assay. Dkk-1 (Dickkopf or Dickkopf-1) is a secreted inhibitor of the Wnt/canonical signaling pathway which mediates a variety of cellular processes. Wnt proteins are extracellular signaling molecules that initiate activation of the canonical pathway by binding low-density lipoprotein receptor-related protein 5&6 (LRP5/6) coreceptors, triggering a cascade of events that stabilize β-catenin. Wnts mediate osteoblast development and cell-to-cell interactions during embryogenesis, and are also responsible for regulating bone mass in adults during normal bone remodeling processes. Wnts are also found to be expressed during the stationary phase of the cell cycle and are thought to control expansion of bone marrow stromal cells preventing overpopulation in marrow. There are at least two families of secreted inhibitors of Wnt signaling: the secreted frizzled-related protein family and the Dickkopf family. The rat Dkk-1 protein consists of 270 amino acids including a 30 amino acid N-terminal signal peptide. Dkk-1 has a predicted molecular weight of 26kD and an apparent molecular weight of approximately 38-42kD by SDS-PAGE and western blot analysis. Dkk-1 disrupts the canonical pathway by binding to the LRP5/6 coreceptors, preventing interaction with Wnt and facilitating degradation of β-catenin, which ultimately results in osteolytic 3

5 breakdown of bone tissue. Wnt signaling dysregulation has been implicated in cancer of the colon and stomach. It has been reported that the Wnt/β-catenin pathway is down regulated by the induction of Dkk-1 expression, a mechanism that is lost in colon cancer, suggesting that Dkk-1 acts as a tumor suppressor gene in this cancer type. Dkk-1 proteins are expressed during log phase of the cell cycle, suggesting that dysregulated Dkk-1 could be responsible for the growth of some malignancies. The Dkk-1 gene has also been determined to be a target of p53, studies suggests it is a necessary requirement for the execution of cell death. It has been reported that elevated Dkk-1 levels in bone marrow, plasma and peripheral blood from patients with multiple myeloma correlated with the gene-expression patterns of Dkk-1, and were associated with the presence of focal bone lesions. A recent study reported serum levels of Dkk-1 in patients with multiple myeloma before autologous stem cell transplantation. The range of Dkk-1 serum levels in this group of patients was ng/ml with a mean of 63.6 ng/ml and a standard deviation of Reports suggest that Dkk-1 may act like a switch on prostate cancer cell activity, causing cells to change from osetoblastic to a highly osteolytic cell line. 4

6 2. Principle of the Assay 1. Samples and standards are added to wells coated with a monoclonal antibody specific for Dkk-1. The plate is then incubated. 2. The plate is washed, leaving only bound Dkk-1 on the plate. A yellow solution of biotinylated polyclonal antibody to Dkk-1 is then added. This binds the Dkk-1 captured on the plate. The plate is then incubated. 3. The plate is washed to remove excess antibody. A blue solution of streptavidin conjugated to horseradish peroxidase is added to each well, binding to the biotinylated antibody. The plate is again incubated. 4. The plate is washed to remove excess HRP conjugate. TMB Substrate solution is added. An HRP-catalyzed reaction generates a blue color in the solution. 5. Stop solution is added to stop the substrate reaction. The resulting yellow color is read at 450 nm. The amount of signal is directly proportional to the level of Dkk-1 in the sample. 5

7 3. Assay Summary Standard or sample are added to plate Incubate Wash Add Antibody Incubate Wash Add Streptavidin HRP Incubate Wash Add Substrate Incubate Read Plate (measure at 450 nm) 6

8 4. Kit Contents Item Description Quantity Dkk-1 Clear Microtiter Plate Assay Buffer 36 Dkk-1 Rat Standard Wash Buffer Concentrate Dkk-1 EIA Antibody Dkk-1 (rat) EIA Conjugate A plate of break-apart strips coated with a mouse monoclonal antibody specific to Dkk-1. Tris buffer containing detergents. Two vials containing 2500pg/vial of recombinant rat Dkk-1. Tris buffered saline containing detergents A yellow solution of biotinylated polyclonal antibody to Dkk-1. A blue solution of streptavidin conjugated to horseradish peroxidase. TMB Substrate A solution of 3,3,5,5 tetramethylbenzidine (TMB) and hydrogen peroxide.. Stop Solution 2 Plate Sealer A 1N solution of hydrochloric acid in water. 1x 96 wells 100 ml 2 vials 100 ml 10 ml 10 ml 10 ml 10 ml 2 units 7

9 5. Storage and Handling All components of this kit, except the Standard, should be stored at 4 C upon receipt. The Standards must be stored at or below - 20 C upon receipt. 6. Additional Materials Required Deionized or distilled water. Precision pipets for volumes between 5 μl and 1000 μl. Repeater pipets for dispensing 100 μl. Disposable beaker for diluting buffer concentrates. Graduated cylinders. A microplate shaker. Lint free paper toweling for blotting. Microplate reader capable of reading at 450 nm. Graph paper for plotting standard curve. 8

10 7. Protocol A. Sample Handling Rat culture fluids, serum, and EDTA plasma are suitable for use in the assay; other sample matrices have not been validated. Samples containing a visible precipitate must be clarified prior to use in the assay. Due to differences in samples, users must determine the optimal sample dilution for their particular experiments. Do not use grossly hemolyzed or lipemic specimens A minimum 1:4 dilution in the assay buffer is required for most serum and plasma samples. Culture media supplemented with 10% fetal bovine serum (FBS) requires a minimum dilution of 1:2 in the assay buffer. This kit also recognizes bovine Dkk-1.* It is recommended that users evaluate each lot of FBS used for the levels of endogenous bovine Dkk-1 present. The user has the option to either dilute the culture fluids to eliminate the endogenous bovine Dkk-1 or subtract the concentration of bovine Dkk-1 contributing to the total Dkk-1 detected in each sample. *Bovine Dkk-1 has 82% sequence identity to rat Dkk-1. Crossreactivity to bovine Dkk-1 is observed. 9

11 B. Protocol for Serum and Plasma 1. Collect whole blood in appropriate tubes for either serum or plasma. 2. Allow serum to clot for 30 minutes. 3. Centrifuge at 1000 x g for 15 minutes at 4 C. 4. Place supernatants in a clean tube. 5. Supernatants may be aliquoted and stored at or below - 20 C, or used immediately in the assay. 6. Avoid repeated freeze-thaw cycles. C. Reagent Preparation 1. Wash Buffer Prepare the wash buffer by diluting 50 ml of the supplied Wash Buffer Concentrate with 950 ml of deionized water. This can be stored at room temperature for 3 months. 2. Preparation of Dkk-1 Standard Curve Add 2 ml of assay buffer to the lyophilized Dkk-1 standard vial and vortex. Wait 5 minutes and vortex again prior to use. Label the vial Standard #1. Label five 12 x 75 mm polypropylene tubes #2 through #6. Pipet 250 µl of the assay buffer into tubes #2 through #6. Add 250 µl of reconstituted Standard #1 to tube #2 and vortex thoroughly. 10

12 Add 250 µl of tube #2 to tube #3 and vortex thoroughly. Continue this for tubes #4 through #6. Diluted standards should be used within 60 minutes of preparation. The concentration of Dkk-1 in tubes is labeled above. D. Assay Procedure Refer to the Assay Layout Sheet to determine the number of wells to be used. Remove the wells not needed for the assay and return them, with the desiccant, to the mylar bag and seal. Store unused wells at 4 C. 1. Pipette 100 μl of the assay buffer into the S0 (0 pg/ml standard) wells. 2. Pipette 100 μl of Standards #1 through #6 to the bottom of the appropriate wells 3. Pipette 100 μl of samples to the bottom of the appropriate wells 4. Seal the plate. Incubate for 1 hour on a plate shaker (~500 rpm) at room temperature.. 5. Empty the contents of the wells and wash by adding 400 µl of wash buffer to every well. Repeat 3 more times for a total of 4 washes. After the final wash, 11

13 empty or aspirate the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. 6. Pipette 100 µl of yellow antibody into each well except the blank. 7. Seal the plate. Incubate for 1 hour on a plate shaker (~500 rpm) at room temperature. 8. Wash as above. 9. Add 100 µl of blue conjugate to each well except the blank. 10. Seal the plate. Incubate for 30 minutes on a plate shaker (~500 rpm) at room temperature. 11. Wash as above. 12. Pipette 100 µl of substrate solution into each well. 13. Incubate for 30 minutes on a plate shaker (~500 rpm) at room temperature. 14. Pipette 100 µl of stop solution into each well. 12

14 15. After blanking the plate reader against the substrate blank, read optical density at 450 nm. If plate reader is not capable of adjusting for the blank, manually subtract the mean OD of the substrate blank from all readings. 13

15 Assay Layout Sheet: A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 Blank Std 3 Std 7 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 Blank Std 3 Std 7 C1 S0 C2 Std 4 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 D1 S0 D2 Std 4 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 E1 Std 1 E2 Std 5 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 F1 Std 1 F2 Std 5 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 G1 Std 2 G2 Std 6 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 H1 Std 2 H2 Std 6 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 14

16 8. Calculation of Results Several options are available for the calculation of the concentration of Dkk-1 in the samples. We recommend that the data be handled by an immunoassay software package utilizing a 4 parameter logistic (4PL) curve fitting program. If data reduction software is not readily available, the concentrations can be calculated as follows: 1. Calculate the average Net OD for each standard and sample by subtracting the average blank OD from the average OD for each standard and sample. Average Net OD = Average OD - Average Blank OD 2. Using linear graph paper, plot the average Net OD for each standard versus Dkk-1 concentration in each standard. Approximate a straight line through the points. The concentration of the unknowns can be determined by interpolation. Samples with concentrations outside of the standard curve range will need to be re-analyzed using a different dilution. 15

17 Typical Results The results shown below are for illustration only and should not be used to calculate results. Sample Net OD Dkk-1 (pg/ml) S S S S S S S

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19 9. Performance Characteristics A. Specificity The cross reactivities for a number of related compounds were determined by diluting cross reactants in the assay buffer at a concentration of 125,000 pg/ml These samples were then measured in the assay. Compound Cross Reactivity rat Dkk-1 100% mouse Dkk-2 mouse Dkk-4 mouse Sgy % 0.03% 0.03% B. Sensitivity The sensitivity or limit of detection of the assay is 26.1 pg/ml. The sensitivity was determined by interpolation at 2 standard deviations above the mean signal at background (0 pg/ml) using data from 10 standard curves. 18

20 C. Parallelism A parallelism experiment was carried out to determine if the recombinant Dkk-1 standard accurately measures Dkk-1 concentrations in biological matrices. To assess parallelism, values for serum were obtained from a standard curve using four parameter logistic curve fitting. The observed concentration was plotted against the dilution factor. Parallelism indicates antibody-binding characteristics of the native and standard proteins are similar, allowing accurate determination of analyte. 19

21 D. Precision Intra-assay precision was determined by assaying 20 replicates of three buffer controls containing Dkk-1 in a single assay. pg/ml %CV Inter-assay precision was determined by measuring buffer controls of varying Dkk-1 concentrations in multiple assays over several days. pg/ml %CV

22 E. Sample Recoveries After diluting each sample matrix to its minimum required dilution, recombinant rat Dkk-1 was spiked at high, medium and low concentrations. To account for any endogenous Dkk-1, an unspiked sample of each matrix was also included. The recovery of the standard in spiked samples was compared to the recovery of identical spikes in the assay buffer. The mean and range of percent recovery at the three concentrations are indicated below for each matrix. Sample Matrix (# of samples) Minimum Required Dilution Spike Concentration (pg/ml) Recovery of Spike (Range) F12K + 10% FBS (n=1) 1: % (n/a) % (n/a) % (n/a) Serum (n=6) 1: % ( %) EDTA plasma (n=6) 1: % (85-119%) % (51-102%) % (76-111%) % (79-110%) % (66-118%) 21

23 F. Dilutional Linearity The minimum required dilution (MRD) for several common samples was determined by serially diluting samples into the assay buffer and identifying the dilution at which linearity is observed. Pools of natural rat serum and Na EDTA plasma were diluted into the assay buffer to produce values within the dynamic range of the assay. Ham s F12k media supplemented with 10% FBS was spiked with recombinant rat Dkk-1 and diluted in assay buffer. At a 1:100 dilution the contribution from bovine Dkk-1 was eliminated. Average % of Expected Dilution Culture Media (F12k + 10% FBS) Serum Na EDTA Plasma Neat 104% (1:100 in Assay Buffer) :2 109% 117% 117% 1:4 87% 97% 102% 1:8 107% 99% 101% 1:16 112% 100% 100% 1:32 100%

24 10. Troubleshooting Weak Color Development How long was the substrate incubation? What were the conditions of the substrate incubation? Were reagents brought to room temperature prior to use? It is possible that Stop Solution was added to the plate without allowing the full substrate incubation. If a plate is left to incubate on a cold lab bench or under a drafty area during ambient incubations, signal values (e.g. optical density) may be lower than expected. It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual. Usually leaving the kit out on the bench top at ambient temperature for about half an hour prior to setting up the assay will be sufficient, when the reagents can be stored at 4 C. Frozen volumes take a little more time to come to room temperature. Do not thaw frozen reagents in a water bath. If a different standard/sample diluent is used (such as culture media) this must also be warmed. 23

25 What were the conditions of the incubations? How was the plate shaken during incubations (if required)? How long after the addition of Stop Solution was the plate read? If the incubation times and temperatures are not observed, this can lead to lower than expected signal values (e.g. optical density). Pay attention that in airconditioned rooms the temperature does not drop below 21 C. If customers do not have a plate shaker, they will often use an orbital flask shaker or some other piece of equipment. This is not a problem as long as the liquid is vigorously displaced about 3/4 of the way up the sides of the wells without coming out. It is very important that the plate is secured into place. If the plate is not shaken and it is required in the procedure, a longer incubation may be necessary to bring the reagents to equilibrium. The plate needs to be read at the correct wavelength as soon as possible after the addition of the Stop Solution. We generally recommend that the plate be read within 10 minutes. 24

26 High Background How was the plate washed? What were the incubation times and temperatures? It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. If the plate was incubated for too long or at a higher than recommended temperature, high background could result. 25

27 Drift Were reagents brought to room temperature prior to use? Was the set-up of the assay interrupted? If the reagents are not at a constant temperature prior to their addition into the wells, the results from one side of the plate to the other can differ depending on the temperatures at addition. If the assay is interrupted at any point during the addition of reagents, it is possible that differing results will be seen before the interruption versus after. The wells that had reagents added before the interruption will have been incubating for longer than those after. 26

28 Poor Precision Were the wells washed properly? All wells receive the same treatment during the wash step. If some are washed less than others, this can translate to poor precision. It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. 27

29 Were the wells aspirated sufficiently after the wash steps? How were reagents pipetted into wells? It is very important that as little Wash Buffer as possible remains in the wells after aspiration. Residual buffer can cause dilution of subsequent reagents. After the last wash step, it is a good idea to hit the plate several times over a piece of paper toweling to remove excess buffer. In order to eliminate precision error, customers need to remember to prerinse all pipet tips used in the assay. We usually recommend that the customer draw up the liquid into the tip and aspirate it three times prior to addition into the well. Regular pipet calibration and maintenance is also essential to ensure that the tips fit properly and that the correct volumes are dispensed. Be sure reagents are not splashed between wells or outside of the wells during pipeting (especially if using repeater pipets). 28

30 Poor Standard Curve What was used as the standard diluent? How was the precision of the standard curve? Were the Blank and NSB values subtracted out? Diluents other than the supplied assay buffer may contain interfering substances that can affect the standard curve. If the %CV values for the standard curve signal values (e.g. optical density) are consistently above 5%, it may be a good idea to pay particular attention to pipeting technique. If the standard curve signal values were acceptable but the sample precision was not, the problem relates to the sample. Also, see recommendations under "Poor Precision". If the net signal values (e.g. optical density) are not used, the signal values will appear higher than those presented in the sample data in the instruction manual. 29

31 How were the standard dilutions prepared? It is important that test tubes of an appropriate size and material are used. Standard dilutions must be properly mixed (e.g. vortexed) while preparing the serial dilutions. It is also crucial that the standard dilutions be prepared and used within the time specified in the product specific instruction manual. Never store unused standard dilutions for a later use. 30

32 Edge Effects Where was the plate incubated? If multiple plates were run, were they stacked on top of each other during incubation? If a non-ambient incubation was required, was the plate properly sealed? Often times the conditions for ambient incubations can be less than ideal. If there is a draft in the area or the plate is incubated on a cold lab bench, this can lead to uneven color development. Multiple plates should only be incubated in a single layer. This will assure that no area of the plate is at a different temperature than any other. Making sure that the plate sealer is tightly covering all of the wells will help to discourage uneven evaporation of the well contents, or condensation for colder incubation conditions. 31

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