ab Endothelin 1 ELISA Kit

Transcription

1 ab Endothelin 1 ELISA Kit Instructions for Use For the quantitative measurement of Endothelin 1 concentrations in serum, plasma and cell culture supernatants. 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 4 3. Assay Summary 5 4. Kit Contents 7 5. Storage and Handling 8 6. Additional Materials Required 8 7. Protocol 9 8. Calculation of Results Performance Characteristics Troubleshooting 24 2

4 1. Introduction Endothelins (ET) were first identified as endothelin-derived relaxation factors then again as an endothelin-derived contracting factor. In 1988, the isolation and sequence of a 21 amino acid peptide was reported and identified by the more common name endothelin. Three distinct endothelin genes encode unique but highly related peptides, ET-1, ET-2, and ET-3. The predominant physical feature of ET s is the central helical core that is stabilized and includes two intra chain disulfide bonds. This conformation is necessary for high affinity binding by the ETA receptor, but is not for binding by ETB. Translated as a pre pro peptide and translocated into circulation as the pro form Big ET-1, bioactive ET-1 is released when Big ET-1 is cleaved by endothelin converting enzymes at the site of action. Once released, ET-1 is able to elicit different vaso-actions depending on which receptor is bound. The basal circulating level of ET-1 is reported to be < 1 to 3 pg/ml but is known to be elevated in atrial and pulmonary hypertensions, atherosclerosis, congestive heart failure, cancer and variably related to lung diseases such as COPD and asthma. 3

5 2. Principle of the Assay The Endothelin 1 (ET-1) Enzyme linked Immunosorbent Assay (ELISA) kit is a complete kit for the quantitative determination of Endothelin 1 in plasma, serum, and culture fluids. Endothelin 1 has identical amino acid sequence for human, mouse, rat, cow, dog, pig, and rabbit. Please read the complete kit insert before performing this assay. Samples and standards are added to wells coated with a monoclonal antibody specific for Endothelin 1. The plate is then incubated. The plate is washed, leaving only bound Endothelin 1 on the plate. A solution of HRP labeled monoclonal antibody to Endothelin 1 is then added. This binds the Endothelin 1 captured on the plate. The plate is then incubated. The plate is washed to remove excess HRP labeled antibody. TMB Substrate solution is added. The substrate generates a blue color when catalyzed by the HRP. 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 Endothelin 1 in the sample. 4

6 3. Assay Summary Refer to the Assay Layout Sheet to determine the number of wells to be used Add 100 μl of the Assay Buffer into the SO (0 pg/ml Standard) wells. Add 100 μl of Standards #1 through #8 into the appropriate wells. Add 100 μl of the Samples into the appropriate wells. Seal the plate. Incubate for 1 hour at RT. Empty the contents of the wells and wash by adding 400 μl of wash solution to every well. Repeat the wash 4 more times for a total of 5 washes. Add 100 μl of diluted antibody into each well except the Blank. Seal the plate. Incubate for 30 minutes at RT. Wash as above. Add 100 μl of substrate solution into each well. Incubate for 30 minutes at RT. Add 100 μl of Stop Solution to each well. This stops the reaction and the plate should be read immediately. 5

7 Blank the plate reader against the Blank wells, read the optical density at 450 nm. 6

8 4. Kit Contents Item Description Quantity Goat anti-mouse IgG Microtiter Plate Assay Buffer 17 Wash Buffer Concentrate Endothelin 1 Standard Antibody Diluent Endothelin 1 Antibody Concentrate A plate using breakapart strips coated with goat antibody specific to mouse IgG. Tris buffered saline containing detergents. Tris buffered saline containing detergents. A solution of 1,000 pg/ml Endothelin 1. Tris buffered saline containing detergents. A solution of HRP labeled rat monoclonal antibody to Endothelin 1. TMB Substrate A solution of 3,3,5,5 tetramethylbenzidine (TMB) and hydrogen peroxide Stop Solution 2 A 1N solution of hydrochloric acid in water. 1x 96 wells 100 ml 100 ml 0.25 ml 10 ml 100 μl 10 ml 10 ml Plate Sealer 3 7

9 5. Storage and Handling All components of this kit are stable at 4 C. 6. Additional Materials Required Deionized or distilled water. Acetic Acid. Methanol. Ethyl acetate. Ammonium bicarbonate. 200 mg C 18 Sep-Pak column. Microcentrifuge. Microcentrifuge tubes. Centrifugal evaporator. Precision pipettes for volumes between 50 μl and 1,000 μl. Repeater pipette for dispensing 100 μl. Disposable beakers for diluting buffer concentrates. Graduated cylinders. Lint-free paper for blotting. Microplate reader capable of reading at 450 nm. 8

10 7. Protocol A. Sample Handling Culture fluids, serum, and plasma are suitable for use in the assay. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic samples. Plasma samples should be drawn into chilled EDTA tubes (1mg/ml blood) containing Aprotinin (500 KIU/ml of blood). Centrifuge the blood at 1,600 x g for 15 minutes at 0 C. Transfer the plasma to a plastic tube and store at 70 C. Aliquot to avoid repeated freeze/thaw cycles. In humans, normal plasma levels of Endothelin 1 have been reported to be in the Range of 1 3 pg/ml. In certain disease states, levels may increase 3 fold or more. Samples with very low levels of Endothelin 1 or with high levels of protein (e.g. serum and plasma), may require extraction for accurate measurement. Extraction of the sample should be carried out using a similar protocol to the one described below. Procedure: 1. Add an equal volume of 20% acetic acid (AA) to the sample. Centrifuge at 3,000 x g for 10 minutes at 4 C to clarify; save the supernatant. 9

11 2. Equilibrate a 200 mg C18 Sep Pak column with one column reservoir volume (CV) 100% methanol (MeOH), followed by one CV water and one CV 10% MeOH. 3. Apply the supernatant to the Sep Pak column and wash with one CV 10% AA. Remove the excess AA by applying reduced pressure. Discard washes. 4. Wash column with two CVs ethyl acetate and remove the excess by applying reduced pressure. 5. Elute the sample slowly by applying 3 ml 100% MeOH/ 0.05 M ammonium bicarbonate (80/20 v/v). Collect the eluant in a plastic tube. 6. Evaporate to dryness using a centrifugal concentrator under vacuum. 7. If samples cannot be assayed immediately, store at 20 C. 8. Reconstitute with at least 250 μl of the assay buffer and measure immediately. Endothelin 1 was spiked into 1ml of sample which was extracted and reconstituted with 0.25 ml of the assay buffer or diluted in the assay buffer and measured in the assay. 10

12 B. Reagent Preparation 1. Endothelin 1 Standard Allow the 1,000 pg/ml Endothelin 1 standard solution to warm to room temperature. Label eight 12 x 75 mm glass tubes #1 through #8. Pipette 450 μl of the assay buffer into tube #1. Pipette 250 μl of the assay buffer into tubes #2 through #8. Add 50 μl of the 1,000 pg/ml standard to tube #1. Vortex thoroughly. Add 250 μl of tube #1 to tube #2 and vortex thoroughly. Add 250 μl of tube #2 to tube #3 and vortex. Continue this for tubes #4 through 8. Diluted standards should be used within 30 minutes of preparation. The concentration of Endothelin 1 in tubes #1 through #8 will be 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 pg/ml respectively. See Assay Layout Sheet for dilution details. 2. Endothelin 1 Antibody Prepare the antibody by diluting 10 μl of the supplied antibody concentrate with 1 ml of antibody diluent for every ml of 1X needed. The diluted antibody must be used within 8 hours. Only prepare what is needed each day. Discard any unused, diluted antibody. 11

13 3. 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. C. Assay Procedure Bring all reagents to room temperature for at least 30 minutes prior to opening. All standards and samples should be run in duplicate. 1. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells with the desiccant back into the pouch and seal the ziploc. Store unused wells at 4 C. 2. Pipette 100 μl of the assay buffer into the SO (0 pg/ml Standard) wells. 3. Pipette 100 μl of Standards #1 through #8 into the bottom of the appropriate wells. 4. Pipette 100 μl of the Samples into the bottom of appropriate wells. 5. Seal the plate. Incubate for 1 hour at room temperature. 6. Empty the contents of the wells and wash by adding 400 μl of wash solution to every well. Repeat the wash 4 more times for a total of 5 Washes. 7. Pipette 100 μl of diluted antibody into each well except the blank. 12

14 8. Seal the plate. Incubate for 30 minutes at room temperature. 9. Wash as above (step 6). 10. Add 100 μl of Substrate solution to every well. Incubate at room temperature for 30 minutes without shaking. 11. Add 100 μl of Stop Solution to every well. This stops the reaction and the plate should be read immediately. 12. Blank the plate reader against the Blank wells, read the optical density at 450 nm. If the plate reader is not able to be blanked against the Blank wells, manually subtract the mean optical density of the Blank wells from all readings. 13

15 Assay Layout sheet: 14

16 9. Calculation of Results Several options are available for the calculation of the concentration of Endothelin 1 in the samples. We recommend that the data be handled by an immunoassay software package utilizing a 4 parameter logistic curve fitting program. If data reduction software is not readily available, the concentration of Endothelin 1 can be calculated as follows: 1. Calculate the average net Optical Density (OD) bound for each standard and sample by subtracting the average NSB OD from the average OD bound: Average Net OD = Average Bound OD - Average NSB OD 2. Calculate the binding of each pair of standard wells as a percentage of the maximum binding wells (Bo), using the following formula: Percent Bound = Net OD/ Net Bo OD x Using Logit-Log paper plot Percent Bound versus Concentration of Endothelin 1 for the standards. Approximate a straight line through the points. The concentration of PGE2 in the unknowns can be determined by interpolation. Samples with concentrations outside of the standard curve range will need to be reanalyzed 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 Endothelin 1 (pg/ml) S S S S S S S S SO Unknown Unknown

18 Typical Standard Curve A typical standard curve is shown below. This curve must not be used to calculate Endothelin 1 concentrations; each user must run a standard curve for each assay. 17

19 Results below demonstrate expected difference in primary incubation options. There is an approximate 67% increase in optical density with the overnight primary incubation. 18

20 10. Performance Characteristics Sensitivity The sensitivity of the assay, defined as the concentration of Endothelin 1 measured at 2 standard deviations from the mean of 24 zeros along the standard curve, was determined to be 0.41 pg/ml. Linearity A buffer sample containing Endothelin 1 was serially diluted 1:2 in the assay buffer and measured in the assay. The results are shown in the table below. Expected (pg/ml) Observed (pg/ml) Recovery (%) Neat pg/ml --- 1: pg/ml pg/ml 93.8% 1: pg/ml pg/ml 95.1% 1: pg/ml 9.97 pg/ml 99.4% 1: pg/ml 5.12 pg/ml 102.0% 1: pg/ml 2.56 pg/ml 102.0% 1: pg/ml 1.49 pg/ml 119.2% 19

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

22 Cross Reactivities The cross reactivities for a number of related compounds were determined by diluting cross reactant in the assay buffer at a concentration of 10,000 pg/ml. Typical expression ratios of immunoreactive Endothelin in normal plasma are: ET-1: Big ET (1:2), ET-1:ET-2 (5:1), and ET-1:ET-3 (2:1). Compound Cross Reactivity Endothelin 1 (ET-1) 100% ET-2 21% ET-3 3.6% Human Big ET-1 <0.1% Rat Big ET-1 <0.1% Human Big ET-2 <0.1% Human Big ET-3 Amide <0.1% 21

23 Sample Values The following samples were tested for the presence of Endothelin 1. Sample # of Samples Range (pg/ml) Mean (pg/ml) tested Human EDTA Plasma Human Serum

24 Sample Recoveries A minimum 1:4 dilution is required for unextracted serum and plasma samples to remove matrix interference in the assay. Culture fluids and extracted samples may be used without dilution. The optimal dilution for a specific experiment should be determined by the investigator. Sample %Recovery Recommended Dilution RPMI + 10% FBS 76 None Human Serum 110 1:4 Human Plasma 104 1:4 Human Serum - extracted Human Plasma - extracted 79 None 72 None 23

25 11. Troubleshooting For technical questions please do not hesitate to contact us by or phone (select contact us on for the phone number for your region). 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. 24

26 What were the conditions of the incubations? How was the plate shaken during incubations (if required)? 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 air-conditioned 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. How long after the addition of Stop Solution was the plate read? 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. 25

27 High Background How was the plate washed? 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. What were the incubation times and temperatures? If the plate was incubated for too long or at a higher than recommended temperature, high background could result. Drift Were reagents brought to room temperature prior to use? 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. 26

28 Was the set-up of the assay interrupted? 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. 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 pipette 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 pipette 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 pipetting (especially if using repeater pipettes). Poor Standard Curve What was used as the standard diluent? Diluents other than the supplied assay buffer may contain interfering substances that can affect the standard curve. 28

30 How was the precision of the standard curve? Were the Blank and NSB values subtracted out? How were the standard dilutions prepared? 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 pipetting 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. 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. 29

31 Edge Effects Where was the plate incubated? 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. 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? 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. 30

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