ELISA/assay

Endotrophin ELISA Kit

DEIA-XYZ75

96T

Endotrophin ELISA Kit

ETP

Endotrophin ELISA Kit

cELISA

ETP

Mouse

Quantitative

serum, plasma

96T

One year from production date. Store refrigerated at 4-8°C.

Endotrophin (mouse): 100%

ETP

Mouse

Endotrophin; ETP; α-endorphin, β-endorphin, γ-endorphin, σ-endorphin, α-neo-endorphin

The Endotrophin EIA Kit is a competitive immunoassay for in vitro quantitative measurement of Endotrophin in mouse serum or plasma.

This ELISA kit is a competitive immunoassay. The antiserum is captured by antibodies coated on a 96-well plate. A constant concentration of Bt-tracer (biotinylated tracer) and varying concentrations of unlabeled standard or sample peptide compete for binding specifically to the antiserum. Captured Bt-tracer is subsequently bound by streptavidinconjugated horseradish peroxidase (SA-HRP), which produces a soluble colored product after a substrate is added. Immunogen: Synthetic peptide H-Thr-Glu-Pro-Leu-Phe-Leu-Thr-Lys-Thr-Asp-Ile-CysLys-Leu-Ser-Arg-Asp-Ala-Gly-Thr-Cys-Val-Asp-Phe-Lys-Leu-Leu-TrpHis-Tyr-Asp-Leu-Glu-Ser-Lys-Ser-Cys-Lys-Arg-Phe-Trp-Tyr-Gly-GlyCys-Gly-Gly-Asn-Glu-Asn-Arg-Phe-His-Ser-Gln-Glu-Glu-Cys-Glu-LysMet-Cys-Ser-Pro-Glu-Leu-Thr-Val-OH (Disulfide bonds, air oxidized) coupled to carrier protein. The standard is used to make a standard curve in the range specified in the kit's datasheet. Standard curves are S-shaped (on a semi-log plot) but for a few kits they appear to be almost linear over the kit's range. The measuring range is the range of standard concentrations near the middle or near the IC50 of the standard curve. Unknown sample concentrations are measured by comparing their absorbance with the standard curve. We include sufficient reagents for 96 determinations.

1. ELISA buffer concentrate (50ml 20× concentrate) 2. 96-well immunoplate with plate sealer 3. Antiserum (lyophilized powder) 4. Standard (1μg lyophilized powder) 5. Biotinylated tracer (lyophilized powder) 6. Streptavidin-HRP (100μl 200x concentrate) 7. TMB substrate stock solution (1.5ml) 8. TMB substrate buffer (15ml citrate buffer) 9. Stop solution 2 N HCl (15 ml)

The following materials are not included but are recommended for EIAH kits. Extraction kit (with 50 Sep-columns and buffers A and B) Buffer A Buffer B Sep-Column (200 mg) Sep-Column adapter Materials 1. 96-well microtiter plate reader set up to measure 450nm and 650nm 2. 96-well plate washer and shaker (optional) 3. Distilled or deionized water, or comparable quality 4. Curve fitting software (optional) 5. Test tubes, pipettes and various other standard laboratory items

Variation • Accuracy • Extraction • Cross-reactivity The kit's IC50, or the shape of the standard curve, may exhibit some variation but this will not affect the kit's accuracy in the measuring range. The kit accurately measures sample peptides if the following conditions are met. A) Both samples and standards must be measured in the same diluent and under the same conditions (same microtiter plate). B) The kit's antiserum must not cross-react appreciably with other factors present in the sample. Cross-reactivity tables are included with each kit. The user may wish to test the cross-reactivity with other peptides. C) The sample peptides must be identical to the kit's standard. Ideally the kit's synthetic standard mimics the natural peptide perfectly. Sometimes, however, natural peptides exist as families of species related by a common or similar sequence. Also, natural peptides may be modified enzymatically or spontaneously, may exist in complexes, and may assume alternative structural forms. In these cases the kit might not measure the exact concentration of a particular natural peptide species, but it may still be used for relative average measurements. D) Sample extraction. Factors present in serum can bind to EIAH kit components. The effects can vary from negligible to complete obliteration of signal. Therefore, sample extraction may be required prior to using EIAH kits. PREPARE SAMPLES 1. Sample extraction. Sample extraction is recommended for serum or plasma samples. It may be less important for tissue culture samples. See "Suggested Protocol for Sample Extraction" below for details. The kit may still be used without extraction but this may cause unexpected results due to the possible binding between serum proteins and kit components. 2. Sample concentration. The concentration of the target molecule must be within the measuring range of the kit (around the IC50). If the concentration range of your sample is difficult to estimate, prepare it at different concentrations such that one of the samples should lie within the measuring range. SUGGESTED PROTOCOL FOR SAMPLE EXTRACTION We have provided an excess amount of standard that you may use to determine if extraction is required. For example, if you are working with serum, you may spike it with known amounts of standard and check if they are accurately determined by the assay with and without extraction. Extraction eliminates potentially interfering substances, such as albumin. Extraction may also be necessary to concentrate the sample to within the measuring range. As with any purification technique, recovery of the desired substance is likely to be incomplete. Therefore, both optimization and quantification of the extraction procedure are recommended for more accurate determinations. While we cannot provide you with extraction optimization and quantification protocols, we have included enough standard in the kit should you wish to use it for this purpose. C 18 Sep-Column Extraction Method The following generic protocol is meant to help users with extracting their samples. It should be applicable to different biological fluids but should not be thought of as an optimized protocol for any particular antigen. Required Materials a. SEP-COLUMN containing 200mg of C 18 b. Buffer A (BUFF-A): 1% trifluoroacetic acid (TFA, HPLC Grade). (Acidifies plasma sample to remove interfering proteins such as albumin) c. Buffer B (BUFF-B): 60% acetonitrile (HPLC Grade), 1% TFA, and 39% distilled water. (Elutes peptide from column) d. You may also consider purchasing Extraction kits, which include SEPcolumns and buffers Withdrawal and Preparation of Plasma Collect blood samples (2 - 6ml) into a chilled syringe and transfer into a polypropylene tube containing EDTA (1mg/ml of blood) as an anticoagulant and Aprotinin (500KIU/ml of blood) as a protease inhibitor at 4°C. Do not use heparinized tubes as they may interfere with the assay. Vacutainers with EDTA are acceptable. Centrifuge blood at 1,600xg for 15 minutes at 4°C. Collect the top (plasma) layer. Proceed to extraction immediately or freeze at -70°C for later use. Extraction Procedure 1. Add an equal amount of Buffer A to the plasma. 2. Centrifuge at 6,000xg to 17,000xg for 20 minutes at 4°C. 3. Transfer supernatant to a new tube discarding any pellet that may be present. 4. Equilibrate a SEP-COLUMN by washing with 1ml Buffer B followed by 3x 3ml Buffer A. 5. Load the plasma solution onto the equilibrated SEP-Column. 6. Slowly wash the column with Buffer A (3 ml, twice) and discard the wash. A light vacuum (10 sec/drop) may be applied to the column. 7. Elute the peptide slowly with Buffer B (3 ml, once) and collect eluant in a polypropylene tube. A light vacuum may be applied as in previous step. 8. Freeze-dry eluant to dryness using a dry ice/methanol bath to freeze the sample and a centrifugal concentrator to evaporate it 9. Dissolve the residue in a suitable volume of ELISA buffer such that the concentration of the substance of interest will fall close to the IC50 (within the measuring range).

ELISA buffer and Diluent. The standards, samples, antiserum and Bt-tracer are always reconstituted and used in ELISA buffer. The standard curve should show similar characteristics as the one from the datasheet. Room Temperature. Reagents, samples, and the plate should be brought to room temperature before use. Shakers. Shakers (optional) may help lower the experimental variation of duplicates (recommended at 60 rpm). Blank Wells. Blanks will give you the background to be subtracted from all readings. These should not be confused with the "S0 Standards" which contain no standard peptide and which will yield the highest readings. Blank readings will not influence concentration calculations – thus, they are optional. Lyophilized kit components should not be re-hydrated until they are needed. Please check the included datasheet for the appropriate protocol. 1. Equilibrate unopened kit components to room temperature. Avoid accumulation of moisture, do not open reagents and immunoplate while they are cold. 2. ELISA buffer. Dilute the ELISA buffer concentrate 1 in 20 with water and mix well. Example: mix the 50ml contained in the kit with 950ml of water. 3. Standard. Add 1ml of ELISA buffer to the vial of lyophilized standard peptide (1μg) and vortex gently. If samples are to be extracted and re-suspended in ELISA buffer as described below, use ELISA buffer as a diluent. Otherwise, we encourage customers to use their own diluent such that standards and samples will be treated equally. 4. Standard curve. Make serial dilutions of the standard to cover the range of this kit. Please check the included datasheet for the appropriate range. 5. Antiserum. Add 5ml of ELISA buffer and vortex. 6. Biotinylated tracer. Add 5ml of ELISA buffer to the vial of lyophilized biotinylated peptide and vortex. Please check the datasheet for exceptions.

1. Into each well of the immunoplate add 50μl standard or sample (in ELISA buffer) 25μl antiserum (in ELISA buffer) Add 75μl ELISA buffer to blank wells. 2. Incubate at room temperature for 1 hour. Shorter incubation may result in lower sensitivity. 3. Rehydrate the Bt-tracer (in ELISA buffer) and add 25μl per well. 4. Incubate at room temperature for 2 hours. 5. Wash immunoplate 5 times with 300μl per well of ELISA buffer. Be careful not to cross-contaminate between wells in the first wash/dispensing cycle. In each wash cycle empty plate contents with a rapid flicking motion of the wrist, then gently blot dry the top of plate on paper towels. Dispense 300μl of ELISA buffer into each well and gently shake for at least a few seconds. Thorough washing is essential. 6. Add 100μl per well of streptavidin-HRP. Tap or centrifuge the SA-HRP vial to collect all liquid contents on the bottom of the vial. Dilute 1/200 in ELISA buffer (60μl in 12ml) and vortex gently. Add 100μl to all the wells, including blanks. 7. Incubate at room temperature for 1 hour. 8. Prepare TMB chromogenic solution immediately before use by mixing 20 parts of the TMB substrate buffer (citrate, brought to room temperature) with 1 part TMB - H 2 O 2 solution (TMB substrate stock). This dilution should be used within 15 minutes after preparation. 9. Wash immunoplate 5 times (see step 5). 10. Add 100μl per well of TMB solution. Add to all the wells, including blanks. 11. Incubate at room temperature (usually 10 to 30 minutes). You may read the developing blue color at 650nm and use the data for your calculations. 12. Terminate reactions by adding 100μl 2N HCl per well. 13. Read absorbance at 450nm within fifteen minutes.

Plot data and calculate results. We recommend that you use curve fitting software for your data analysis. Plate readers often include such software packages. This is, however, not essential and you may opt to plot manually on semi-log paper. You can also use a spreadsheet program. Should you need help with the latter method we recommend the following procedure. Set up a spreadsheet as shown below (note that the values on the spreadsheet are merely illustrative and are not necessarily typical for this particular kit). If you e-mail us (contact information on front cover) we will be happy to send you the actual working Excel spreadsheet shown below. Set up an 8 × 12 area to match the layout of the plate and copy the plate reader data in it. Calculate the average of the blanks on another cell as indicated by the arrows starting from cells A1 and A2. Enter the concentration of the standards (see under ng/ml in figure). Calculate the average of the ODs of the standards and subtract the background (blank) as indicated by the arrows for the last standard (cells H1 and H2). Make a standard curve by plotting the OD readings (minus the blank average) against the standard concentrations in ng/ml. Use the equation shown below to calculate the values on the "FIT" column and plot a smooth line of FIT values versus standard concentrations. Then change the parameters a (max), b (slope), c (IC50), and d (min), until you are satisfied that fit is good. y = [ (a-d) / (1 + (x/c) b ) ] + d Next, calculate the average of your sample readings and subtract the blank average (see arrows starting from A3 and A4, and the arrows leading to "Average"). Finally, you may isolate x in the equation above to calculate the concentrations in ng/ml for all your samples: x = c ( (y-a) / (d-y) ) 1/b Caution: when you calculate sample concentrations using the "reverse" equation if y = d or y > a or y < d, the reading is out of range and the calculation will yield an error or a meaningless negative concentration.

AVERAGE IC50: 3 ng/ml

0-100ng/ml

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet