Manual - RayBiotech, Inc.

RayBio® Human/Mouse/Rat
Somatostatin Enzyme Immunoassay Kit
Catalog #: EIA-SOM, EIAM-SOM, EIAR-SOM
User Manual
Last revised December 1, 2015
Caution:
Extraordinarily useful information enclosed
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1
Table of Contents
Section
Page #
I.
Introduction
3
II.
General Description
4
III.
How It Works
4
IV.
Storage
5
V.
Reagents
5
VI.
Additional Materials Required
6
VII.
Reagent Preparation
A. Preparation of Plate and Anti-Somatostatin Antibody
B. Preparation of Biotinylated Peptide (Item F)
C. Preparation of Standards
D. Preparation of Positive Control
E. Preparation of Samples
F. Preparation of Wash Buffer and HRP-Strep
6
6
7
8
9
9
10
VIII.
Assay Procedure
10
IX.
Assay Procedure Summary
11
X.
Calculation of Results
A. Typical Data
B. Sensitivity
C. Detection Range
D. Reproducibility
E. Assay Diagram
12
12
12
12
12
13
XI.
Specificity
14
XII.
Select Publications
14
XIII.
Troubleshooting Guide
15
Please read the entire manual carefully before starting your experiment
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I. Introduction
Somatostatin, also known as growth hormone-inhibiting hormone (GHIH) that
regulates the endocrine system and affects neurotransmission and cell proliferation
via interaction with G protein-coupled somatostatin receptors and inhibition of the
release of numerous secondary hormones. Somatostatin regulates insulin and
glucagon.
Somatostatin is secreted in several locations in the digestive system: stomach,
intestine and delta cells of the pancreas. In the stomach, somatostatin acts on the
acid-producing parietal cells via G-coupled receptor to reduce secretion.
Somatostatin also indirectly decreases stomach acid production by preventing the
release of other hormones including gastrin, secretin and histamine which
effectively slows down the digestive process.
Somatostatin has two active forms produced by alternative cleavage of a single
preproprotein: one of 14 amino acids, the other of 28 amino acids. In all
vertebrates, there exist six different somatostatin genes that have been named
SS1, SS2, SS3, SS4, SS5, and SS6. The six different genes along with the five
different somatostatin receptors allow somatostatin to possess a large range of
functions. Humans have only one somatostatin gene, SST.
3
II. General Description
The RayBio® Somatostatin Enzyme Immunoassay (EIA) Kit is an in vitro
quantitative assay for detecting Somatostatin peptide based on the competitive
enzyme immunoassay principle.
In this assay, a biotinylated Somatostatin peptide is spiked into the samples and
standards. The samples and standards are then added to the plate, where the
biotinylated Somatostatin peptide competes with endogenous (unlabeled)
Somatostatin for binding to the anti-Somatostatin antibody. After a wash step, any
bound biotinylated Somatostatin then interacts with horseradish peroxidase (HRP)streptavidin, which catalyzes a color development reaction. The intensity of the
colorimetric signal is directly proportional to the amount of captured biotinylated
Somatostatin peptide and inversely proportional to the amount of endogenous
Somatostatin in the standard or samples. A standard curve of known concentration
of Somatostatin peptide can be established and the concentration of Somatostatin
peptide in the samples can be calculated accordingly.
III. How It Works
4
IV. Storage
The entire kit may be stored at -20°C to -80°C for up to 6 months from the date of
shipment. For extended storage, it is recommended to store at -80°C. Avoid
repeated freeze-thaw cycles. For prepared reagent storage, see table below.
V. Reagents
Component
Size / Description
Storage / Stability
After Preparation
Somatostatin Microplate
(Item A)
96 wells (12 strips x 8 wells) coated with
secondary antibody.
1 month at 4°C*
Wash Buffer Concentrate
(20X) (Item B)
25 ml of 20X concentrated solution.
1 month at 4°C
Standard Somatostatin
Peptide (Item C)
2 vials of Somatostatin Peptide. 1 vial is enough
to run each standard in duplicate.
The first standard:
2-3 days at 4°C
Additional dilutions:
Do not store
Anti-Somatostatin Polyclonal
Antibody (Item N)
2 vials of anti-Somatostatin.
1 month at 4°C
5X Assay Diluent B (Item E)
15 ml of 5X concentrated buffer. Diluent for both
standards and samples including serum, plasma,
cell culture media or other sample types.
1 month at 4°C
Biotinylated Somatostatin
Peptide (Item F)
2 vials of Biotinylated Somatostatin Peptide, 1
vial is enough to assay the whole plate.
2-3 days at 4°C
HRP-Streptavidin
Concentrate (Item G)
600 µl 800X concentrated HRP-conjugated
streptavidin.
Do not store and
reuse
Positive Control (Item M)
1 vial of Positive Control.
2-3 days at 4°C
TMB One-Step Substrate
Reagent (Item H)
12 ml of 3,3,5,5'-tetramethylbenzidine (TMB) in
buffer solution.
N/A
Stop Solution (Item I)
8 ml of 0.2 M sulfuric acid.
N/A
*Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
5
VI. Additional Materials Required
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Microplate reader capable of measuring absorbance at 450 nm
Precision pipettes to deliver 2 µl to 1 ml volumes
Adjustable 1-25 ml pipettes for reagent preparation
100 ml and 1 liter graduated cylinders
Absorbent paper
Distilled or deionized water
SigmaPlot software (or other software which can perform four-parameter
logistic regression models)
Tubes to prepare standard or sample dilutions
Orbital shaker
Aluminum foil
Plastic wrap
VII. Reagent Preparation
Keep kit reagents on ice during reagent preparation steps.
A. Preparation of Plate and Anti-Somatostatin Antibody
1. Equilibrate plate to room temperature before opening the sealed pouch.
2. Label removable 8-well strips as appropriate for your experiment.
3. 5X Assay Diluent B (Item E) should be diluted 5-fold with deionized or distilled
water.
4. Briefly centrifuge the anti-Somatostatin antibody vial (Item N) Then add 50 µl
of 1X Assay Diluent B to the vial to prepare the antibody concentrate. Pipette
up and down to mix gently.
5. The antibody concentrate should then be diluted 100-fold with 1X Assay
Diluent B. This is your anti-Somatostatin antibody working solution, which will
be used in step 2 of Assay Procedure (Section VIII).
Note: The following steps may be done during the antibody incubation procedure
(step 2 of Assay Procedure)
6
B. Preparation of Biotinylated Somatostatin (Item F)
5. Briefly centrifuge the vial of Biotinylated Somatostatin (Item F) before use.
6. See the image below for proper preparation of Item F. Transfer the entire
contents of the Item F vial into a tube containing 10 ml of 1X Assay Diluent B.
This is your Working Stock of Item F. Pipette up and down to mix gently.
The final concentration of biotinylated Somatostatin will be 20 ng/ml.
a. Second Dilution of Item F for Standards: Add 2 ml of Working Stock Item
F to 2 ml of 1X Assay Diluent B. The final concentration of biotinylated
Somatostatin will be 10 ng/ml.
b. Second Dilution of Item F for Positive Control: Add 100 µl of Working
Stock Item F to 100 µl of the prepared Positive Control (Item M). (See
section D for Positive Control preparation) The final concentration of
biotinylated Somatostatin will be 10 ng/ml.
c. Second Dilution of Item F for samples: Add 125 µl of Working Stock Item
F to 125 µl of prepared sample (see section E for sample preparation).
This is a 2-fold dilution of your sample. The final concentration of
biotinylated Somatostatin will be 10 ng/ml.
7
C. Preparation of Standards
7. Label 6 microtubes with the following concentrations: 1000 ng/ml, 100 ng/ml,
10ng/ml, 1 ng/ml, 100 pg/ml and 0 pg/ml. Pipette 450 µl of biotinylated
Somatostatin Item F working solution (prepared in step 6a) into each tube,
except the 1,000 ng/ml (leave this one empty).
It is very important to make sure the concentration of biotinylated Somatostatin is
10 ng/ml in all standards.
8. Briefly centrifuge the vial of Somatostatin Standard (Item C). Pipette 8 µl of
Item C and 792 µl of 10 ng/ml biotinylated Somatostatin working solution
(prepared in step 6a) into the tube labeled 1000 ng/ml. Mix thoroughly. This
solution serves as the first standard (1,000 ng/ml Somatostatin standard, 10
ng/ml biotinylated Somatostatin).
9. To make the 100 ng/ml standard, pipette 50 µl of the 1000 ng/ml Somatostatin
standard into the tube labeled 100 ng/ml. Mix thoroughly.
10. Repeat this step with each successive concentration, preparing a dilution
series as shown in the illustration below. Each time, use 450 µl of biotinylated
Somatostatin and 50 µl of the prior concentration until the 100 pg/ml is
reached. Mix each tube thoroughly before the next transfer.
8
D. Positive Control Preparation
11. Briefly centrifuge the Positive Control vial (Item M).
12. Refer to step 6b. This is a 2-fold dilution of the Positive Control. The final
concentration of biotinylated Somatostatin should still be 10 ng/ml.
The Positive Control is a cell culture media sample that serves as a system control
to verify that the kit components are working. The resulting OD will not be used in
any calculations; if no positive competition is observed please contact RayBiotech
Technical Support. The Positive Control may be diluted further if desired, but be
sure the final concentration of biotinylated Somatostatin is 10 ng/ml.
E. Sample Preparation
13. If you wish to perform a 2-fold dilution of your sample, proceed to step 6c. If
you wish to perform a higher dilution of your sample, dilute your sample with
1X Assay Diluent B before performing step 6c.
EXAMPLE (to make a 4-fold dilution of sample):
a. Dilute sample 2-fold (62.5 µl of sample + 62.5 µl of 1X Assay Diluent B.).
b. Perform step 6c (125 µl of working solution Item F + 125 µl of sample
prepared above).
The total volume is 250 µl, enough for duplicate wells on the microplate.
It is very important to make sure the final concentration of the biotinylated
Somatostatin is 10 ng/ml.
Note: Optimal sample dilution factors should be determined empirically, however you
may contact technical support (888-494-8555; techsupport@raybiotech.com) to
obtain recommended dilution factors for serum.
9
F. Preparation of Wash Buffer and HRP
14. If Item B (20X Wash Concentrate) contains visible crystals, warm to room
temperature and mix gently until dissolved.
15. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield
400 ml of 1X Wash Buffer.
16. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use.
17. Dilute the HRP-Streptavidin concentrate 800-fold with 1X Assay Diluent B.
VIII. Assay Procedure
1. Keep kit reagents on ice during reagent preparation steps. It is recommended
that all standards and samples be run at least in duplicate.
2. Add 100 µl of Anti-Somatostatin Antibody (Item N) (See Reagent Preparation
step 3) to each well. Incubate for 1.5 hours at room temperature with gentle
shaking (1-2 cycle/sec). You may also incubate overnight at 4ºC.
3. Discard the solution and wash wells 4 times with 1X Wash Solution Buffer (200300 µl each). Washing may be done with a multichannel pipette or an automated
plate washer. Complete removal of liquid at each step is essential to good assay
performance. After the last wash, remove any remaining Wash Buffer by
aspirating or decanting. Invert the plate and blot it against clean paper towels.
4. Add 100 µl of each standard (see Reagent Preparation Section C), Positive
Control (see Reagent Preparation Section D) and sample (see Reagent
Preparation Section E) in appropriate wells. Be sure to include a blank well
(Assay Diluent only). Cover wells and incubate for 2.5 hours at room
temperature with gentle shaking (1-2 cycles/sec) overnight or at 4ºC.
5. Discard the solution and wash 4 times as directed in Step 3.
10
6. Add 100 µl of prepared HRP-Streptavidin solution (see Reagent Preparation
step 7) to each well. Incubate for 45 minutes at room temperature with gentle
shaking. It is recommended that incubation time should not be shorter or longer
than 45 minutes.
7. Discard the solution and wash 4 times as directed in Step 3.
8. Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate
for 30 minutes at room temperature in the dark with gentle shaking (1-2
cycles/sec).
9. Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately.
IX. Assay Procedure Summary
1. Prepare all reagents, samples and standards as instructed.
2. Add 100 µl anti-Somatostatin to each well. Incubate 1.5 hours at room
temperature or overnight at 4ºC.
3. Add 100 µl standard or sample to each well. Incubate 2.5 hours at room
temperature or overnight at 4ºC.
4. Add 100 µl prepared Streptavidin solution. Incubate 45 minutes at room
temperature.
5. Add 100 µl TMB One-Step Substrate Reagent to each well. Incubate 30 minutes
at room temperature.
6. Add 50 µl Stop Solution to each well. Read at 450 nm immediately.
11
X. Calculation of Results
Calculate the mean absorbance for each set of duplicate stands, controls, and
samples and subtract the blank optical density. Plot the standard curve using
SigmaPlot software (or other software which can perform four-parameter logistic
regression models), with standard concentration on the x-axis and percentage of
absorbance (see calculation below) on the y-axis. Draw the best-fit curve through the
standard points.
Percentage absorbance = (B-blank OD)/B 0-blank OD) where
B = OD of sample or standard and
B0 = OD of zero standard (total binding)
A. Typical Data
These standard curves are for demonstration only. A standard curve must be run with
each assay.
B. Sensitivity
The minimum detectable concentrations of Somatostatin is
C. Detection Range
0.1-1,000 ng/ml
D. Reproducibility
Intra-Assay: CV<10%
Inter-Assay: CV<15%
E. Assay Diagram
12
Recommended Plate Layout:
Key:
Blank = Buffer Only
Total Binding = Biotin- Somatostatin only
Standard 1 = 1000 ng/ml
Standard 2 = 100 ng/ml
Standard 3 = 10 ng/ml
Standard 4 = 1 ng/ml
Standard 5 = 100 pg/ml
Pos Control = Biotin with Item M
13
XI. Specificity
This EIA kit is designed to detect pro-Somatostatin, not active Somatostatin.
XIV. Select EIA Publications
1. Plum L, Lin HV, Dutia R, Tanaka J, Aizawa KS, et al. The Obesity Susceptibility
Gene Carboxypeptidase E Links FoxO1 Signaling in Hypothalamic Proopiomelanocortin Neurons with Regulation of Food Intake. Nature Med.
2009;15(10):1195-1201. (Ghrelin EIA, EIA-GHR-1)
2. Hug C, Lodish HF. Visfatin: a new adipokine. Science. 2005; 307(5708):366-7.
3. Kim MK. Crystal structure of visfatin/pre-B cell colony-enhancing factor
1/nicotinamide phosphoribosyltransferase, free and in complex with the anticancer agent FK-866. J Mol Biol. 2006; 362(1):66-77.
4. Revollo, J.R., et al. The NAD biosynthesis pathway mediated by nicotinamide
phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J. Biol.
Chem. 2004; 279: 50754-50763.
5. Oh-I S, Shimizu H, Satoh T, et al. Identification of nesfatin-1 as a satiety
molecule in the hypothalamus. Nature 2006; 443 (7112): 709-12.
6. Zhang J, Ren P, Avsian-Kretchmer O, Luo C, Rauch R, Klein C, Hsueh A.
Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on
food intake. Science 2005; 310 (5750): 996-9.
7. Cummings D, Weigle D, Frayo R, Breen P, Ma M, Dellinger E, Purnell J. Plasma
ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J
Med 2002; 346 (21): 1623-30.
8. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature
2002; 407 (6806): 908-913.9. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo
H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from
stomach. Nature 1999; 402 (6762): 656-60.
14
XIII. Troubleshooting Guide
Problem
Cause
Solution
Inaccurate pipetting
Improper standard dilution
Check pipettes
Briefly centrifuge Item C and dissolve
the powder thoroughly by gently
mixing
Low signal
Improper preparation of
standard and/or
biotinylated antibody
Too brief incubation times
Inadequate reagent
volumes or improper dilution
Briefly spin down vials before
opening. Dissolve the powder
thoroughly.
Ensure sufficient incubation time;
assay procedure step 2 may be done
overnight
Check pipettes and ensure correct
preparation
Large CV
Inaccurate pipetting
Air bubbles in wells
Check pipettes
Remove bubbles in wells
High
background
Plate is insufficiently
washed
Contaminated wash buffer
Review the manual for proper wash.
If using a plate washer, ensure that
all ports are unobstructed.
Make fresh wash buffer
Improper storage of the
ELISA kit
Stop solution
Follow storage recomendations in
sections IV and V. Keep substrate
solution protected from light.
Add stop solution to each well before
reading plate
Poor standard
curve
Low sensitivity
15
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©2015 RayBiotech, Inc
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