Oxytocin RIA KIT,进口催产素放免试剂盒
Oxytocin (Human, Rat, Mouse, Bovine)
RIA Kit
(Range: 1.25-160 pg/ml)
Introduction
Contents:
This kit is designed to measure a specific peptide and its related
peptides by a competitive radioimmunoassay method. It is intended for in vitro study only. The antibody used for this assay was
raised against a synthetic form of the peptide.
1. RIA buffer 50 ml (4x concentrate) (large bottle, silver
cap)
2. Standard Peptide 12.8 µg
(lyophilized powder in an eppendorf tube, purple cap)
3. Rabbit antibody specific for the peptide
(lyophilized powder, blue cap)
4. 125I-peptide, 1.5 µCi
(lyophilized powder in an eppendorf tube, red cap)
5. Goat Anti-Rabbit IgG Serum (GAR)
(lyophilized powder, gold cap)
6. Normal Rabbit Serum (NRS)
(lyophilized powder, green cap)
7. Positive Control (small bottle, silver cap)
(lyophilized powder in an eppendorf tube)
8. General Protocol, 1 booklet
Materials for extraction are not included.
.
PAGE 2
Storage
General Information
Sample Preparation
This kit contains reagents sufficient for 125 RIA tubes. 125I-peptide
expires in approximately 6 weeks. Store at -20°C upon receipt. We
strongly recommend that this kit be used as soon as possible upon
receiving. All solutions should be used on the same day as rehydration.
The assay is based upon the competition of hot 125I-peptides and
cold peptides (standard or unknown) binding to the limited quantity of antibodies specific for the peptides in each reaction mixture.
As the concentration of standard or unknown sample in the reaction increases, the amount of 125I-peptide able to bind to the antibody decreases. By measuring the amount of 125I-peptide bound as
a function of the concentration of the peptide (in standard reaction
mixtures), it is possible to construct a “standard curve” from which
the concentration of the peptide in the unknown sample can be determined. The assay requires two overnight incubations, so please
plan accordingly.
Plasma, serum, culture media, tissue homogenate, CSF, urine or
any biological fluid can be assay as long as the level of peptide in
the sample is high enough for the sensitivity of the kit to detect.
Blood Collection: See page 10.
PAGE 3
Radio Immunoassay Kit Quick Guide
Add standards, samples and antibodies
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Vortex and incubate for 16-24 hours at 4ºC
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Add 125I-peptides
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Vortex and incubate for another 16-24 hours at 4ºC
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Add GAR and NRS (except TC tubes)
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Vortex and incubate at room temperature for 90 minutes
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Add RIA buffer (except TC tubes)
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Vortex and centrifuge for 20 minutes at 3,000 rpm / 1,700 x g
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Aspirate off the supernatant (except TC tubes)
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Count assay tubes
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Calculation of results
PAGE 4
General procedure for utilization of the ria kit:
1. Dilute the RIA buffer (4X concentrate) (large bottle, silver cap)
with 150 ml of distilled water. This buffer will be used to reconstitute all of the other compounds in this kit and should be
used for dilution of samples if needed.
2. Reconstitute the standard peptide (purple cap) with 1 ml of RIA
buffer. Vortex at least two minutes until ALL the peptide powder is completely dissolved in the eppendorf tube.
Note: Before adding buffer, carefully examine the eppendorf
tube containing the standard. During shipping, part or all
of the lyophilized standard may have come loose from
the bottom of the tube causing it to stick to the cap or
walls of the tube. Gently tap or centrifuge the tube to
dislodge powder from the cap or walls. Carefully open
the tube and add buffer.
3. Reconstitute the antibody (blue cap) with 13 ml of RIA buffer
and vortex.
4. Reconstitute the Positive Control (small bottle, silver cap) with
1ml of RIA buffer and vortex the eppendorph tube.
5. Reconstitute unknown samples with RIA buffer to a concentration that will allow their values to fall within the linear range of
the standard curve (we cannot ensure success with other buffers that have not been tested). (Refer to step 7 on page 11).
Note: The remaining reagents are not required at this time and
should be stored in their lyophilized state until needed.
990
µl
990
µl
875
µl
500
µl
500
µl
10µl 10µl
12.8
μg/ml
128,000
pg/ml
1,280
pg/ml
160
pg/ml
80
pg/ml
2.5
pg/ml
500µl 500µl 125µl
G B A 1 0 Stock
500
µl
500µl
H
1.25
pg/ml
H
500µl
PAGE 5
6. Prepare dilutions of the standard as shown in Page 4 and
Table 1 below. Vortex each tube and switch each tip between
dilutions.
Table 1: Standard Dilutions
Tube RIA Buffer Standard Std. Conc.
Stock 1ml Powder 12.8 μg/ml
0 990 µl 10 µl of Stock 128,000 pg/ml
1 990 µl 10 µl of 0 1,280 pg/ml
A 875 µl 125 µl of 1 160 pg/ml
B 500 µl 500 µl of A 80 pg/ml
C 500 µl 500 µl of B 40 pg/ml
D 500 µl 500 µl of C 20 pg/ml
E 500 µl 500 µl of D 10 pg/ml
F 500 µl 500 µl of E 5 pg/ml
G 500 µl 500 µl of F 2.5 pg/ml
H 500 µl 500 µl of G 1.25 pg/ml
7. Set up RIA reactions (see Table 2 on page 6) in up to 125
12 mm x 75 mm polystyrene tubes.(DO NOT USE GLASS
TUBES)
a) Number tubes TC-1, TC-2, NSB-1, NSB-2, TB-1, TB-2
and #7 - #22 for the standards.
b) Number tubes #23, #24 for the positive controls.
c) Number tubes #25 up to #125 for the unknown samples.
d) Pipette 200 µl of RIA buffer into each NSB tube.
e) Pipette 100 µl of RIA buffer into each TB tube.
f) Pipette 100 µl of standards H through A into duplicate
tubes #7-#22.
Note: The tubes should be prepared in reverse order of serial
dilution so that the concentration increases as the number
of the tube increases. For example: Begin by pipetting
100 µl of standard H into tubes #7 & #8, then proceed
to standard G into #9 & #10...
g) Pipette 100 µl of positive control into tubes #23 & #24.
h) Pipette 100 µl of unknown sample into duplicate tubes:
tube #25 and up.
PAGE 6
i) Pipette 100 µl of antibody into all tubes
EXCEPT TC AND NSB TUBES.
j) Vortex the contents of each tube.
k) Cover and incubate all tubes at 4°C for 16-24 hours.
Tube Contents RIA
Buffer
Std or
Samples
Antibody Working
Tracer Solution (WTS)
TC-1 &2 Total Counts 100 µl
NSB-1 & 2 Non-specific
binding
200 µl 100 µl
TB-1 & 2 Total
binding
100 µl 100 µl 100 µl
7, 8 H Standard 100 µl 100 µl 100 µl
9, 10 G Standard 100 µl 100 µl 100 µl
11, 12 F Standard 100 µl 100 µl 100 µl
13, 14 E Standard 100 µl 100 µl 100 µl
15, 16 D Standard 100 µl 100 µl 100 µl
17, 18 C Standard 100 µl 100 µl 100 µl
19, 20 B Standard 100 µl 100 µl 100 µl
21, 22 A Standard 100 µl 100 µl 100 µl
23, 24 Positive
Control
100 µl P.C 100 µl 100 µl
25, 26 Sample 1 100 µl 100 µl 100 µl
27, 28 Sample 2 100 µl 100 µl 100 µl
Etc. Etc. 100 µl 100 µl
(After 16-24 hours)
8. a) Add 1 ml RIA buffer into the 125I-peptide in the eppendorf tube
(red cap) and vortex. This is the Stock Tracer Solution (STS).
Take 10 µl of STS and check its concentration (cpm/µl) using a
γ-counter.
b) Prepare 13 ml RIA buffer in a polystyrene container. Add an
adequate amount of STS into this container so that the concentration is 8,000-10,000 cpm/100µl. Confirm the concentration
with a γ-counter. This is the Working Tracer Solution (WTS).
c) Add 100 µl of the WTS to each tube.
Table 2: Contents in Each Tube for Incubation
PAGE 7
9. Vortex the contents in each tube.
10. Cover and incubate all tubes for another 16-24 hours at 4°C.
(After 16-24 hours)
11. Reconstitute the Goat Anti-Rabbit IgG serum (GAR)
(gold cap) with 13 ml of RIA buffer.
12. Reconstitute the Normal Rabbit Serum (NRS) (green cap)
with 13 ml of RIA buffer.
Note: The Total Count Tubes (TC) are not involved in the
following reactions.
13. Add 100 µl of GAR to each tube except the TC tubes.
14. Add 100 µl of NRS to each tube except the TC tubes.
15. Vortex the contents of each tube. Incubate all tubes at room
temperature for at least 90 minutes
16. Add 500 µl of RIA buffer to each tube (except the TC tubes)
and vortex.
17. Centrifuge all tubes (except the TC tubes) at 3,000 rpm
(approx. 1700 x g) for at least 20 minutes at 4°C.
18. Carefully aspirate ALL the supernatant (without touching
the pellet) immediately following centrifugation (do not
decant as the pellet might be lost or excess liquid could be
left). DO NOT ASPIRATE THE TC TUBES.
Note: For best results, the supernatant should be immediately
aspirated after centrifugation. If the pellet sits for more
than 15-30 minutes, it may become detached and make
aspiration difficult. Do not aspirate any solids.
19. Use a γ-counter to count the cpm of the pellet.
PAGE 8
Oxytocin RIA KIT,进口催产素放免试剂盒
Calculations:
1. Calculate the average NSB and label it as NSB using cpm.
2. Calculate the average TB and label it as TB using cpm.
3. Use the following equation to find B0
: B0
= TB-NSB
4. Use the following calculation to determine the B/B0
(%) for
paired standards and unknown samples:
a) Example for standard H:
B/B0
(%) = (Avg. cpm Std. H) - (NSB)
B0
b) Standards G through A (tubes #9-#22), Positive Controls
(tubes #23 & #24) and the unknown samples (tubes #25
up to #125) are handled as shown above for standard H.
5. Examples of Tabulated Data:
Tube Samples Peptide Average
cpm
B/B0
(%)
TC-1,2 9,000
NSB-1,2 150
TB-1,2 0 pg/ml 4,000 100
7,8 H Standard 1.25 pg/ml 3,471 93.3
9,10 G Standard 2.5 pg/ml 2,287 55.5
21,22 A Standard 160 pg/ml 420 7.0
23,24 Positive
Control
? 2,171 52.5
25,26 Sample 1 ? 976 21.5
27,28 Sample 2 ? 1,383 32.0
x 100%
Table 3: Tabulated Data After Calculation
PAGE 9
Examples of Tabulated Data Continued:
Total Count (Total activity) (cpm/100µl) = 9,000 cpm
NSB = 150 cpm
TB = 4,000 cpm
B0
= 4,000 cpm - 150 cpm = 3850 cpm
6. On semi log graph paper, plot B/B0
(%) (in decimal scale )
versus the standard peptide concentrations (in log scale).
a) Label the concentrations of standard H through A (1.25-
160 pg/ml) on the X-axis (log scale).
b) Label B/B0
(%) (0 to 100%) on the Y-axis (decimal
scale)
c) Plot B/B0
(%) for each standard concentration directly
above its X-axis designation.
d) Draw the “Best-Fit” curve.
7. Determination of the concentration of peptide in unknown
samples.
a) Using B/B0
(%) calculated for each unknown sample,
read across the graph to the point of intersection with
the “Best-Fit” curve.
b) The corresponding X-axis coordinate is equivalent to the
concentration of peptide (pg/ml) in the assayed sample.
c) To calculate the amount of peptide in the original
sample, multiply the concentration of the assayed sample
by any dilution factor used to prepare the sample.
8. Conversion of units:
pg/ml x 1000 ÷ Mol. Wt. = PMole/L
PAGE 10
Suggested method for the extraction of Peptides from plasma
Blood Withdrawal:
Collect blood samples into Lavender Vacutainer tubes (Cat. No.
VT-6450) which contain EDTA. Each tube can collect 7ml of blood/
tube. Gently rock the Lavender Vacutainer tubes several times immediately after collection of blood for anti-coagulation. Transfer
the blood from the Lavender Vacutainer tubes to centrifuge tubes
containing aprotinin (Cat. No. RK-APRO) (0.6 TIU/ml of blood)
and gently rock several times to inhibit the activity of proteinases.
Centrifuge the blood at 1600 x g for 15 minutes at 4ºC and collect
the plasma. Plasma kept at -70ºC is stable for up to one month.
PAGE 11
References:
1. Berson, S.A. and Yalow, R.S. Kinetics of reaction between
insulin and insulin binding antibody. J. Clin. Invest 36:873,
(1957).
2. Patrono, C. and Peskar, B.A., (eds) Radioimmunoassay in basic and clinical pharmacology. Heidelberg, Springer-Verlag,
(1987).
3. Reuter, A., Vrindts-Gevaerts, Y., Meuleman-Gathy, R., Joris,
J., Chretien, M. and Franchimont, P. A Radioimmunoassay for
Beta-Endorphins. (BETA-END) and (BETA-LPH) in Plasma.
Horm Res 25:236, (1987).
4. Dwenger, A. Radioimmunoassay: An Overview, J.Clin. Biochem. 22: 883, (1984)
5. Wang, Y.N., Chou J., Chang, D., Chang, J.K., Avila, C. and
Romero, R. Endothelin-1 in Human Plasma and Amniotic Fluid. In Endothelin-Derived Contracting Factors, edited by G.
Rubanyi and P. Vanchoutte, Karger, Basel, pg. 143, (1990).
CAUTION: SOME REAGENTS IN THIS KIT CONTAIN SODIUM
AZIDE WHICH MAY REACT WITH LEAD AND COPPER PLUMBING TO FORM EXPLOSIVE METAL AZIDES. FLUSH WITH LARGE
VOLUMES OF WATER DURING DISPOSAL.
PAGE 12
Instructions for possession, handling and use of
radioactive material
Precautions in Handling Radioactive Material:
The user should store the by-product material, until used, in the
original shipping container or in a container providing equivalent
radiation protection.
When Handling Radioactive Materials: there should be no drinking, eating or smoking; hands should be covered with gloves, and
thoroughly washed after; do not pipette by mouth.
Spills must be quickly and thoroughly cleaned up. Surfaces involved should be washed with an alkali detergent (alconox or the
equivalent).
Persons under 18 should not be permitted to handle radioactive
material or enter radioactive areas.
Disposal:
Radioactive waste should be disposed of in compliance with Federal, State, and Local Government regulations. Agencies that can
be consulted include the Environmental Protection Agency (EPA),
the Nuclear Regulatory Commission (NRC), the Department of
Energy (DOE), and the Department of Transportation.
THIS PACKAGE CONFORMS TO THE CONDITIONS AND LIMITATIONS SPECIFIED IN 49 CFR173.421 FOR EXCEPTED RADIOACTIVE MATERIAL LIMITED QUANTITY, N.O.S. UN2910.
PAGE 13
Notes:
Oxytocin RIA KIT,进口催产素放免试剂盒
原创作者:上海信帆生物科技有限公司