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神经肽Y放免试剂盒，NPY放免试剂盒代测
NPY (Human, Rat, Mouse) - RIA
Kit
(range: 10-1280 pg/ml)
introduction
contents:
This kit is designed to measure a specific peptide and its related
peptides by a competitive radioimmunoassay. It is intended for in
vitro protocols only. The antiserum used for this assay was raised
against a synthetic form of the peptide.
1. RIA buffer, 50ml (4x concentrate)
2. Standard peptide, 12.8 μg (lyophilized powder)
3. Rabbit antiserum specific for the peptide, 13 ml
(lyophilized powder)
4. 125I-peptide, 1.5 μCi (lyophilized powder)
5. Goat Anti-Rabbit IgG Serum (GAR), 13 ml (lyophilized
powder)
6. Normal Rabbit Serum (NRS), 13ml (lyophilized powder)
7. Positive Control (lyophilized powder)
8. Instructions, 1 booklet
Materials for extraction are not included.
If needed, extraction procedure for plasma is provided.
Note: Phoenix Pharmaceuticals, Inc. guarantees that its
products conform to the information contained in this
publication. The purchaser must determine the suitability
of the product for their particular needs and establish
optimum sample concentration.
PAGE 1
PAGE 2
Storage
General Information
Assay Conditions
This kit contains reagents sufficient for 125 RIA tubes. 125I-peptide
expires in approximay 6 weeks. Store at -20°C upon receipt. We
strongly recommend that this kit be used as soon as possible upon
receiving it. All solutions should be used on the same day as rehydration.
The assay is based upon the competition of 125I-peptide and peptide
(standard or unknown) binding to the limited quantity of antibodies
specific for peptide in each reaction mixture. As the quantity of
standard or unknown sample in the reaction increases, the amount
of 125I-peptide able to bind to the antibody is decreased. By measuring
the amount of 125I-peptide bound as a function of the concentration
of peptide (in standard reaction mixtures), it is possible to construct
a “standard curve” from which the concentration of peptide
in the unknown sample can be determined. The assay requires two
overnight incubations, so plan accordingly.
Plasma, serum, culture media, tissue homogenate, CSF, urine or
any biological fluid can be assay as long as the level of sample is
high enough for the sensitivity of the kit to detect.
Blood Collection: See page 10.
Plasma Extraction: Extraction is strongly recommended but not
required. It is up to the discretion of the paper reviewers. See page
10.
Tissue Extraction Method: Visit www.phoenixpeptide。。ｃｏｍ and
click on the link, “Sample Preparation”, for more information.
PAGE 3
General procedure for utilization of the ria kit:
1. Dilute the RIA buffer (4X concentrate) with 150ml 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 with 1ml of RIA buffer.
Mix well and store on ice.
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.
After adding the RIA buffer, vortex for approximay 2
minutes until ALL the peptide powder is compley
dissolved. For hydrophobic and hard-to-dissolve
peptides, longer vortexing may be required.
3. Reconstitute the rabbit anti-peptide serum with 13ml of RIA
buffer, mix well and store on ice.
Note: The remaining reagents are not required at this time and
should be stored in their lyophilized state until needed.
4. Reconstitute the Positive Controls with 1ml of RIA buffer.
Mix well and store on ice.
5. Reconstitute samples with RIA buffer (we cannot ensure success
with other buffers since they have not been tested).
PAGE 4
6. Prepare dilutions of the standard as below:
Tube Sample RIA Buffer Std. Conc.
Stock Powder 1.0ml 12.8 μg/ml
0 10 μl of Stock 990 μl 128,000 pg/ml
A 10 μl of 0 990 μl 1,280 pg/ml
B 500 μl of A 500 μl 640 pg/ml
C 500 μl of B 500 μl 320 pg/ml
D 500 μl of C 500 μl 160 pg/ml
E 500 μl of D 500 μl 80 pg/ml
F 500 μl of E 500 μl 40 pg/ml
G 500 μl of F 500 μl 20 pg/ml
H 500 μl of G 500 μl 10 pg/ml
Table 1: Standard Dilutions
7. Set up initial RIA reactions (see diagram on page 5) in 12 x
75 mm polystyrene 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 unkown 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: Reverse the order of preparation so that the concentration
increases as the number of the tube increases. For
example: Pipette 100 μl of standard H into tubes #7 & #8.
g) Pipette 100 μl of positive control in tubes #23 & #24.
h) Pipette 100 μl of unknown sample into duplicate tubes:
tube #25 and up.
PAGE 5
i) Pipette 100 μl of primary antibody (rabbit anti-peptide
serum) into all tubes EXCEPT TC AND NSB TUBES.
j) Vortex the contents of each tube.
k) Cover and incubate all tubes for 16-24 hours at 4°C.
8. Reconstitute the 125I-peptide with 13ml of RIA buffer and mix
well to make tracer solution. Please check the concentration
of this tracer solution and adjust it with RIA buffer until the
concentration (total activity) is 8,000-10,000 cpm/100 μl.
9. Add 100 μl of the tracer solution to each tube.
10. Vortex the contents in each tube.
11. Cover and incubate all tubes for 16-24 hours at 4°C.
Tube Contents RIA
Buffer
STD or
Samples
Primary
Antibody
125I
Peptide
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
Table 2: Contents before Incubation
PAGE 6
12. Reconstitute the Goat Anti-Rabbit IgG serum (GAR) with
13ml of RIA buffer
13. Reconstitute the Normal Rabbit Serum(NRS) with 13ml of
RIA buffer.
14. Add 100 μl of GAR to each tube except the TC tubes.
15. Add 100 μl of NRS to each tube except the TC tubes.
16. Vortex the contents of each tube. Incubate all tubes at room
temperature for 90 minutes
17. Add 500 μl of RIA buffer to each tube except the TC tubes
and vortex.
18. Centrifuge all tubes (except the TC tubes) at 3,000 rpm
(approx. 1700 x g) for 20 minutes at 4°C.
19. Carefully aspirate ALL the supernatant (without touching
the pellet) immediay 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 immediay
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.
20. Use a γ-counter to count the cpm of the pellet.
PAGE 7
Calculations:
1. Using cpm, calculate the average NSB and label this as NSB.
2. Using cpm, calculate the average TB and label this as TB.
3. To find B0 use the following equation: B0 = TB-NSB
4. To determine the B/B0 (%) for paired standards and unknown
samples use the following calculation:
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 10 pg/ml 3,471 93.3
9,10 G Standard 20 pg/ml 2,287 55.5
▲│▼
▲│▼
▲│▼
▲│▼
▲│▼
21,22 A Standard 1280 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 8
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 (10-
1280 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:
100 pg/ml x [1000 ÷ Mol. Wt.] = fmole/ml (PMole/L, pM)
PAGE 9
Summary of Assay Protocol
Add sample or standard and antibody
▼
Vortex and incubate 16-24 hours at 4ºC
▼
Add 125I-peptide
▼
Vortex and incubate for 16-24 hours at 4ºC
▼
Add GAR and NRS
▼
Vortex and incubate at room temperature for 90 minutes
▼
Add RIA buffer
▼
Vortex and centrifuge for 20 minutes at 1,700 x g
▼
Aspirate off the supernatant (except TC tubes)
▼
Count assay tubes
▼
Calculation of results
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 immediay 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.
Elution Solvents:
1. Buffer A (Cat. No. RK-BA-1)
2. Buffer B (Cat. No. RK-BB-1)
Extraction of Peptides from Plasma:
1. Acidify the plasma with an equal amount of buffer A. For
example, if you are using 1ml of plasma, add 1ml of buffer A.
Mix and centrifuge at 6,000 to 17,000 x g for 20 minutes at
4ºC. Keep the supernatant.
2. Equilibrate the SEP-COLUMN containing 200mg of C18 (Cat.
No. RK-SEPCOL-1) by washing with buffer B (1ml, once)
followed by buffer A (3ml, 3 times)
Note: From steps 3-5, no pressure should be applied to the
column.
PAGE 11
3. Load the acidified plasma solution onto the pre-treated C-18
SEP-COLUMN.
4. Slowly wash the column with buffer A (3ml, twice) and
discard the wash.
5. Elute the peptide slowly with buffer B (3ml, once) and collect
eluant into a polystyrene tube.
6. Evaporate eluant to dryness in a centrifugal concentrator or
by a suitable substitute method
7. Dissolve the residue in RIA buffer for radioimmunoassay as
follows: For normal subjects, dissolve in 250μl of RIA buffer
for a two-tube assay. Aliquot 100μl into each tube (50μl is
left over). Please note, at this point the sample has been
concentrated by 4. If each tube is found to contain 100pg/ml
of the peptide, then the total level of peptide in plasma =
100pg/ml ÷ 4 = 25pg/ml (where the concentration factor is 4).
If upon assay the peptide value exceeds or does not fall in the
range of detection, dilute or concentrate the sample
accordingly.
PAGE 12
Tips for extraction of plasma:
When using SEP-COLUMN for the first time, use the enclosed
bulb to apply pressure to the column after addition of 1ml of buffer
B to facilitate flow. From steps 3-5, no pressure should be applied.
Ensure there is a constant flow for all solutions during the extraction
procedure. Do not allow air bubbles to enter the C-18 matrix
for optimal sample processing and recovery.
Drying Sample After Extraction:
A combination of centrifugal concentrator (i.e Speedvac) and
a lyophilizer (freeze-dryer) produces the best results for drying
the sample after extraction. First, use a Speedvac to dry sample
for approximay 15 minutes to remove the organic layer. Then
snap-freeze the remaining sample, and freeze-dry overnight using
a lyophilizer. This two-step procedure produces a more consistent
fluffy powder that is easier to rehydrate than a sample dried only
with a centrifugal concentrator. However, if a centrifugal concentrator
is not accessible, freeze-drying overnight using a lyophilizer
will be sufficient.
PAGE 13
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 WASTE DURING DISPOSAL.
PAGE 14
Instructions for possession, handling and use of
radioactive material
This radioactive material shall only be received, acquired, possessed
and used by physicians and veterinarians in clinical laboratories
or hospitals for in vitro laboratory tests. Its use should not
involve internal or external administration of the material and radiation
therefrom to human beings or animals. Its receipt, acquisition,
possession, use and transfer are subject to the regulations and
general license requirements of the U.S. Nuclear Regulatory Commission
or of a State with which the Commission has entered into
an agreement for the exercise of regulatory authority.
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. There should be no drinking, eating or smoking
while radioactive material is being handled. Hands should be
covered with gloves during, and thoroughly washed after the handling
of radioactive material. When handling radioactive material
do not pipette by mouth. Spills must be quickly and thoroughly
cleaned up. Surfaces involves 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:
Used radioactive test solutions must be disposed of by flushing
down a laboratory sink drain with copious amounts of water. Radioactive
waste should be disposed of in compliance with Federal,
State, and Local Government regulations.
THIS PACKAGE CONFORMS TO THE CONDITIONS AND LIMITATIONS
SPECIFIED IN 49 CFR173.421 FOR EXCEPTED RADIOACTIVE
MATERIAL LIMITED QUANTITY, N.O.S. UN2910.
for research only
not for use in diagnostic
procedures
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