YK340 Substance P (Saliva) EIA

FOR LABORATORY USE ONLYYANAIHARA INSTITUTE INC.
2480 - 1 AWAKURA, FUJINOMIYA-SHISHIZUOKA, JAPAN 418 - 0011

ContentsⅠ．

Introduction

2Ⅱ．

Characteristics

3Ⅲ．

Composition

4Ⅳ．

Method

5-7

Ⅴ．

Notes

7-8

Ⅵ．

Performance Characteristics

8-10

Ⅶ．

Stability and Storage

10Ⅷ.

References

11- Please read all the package insert carefully before beginning the assay -1

YK340 Substance P (Saliva) EIA Kit
Ⅰ. Introduction
Substance P is a peptide composed of a chain of 11 amino acid residues, which is one of a member ofneuropeptides with similar structure called tachykinin.
Substance P was first found to function as a neurotransmitter, and subsequent research has shown that it
is expressed not only in the central and peripheral nervous systems, but also in peripheral non-nervous
cells, and is now involved in a wide range of physiological functions. It is also known to be associatedwith various pathologies.
Pneumonia is considered to be one of the major causes of death among the elderly in Japan, anddecreased secretion of substance P leads to a decrease in normal swallowing reflex and cough reflex,
which increases silent aspiration, it has been pointed out that the risk of aspiration pneumonia isincreased as a result.
Saliva can be collected easily and noninvasively, and Substance P concentration in saliva is considered to
be a useful biomarker.
This kit can specifically and easily measure Substance P present in saliva.

YK340 Substance P (Saliva) EIA Kit

Contents▼ The assay kit can measure Substance P in saliva
within the range of 6.859-5,000 pg/mL▼ The assay is completed within 18 hr.+1.5 hr.
▼ With one assay kit, 40 samples can be measured
in duplicate.
▼ Test sample: Human saliva
Sample volume: 50 μL▼ The 96-wells plate in kit is consisted by 8-wells
strips, and the strips can be used separately.
▼ Stability and storage
Store all of the components at 2-8ºC.
The kit is stable under the condition for 18 months
from the date of manufacturing.
The expiry date is stated on the label of kit.

21)
2)
3)
4)
5)
6)
7)
8)
9)

Antibody coated plate
StandardLabeled antigen
Antibody solution
SA-HRP solution
Enzyme substrate solution (TMB)
Stopping solutionBuffer solution
Washing solution(concentrated)
10) Adhesive foil

Ⅱ. Characteristics
This EIA kit is used for quantitative determination of Substance P in saliva. The kit is characterized
by sensitive quantification and high specificity. In addition, it has no influence by other constituents insamples. Substance P standard of this kit is a highly purified synthetic product.
< Assay principle >This EIA kit for determination of Substance P is based on a competitive enzyme immunoassay using
combination of highly specific antibody to Substance P and biotin-avidin affinity system. To the wellof the plate coated with goat anti rabbit IgG, Substance P standard or samples, labeled antigen andSubstance P antibody are added for competitive immunoreaction. After incubation and plate washing,horse radish peroxidase (HRP) labeled streptavidin (SA) is added to form HRP labeledSA-biotinylated Substance P - antibody complex on the surface of the wells. Finally, HRP enzyme
activity is determined by 3,3’,5,5’-Tetramethylbenzidine (TMB) and the concentration of Substance P
is calculated.

3Ⅲ. Composition

ComponentFormQuantity

Main Ingredient1.

Antibody coated plate

microtiterplate

1 plate (96 wells)

Goat anti rabbit IgG antibody

2.

Standardlyophilized

1 vial (5,000 pg)

Substance P (1-11) amide

3.

Labeled antigen

lyophilized

1 vialBiotinylated Substance P

4.

Antibody solution

liquid1 bottle (7 mL)

Rabbit anti Substance P (1-11)
amide antibody

5.

SA-HRP solution

liquid1 bottle (12 mL)

Horseradish peroxidase labeledstreptavidin

6.

Enzyme substratesolution (TMB)

liquid1 bottle (12 mL)

3,3’,5,5’-Tetramethylbenzidine(TMB)

7.

Stopping solution

liquid1 bottle (12 mL)

1M H2SO48.

Buffer solution

liquid1 bottle (30 mL)

Phosphate buffer

9.

Washing solution(concentrated)liquid1 bottle (50 mL)

Concentrated saline

10.

Adhesive foil

3 pieces

4Ⅳ. Method< Equipment required >1. Photometer for microtiter plate (plate reader) which can read extinction 3.0 at 450nm
2. Microtiter plate shaker
3. Washing device for microtiter plate and dispenser with aspiration system
4. Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
5. Glass test tubes for preparation of standard solution and saliva sample dilution
6. Graduated cylinder (1,000 mL)
7. Distilled water or deionized water<Preparation of saliva sample>
To collect saliva, use an oral swab + storage tube (Funakoshi Co., Ltd. product code 5001.02 + 5001.05).
Collect saliva according to the instruction manual of the collection device. Collected saliva samples
should be frozen at or below -30°C immediately. (If possible, cryopreservation of saliva samples isrecommended at -80°C.) These samples should be brought back to room temperature (20-30°C), andthen be centrifuged (3000 rpm x 15 min.) before starting assay.

< Preparatory work >1. Preparation of labeled antigen solution:
Reconstitute labeled antigen with 7 mL of buffer solution.
2. Preparation of standard solution:
Reconstitute the standard with 1.0 mL of buffer solution, which affords 5000 pg/mL standardsolution. The reconstituted standard solution (0.1 ml) is diluted with 0.2 mL of buffer solution,
which yields 1666.667 pg/mL standard solution. Repeat the dilution procedure to make each of555.556, 185.185, 61.728, 20.576 and 6.859 pg/mL standard solutions. Buffer solution itself is used
as 0 pg/mL.
3. Preparation of washing solution:
Dilute 50 mL of washing solution (concentrated) to 1,000mL with distilled water.
4. Other reagents are ready for use.

5

<Dilution of saliva sample>
Thaw frozen saliva samples at room temperature. Centrifuge (3,000 rpm, 15 minutes) and well mix
collected samples. Dilute these saliva samples with buffer 4 times.
< Example of dilution >Add 150 L of buffer solution into an appropriate tube. Pipette 50 L of saliva into the tube and vortex
to mix.
< Procedure >1. Bring all the reagents and samples to room temperature (20-30ºC) at least 1 hour before starting
assay.
2. Add 0.35 mL/well of washing solution into the wells and aspirate washing solution in the wells.
Repeat this washing procedure further twice (total 3 times). Finally, invert the plate and tap it onto
an absorbent surface, such as paper toweling, to ensure blotting free of most residual washingsolution.
3. Fill 50 L of each of standard solutions (0, 6.859, 20.576, 61.728, 185.185, 555.556, 1666.667,
5000 pg/mL) or samples into the wells first, then introduce 50 L of labeled antigen solution andfinally add 50 L of antibody solution into the wells.
4. Cover the plate with adhesive foil and incubate it at room temperature for 18 hours. During theincubation, the plate should be shaken with a plate shaker (approximately 100 rpm).
5. After incubation, take off the adhesive foil, aspirate and wash the wells 5 times with 0.35 mL/wellof washing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper
toweling, to ensure blotting free of most residual washing solution.
6. Add 100 L of SA-HRP solution into each of the wells.
7. Cover the plate with adhesive foil and incubate it at room temperature for 1 hour. During theincubation, the plate should be shaken with a plate shaker (approximately 100 rpm).
8. Take off the adhesive foil, aspirate and wash the wells 5 times with approximately 0.35 mL/well ofwashing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper
toweling, to ensure blotting free of most residual washing solution.
9. Add 100 L of Enzyme substrate solution (TMB) to each of the well, cover the plate with adhesive
foil and keep it for 30 minutes at room temperature in a dark place for color reaction (keep still,plate shaker not need).
10. Add 100 L of stopping solution into the wells to stop color reaction.
11. Read the optical absorbance of the solution in the wells at 450 nm/620 nm. The dose-response curveof this assay fits best to a 5 (or 4)-parameter logistic equation. The results of unknown samples can6be calculated with any computer program having a 5 (or 4)-parameter logistic function. Otherwise
calculate mean absorbance values of wells containing standards and plot a standard curve on semi
logarithmic graph paper (abscissa: concentration of standard; ordinate: absorbance values). Use theaverage absorbance of each sample to determine the corresponding value by simple interpolationfrom this standard curve.
Multiply concentrations of unknown samples by 4 times to obtain the final concentration ofSubstance P.
V. Notes
1.

The measurement is affected by surfactants, so be careful not to contaminate such as toothpaste or
mouthwash into saliva sample. It has been confirmed that there is a tendency for high absorbance at
the measurement, due to the interference effect of the surfactants.

2.

Saliva samples should be divided into test tubes in small amount and frozen at or below -30°C.
Avoid repeated freezing and thawing of samples. If possible, cryopreservation of saliva samples isrecommended at -80°C.

3.

Precipitation may occur in buffer solution and antibody solution, please tipping agitation before use.
The precipitate has no effect on the measurement results.

4.

Standard solutions should be prepared immediately before use. This kit can be used dividedly in
strips of the plate. In such case, the rest of reconstituted reagent (standard and labeled antigen)should be stored at 4°C. The reconstituted standard (5,000 pg/mL) and the labeled antigen are stablefor 4 weeks when stored refrigerated at 4°C. If stored these reagents frozen at -30°C, theperformance is stable for 4 weeks, but many precipitate are generated with freezing, it is notrecommended to store reagents frozen.

5.

During storage of washing solution (concentrated) at 2-8°C, precipitates may be observed, however,they will be dissolved when diluted.

6.

Pipetting operations may affect the precision of the assay, so that pipette standard solutions or
samples precisely into each well of plate. In addition, use clean test tubes or vessels in assay anduse new tip for each standard or sample to avoid cross contamination.

7.

When sample concentration exceeds 5,000 pg/mL, it needs to be diluted with buffer solution toproper concentration.

8.

During the incubation except the color reaction, the plate should be shaken gently by a plate shaker
to promote immunoreaction (approximately 100 rpm).

9.

Perform all the determination in duplicate.

10. Read plate optical absorbance of reaction solution in wells as soon as possible after stop color

7

reaction.
11. To quantitate accurately, always run a standard curve when testing samples.
12. Protect reagents from strong light (e.g. direct sunlight) during storage and assay.
13. Satisfactory performance of the test is guaranteed only when reagents are used from combination
pack with identical lot number.
Ⅵ. Performance Characteristics<Analytical Recovery><Human saliva A>
Added Substance P(pg/mL)0152001,000<Human saliva B>
Added Substance P(pg/mL)0152001,000

Observed(pg/mL)
21.6
36.1
240.9
1004.1
Observed(pg/mL)
42.1
64.3
273.4
1033.6

8

Expected(pg/mL)

Recovery
(%)

36.6
221.6
1021.6

98.6
108.7
98.3

Expected(pg/mL)

Recovery
(%)

57.1
242.1
1042.1

112.6
112.9
99.2

<Human saliva C>
Added Substance P(pg/mL)0152001,000<Human saliva D>
Added Substance P(pg/mL)0152001,000<Human saliva E>
Added Substance P(pg/mL)0152001,000

Observed(pg/mL)
25.2
42.6
272.2
1020.9

Expected(pg/mL)

Recovery
(%)

40.2
225.2
1025.2

106.0
120.9
99.6

Observed(pg/mL)
11.7
27.4
232.2
1068.0

Expected(pg/mL)

Recovery
(%)

26.7
211.7
1011.7

102.6
109.7
105.6

Observed(pg/mL)
20.6
41.9
277.4
1249.8

Expected(pg/mL)

Recovery
(%)

35.6
220.6
1020.6117.7
125.8
122.5<Dilution test >Let x1 be a saliva sample before dilute preparation. x3 is a reference value.
< Human saliva A>
Sample preparation
Observed
Expected(pg/mL)(pg/mL)
x3
36.1
108.3
x4
22.0
88.0x6
13.7
82.2
x8
10.0
80.0
< Human saliva B>
Sample preparationx3x4x6x8< Human saliva C>
Sample preparationx3x4x6x8% of Expected
(%)
123.1
93.4
90.9

Observed(pg/mL)
61.4
41.5
27.3
21.0

Expected(pg/mL)
184.2
166.0
163.8
168.0

% of Expected
(%)
111.0

Observed(pg/mL)
43.9
27.5
17.4
13.2

Expected(pg/mL)
131.7
110.0
104.4
105.6

% of Expected(%)
119.7

998.7
101.2

94.996.0

< Human saliva D>
Sample preparationx3x4x6x8<Human saliva E>
Sample preparationx3x4x6x8

Observed(pg/mL)
22.4
15.5
9.6
6.9

Expected(pg/mL)
67.2
62.0
57.6
55.2

% of Expected
(%)
108.4

Observed(pg/mL)
32.7
22.2
13.6
11.7

Expected(pg/mL)
98.1
88.8
81.6
93.6

% of Expected(%)
110.5

92.989.0

91.9
105.4

< Precision and reproducibility >Test sample
Human saliva

Intra-assay CV (%)
2.8-5.6

Inter-assay CV (%)
6.1-10.9<Crossreactivity>
Crossreactivity（％）
< 0.1
86.5
99.1
48.8
0.3
0.1
86.2

Related peptides
Substance P (1-4)
Substance P (3-11) amide
Substance P (4-11) amide
Substance P (7-11) amide
Neurokinin A
Neurokinin B
Hemokinin-1 (human)

Ⅶ. Stability and Storage< Storage >< Shelf life >< Package >Store all of the components at 2-8ºC.
The kit is stable under the condition for 18 months from the date of manufacturing.
The expiry date is stated on the package.
For 96 tests per one kit.

10

Ⅷ. References
1. U.S.v. Euler and J.H.Gaddum (1931) An unidentified depressor substance in certain tissue extracts.
The Journal of Physiology 72 (1): 74-87
2. M.M. Chang and S.E. Leeman (1970) Isolation of a sialogogic peptide from bovine hypothalamic
tissue and its characterization as substance P. Journal of Biological Chemistry 245: 4784-4790
3. A. Kuwahara, N. Yanaihara et al. (1983) Distribution of neurons containing immunoreactivity forgastrin-releasing peptide (GRP), substance P, and vasoactive intestinal polypeptide (VIP) in the rat
gastric wall. Biomedical Research 4: 473-478
4. Y. Tsutsumi, N. Yanaihara et al. (1984) Histochemical studies of metaplastic lesions in the humangallbladder. Archives of Pathology and Laboratory Medicine 108: 917-921
5. T. Uchida, N. Yanaihara et al. (1985) Occurrence and projections of three subclasses of metenkephalin-arg6-gly7-leu8 neurons in the guinea-pig duodenum: immunoelectron microscopic study
on the co-storage of met enkephalin-arg6-gly7-leu8 with substance P or PHI (1-15). Biomedical
Research 6: 415-422
6. Y. Tsutsumi (1990) Immunohistochemical analysis of neuroendocrine substances in non neoplastic
lung and in neuroendocrine lung tumors. In: Endocrine Pathology Update (Eds. J. Lechago and T.
Kameya) Field & Wood Philadelphia 1: 189-213
7. M. Yazawa, N. Yanaihara et al. (1990) Immunohistochemical demonstrations of peptide histidine
methionine and substance P immunoreactive nerves in endoscopically biopsied rectal mucosa. TheAutonomic Nervous System 27: 130-136
8. H. Suzuki (2002) Tachykininergic neurotransmission in the central nervous system. J Nippon Med
Sch. 69 (4): 322-7
9. M.Paul E. et al. (2003) Aspiration pneumonia and dysphagia in the elderly. Chest. 124: 328-336<Manufacturer>
Yanaihara Institute Inc.
2480-1 Awakura, Fujinomiya-shi
Shizuoka, Japan 418-0011
TEL: +81-544-22-2771 FAX: +81-544-22-2770
Website: http://www.yanaihara.co.jp E-mail: ask@yanaihara.co.jp
Update at Dec. 27, 202211