Document#: IM-KHL-130903E

JaICA

FOR RESEARCH USE ONLY

Japan Institute for the Control of Aging

Catalog #KHL-700/E

Hexanoyl-Lys adduct (HEL) ELISA kit
Read entire insert before use.
Hexanoyl-Lys adduct (HEL) is formed by the reaction of linoleic acid hydroperoxide and Lysine, and a biomarker
for oxidative stress. HEL ELISA kit is a competitive enzyme-linked immunosorbent assay for quantitative
measurement of hexanoyl-Lys adduct. This kit is based on the monoclonal antibody clone 5H4, which is specificfor HEL. Suitable for urine, serum and other biological samples. For research use only. Not for diagnostic nor
medical use.

1. Kit contents.
①HEL Microtiter Plate②Primary Antibody③Secondary Antibody④Secondary Antibody Buffer⑤Chromogen⑥Chromogen Buffer⑦Washing Buffer (5X)
⑧Stop Solution⑨Standard A-F⑩Plate Seal

: Precoated with HEL (8x12 wells, Split Type)
1 Plate: Monoclonal Antibody specific for HEL
1 Vial (7mL): HRP-Conjugated Anti Mouse IgG Antibody
1 Vial: Phosphate Buffered Saline
1 Vial (12mL): 3,3',5,5'-Tetramethylbenzidine
1 Vial (250 μL): Hydrogen Peroxide/ Citrate-Phosphate Buffer
1 Vial (12mL): Concentrated Phosphate Buffered Saline
2 Vials (25mL X 2)
: 1M Phosphoric acid
1 Vial (12mL): Bz-Gly-Hexanoyl-Lys
1 Vial each(500μL)(A: 2.6, B:7.7, C:22.7, D:69.7, E:207, F:624 nmol/L）: Adhesive seal to prevent evaporation
2 Sheets*Storage conditions: Store at 2-8℃. Don’t freeze.
*Expiration: 2 years. After the vials are opened, the kit should be used in one week.
*Measuring range : 2 – 700 nmol/L

2. Required but not provided.
A) Distilled water (Preparation of washing solution)B) 50 micro L micropipettor and pipette tips
C) 8-channel (50-200 micro L) micropipettor and tips.
D) Reagent trays for 8-channel micropipettor.
E) 4-7 degree C incubator.
F) Microtiter plate reader (measuring wavelength 450 nm).

3. Principles of the procedure.
(Fig.1)

1) Prepare microtiter plate precoated with hexanoy-Lys adduct (HEL).

①2) Add HELstandard solution or sample to microtiter plate well, and subsequently add
anti HEL monoclonal antibody. The HEL in the standard or sample competes with theHEL on the well surface for the anti HEL antibody. As a result, higher concentration ofHEL in sample will result in reduced binding of the antibody bound to the surface ofthe well.

②3) The antibody bound to the HEL in sample, is removed from the well by washing.
While the antibody bound to precoated HEL remain on the surface of the well.

③4) Peroxidase-conjugated secondary antibody is added to the well, and binds to the anti
HEL antibody.

④5) Unbound secondary antibody is removed by washing.

⑥⑤6) Addition of the chromatic reagent results in the development of color in proportion tothe amount of antibody bound to the well. The reaction is terminated by stop solution.
Absorbance at 450 nm is measured using microtiter plate reader.
7) Make a calibration curve from the absorbance data of standards, and calculate theconcentration of HEL in the sample.

4. Sample pretreatment.
A. Urine sample
Dilution of samples for at least 4 times using phosphate buffered saline (PBS at pH7.4). For urine
sample from experimental animal such as dogs and cats, 10 times or 20 times dilution isrecommended. If insoluble materials are observed, remove them by centrifugal machine. If the urine
contains proteins, treat the urine by the same procedure as for serum sample.
B. Serum sample
1) Prepare “Enzyme reagent”, by dissolving 14 mg/mL of alpha-chymotrypsin in PBS (pH7.4).
2) Dilute the serum sample for at least 2 times using PBS (pH7.4).
3) Mix 300 micro L of diluted sample and 60 micro L of “Enzyme reagent”, and incubate at 37
degree C for over night.
4) Filtrate using ultra filter with cut-off molecular weight 10kDa (for example Microcon YM-10,Millipore), and remove enzymes. Apply the filtrate to ELISA.
*This is an example of procedure. Please investigate optimum condition depending on the sample.

5. Procedure.
Bring all reagents, samples and microtiter plate to room temperature before use.
A) Pick out ①Microtiter plate from the bag. To use some wells at the next experiment, remove the well splits
from the frame, put it into the bag, and store at 4 degree C. It will be available for 1 week.
B) Prepare Washing solution by mixing one bottle of ⑦Washing buffer(x5) and 100 mL of distilled water.
C) Add 50 micro L of ⑨Standards(A～F) or sample per well. For the Blank well, add 100 micro L of Washingsolution. The typical layout of microtiter plate is shown in Fig.2.
D) Add 50 micro L of ②Primary Antibody to all well except Blank well. Seal the microtiter plate tightly with ⑩Plate Seal. Mix gently by shaking the microtiter plate horizontally. Incubate at 4 - 7 degree C for over night.
E) Reconstitute ③Secondary Antibody with one bottle of ④Secondary Antibody Buffer. This is stable for 1
week at 4 degree C.
F) Remove the plate seal, and pour off the contents of mictotiter plate by turn the plate upside down. The use ofaspirator is not recommended. Remove the remaining solution by blotting the plate against clean paper towel.
Add 250 micro L of Washing solution to each well, mix gently by horizontal shaking, and remove the contents
similarly. Repeat washing procedure twice and remove the remaining solution of the well.
G) Add 100 micro L of Secondary Antibody to all well. Seal the microtiter plate tightly with ⑩Plate Seal. Mix
gently by shaking the microtiter plate horizontally. Incubate at room temperature (20-24 degree C) for 1 hour.
H) Prepare Chromogen solution. Add 120 micro L of ⑤Chromogen to ⑥Chromogen Buffer bottle. Please notethat Chromogen solution should be prepared just before use. Alternatively, dilute ⑤Chromogen with 100
volumes of ⑥Chromogen Buffer.
I) Remove the plate seal, and wash the plate as mentioned at STEP F for 3 times. Remove the remaining
solution of the well.
J) Add 100 micro L of Chromogen solution to all well, and incubate at room temperature for 15 minutes in thedark.
K) Add 100 micro L of ⑧Stop Solution to all well, mix gently, wait for 3 minutes, and measure the absorbanceat 450 nm.

(Fig.2 Typical layout of microtiter plate for triplicate assay.)
To avoid edge effects, the use of outmost wells (Rows A and H, marked as “X”) is notrecommended. 54 wells(18 samples x 3) are applicable for test samples.
12
3
4
5
6
7
8
9101112A
Blank wells(n=3)
X
X
X
X
X
X
X
X
X
⑨Standard-A
Sample-1
Sample-7
Sample-13
B
Sample-2
Sample-8
Sample-14⑨Standard-B
C
Sample-3
Sample-9
Sample-15⑨Standard-C
D
Sample-4
Sample-10
Sample-16⑨Standard-D
E
Sample-5
Sample-11
Sample-17⑨Standard-E
F
Sample-6
Sample-12
Sample-18⑨Standard-F
GH
X
X
X
X
X
X
X
X
X
X
X
X

6. Procedure flow chart.
Samples / ⑨Standards
50μL/well
50μL

Incubation②Primary Antibody

4 - 7℃ Over nightPreparation ofSecondary Antibody

Wash③Secondary Antibody④Secondary Antibody Buffer

Preparation ofWashing solution⑦Washing Buffer(5X): 1 vial
Distilled water:
100 mL
Remove remaining solution.
Use of aspirator is not recommended.
Take care for well-to-well contamination.

(250μL x 3 times)

100μL

Incubation
Room temperaturefor 1 hourPreparation ofChromogen solution

Wash⑤Chromogen: 120μL⑥Chromogen Buffer: 1 vial

(250μL x 3 times)
Remove remaining solution.
Use of aspirator is not recommended.

*Prepare just before use.
100μL

Incubation
Room temperature for 15 minutes
in the dark.

⑧Stop solution

100μL

Absorbance at 450nm

Calibration curve.
Calculate HEL conc.

7. Standard curve and calculations.
Generate the standard curve by plotting absorbance vertical axis and log of concentration as thehorizontal axis. An example is shown in Fig.3. Any smooth curve fit may be applicable. Please note thatthe standard curve should be established for every assay.
[Fig.3] A standard curve example.
1.4

Absorbance at 450 nm

1.2⑨HEL Standard-A-B-C-D-E-F

1.0
0.8
0.6

HEL conc.
（ｎmol/L）
2.6
7.7
22.7
69.7
207624

Absorbance(450nm)
1.197
1.070
0.895
0.586
0.305
0.166

0.4
0.2
0.0110100

Conc. of HEL standard （nmol/L）

1000

8. References.
1) Yoji Kato, Yoko Mori, Yuko Makino, Yasujiro Morimitsu, Sadayuki Hiroi, Toshitsugu Ishikawa, ToshihikoOsawa.
Formation of Nε-(hexanonyl) lysine in protein exposed to lipid hydroperoxide.
Journal of Biological Chemistry, Vol. 274, No. 29, pp. 20406 – 20414, 19992) Yoji Kato, Yoshiaki Miyake, Kanefumi Yamamoto, Yoshiharu Shimomura, Hirotomo Ochi, Yoko Mori,Toshihiko Osawa.
Preparation of a monoclonal antibody to Nε-(hexanonyl) lysine: applycation to the evaluation of protective
effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle.
Biochem. Biophys. Res. Commun., Vol. 274(2), pp389-393, 2000

9. Technical support.
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