Document#: IM-KDT-141016E

FOR RESEARCH USE ONLY

Catalog #KDT-010/E

Dityrosine (DT) ELISA kit
Dityrosine (DT) is known to be formed when tyrosine is damaged by free radicals, and is reported to exist in various tissues
and is also detected in urine. DT ELISA kit is designed for quantitative measurement of DT especially in urine samples.
For research use only. Not for diagnostic nor medical use.

1. Kit contents.
1) DT Microtiter Plate: Pre-coated with DT (8x 12 wells, split type)1 plate2) Primary Antibody: Anti DT monoclonal antibody (ready to use)
1 vial (7 mL)3) Secondary Antibody: HRP-conjugated anti-mouse antibody
1 vial4) Secondary Antibody Solution: Phosphate Buffered Saline
1 vial (12 mL)5) TMB Substrate: Chromogen (ready to use)
1 vial (12 mL)
6) Stop Solution: 1.96% Sulfuric acid (ready to use)
1 vial (12 mL)
7) Washing Solution (x5)
: Concentrated wash buffer
3 vials (25 mL x 3)
8) DT Standards: DT standards (ready to use)
1 vial each (0.5 mL)(A: 0.05, B: 0.2, C: 0.5, D: 2, E: 5, F: 12 µmol/L)
9) Plate seal
：2 sheets*Storage conditions: Store at 2-8°C. Don’t freeze.
*Expiration: See the outer box label. After the vials are opened, the kit should be used in one week.
*Measuring range : 0.05 - 12 µmol/L

2. Required but not provided.
1) Distilled water (Preparation of washing solution)2) Saline (0.9％ NaCl in distilled water）for sample dilution3) 50 micro L micropipettor and pipette tips
4) 8-channel (50-200 µL) micropipettor and tips.
5) Reagent trays for 8-channel micropipettor.
6) 4-10 °C incubator.
7) Microtiter plate reader (measuring wavelength 450 nm).

3. Principles of the procedure.
[Fig.1]

1) Prepare microtiter plate pre-coated with dityrosine (DT).

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

②3) The antibody bound to the DT in sample, is removed from the well by washing. While
the antibody bound to pre-coated DT remain on the surface of the well.

③④⑥4) Peroxidase-conjugated secondary antibody is added to the well, and binds to the anti
DT antibody.
5) Unbound secondary antibody is removed by washing.

⑤6) Addition of the chromatic reagent results in the development of color in proportion to
the 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) Draw a calibration curve from the absorbance data of standards, and calculate theconcentration of DT in the sample.

4. Sample pretreatment.
Application to human urine:
Mix with 3 volumes of saline before assay. Insoluble materials should be removed by centrifugation.
Please note that samples must be diluted with saline (0.9% NaCl in distilled water).
Washing buffer and PBS can’t be used for sample dilution.

5. Assay Procedure.
Bring all reagents, samples and microtiter plate to room temperature before use. Pick out ①Microtiterplate 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 °C. ①Microtiter plate is stable for 1 week after opening the bag.
A) Mix 1 vial of ⑦Washing solution with 100 mL of distilled water, and prepare working washing buffer.
B) Put ①Microtiter plate from the bag. Inside wells of ①Microtiter plate, glycerol is coated as a preservative.
Remove seal from wells before use.
C) Pour 250 µL of working washing buffer, stand for 3 minutes at room temperature, and remove buffer fromwells. Remove the remaining solution by tapping the plate against clean paper towel. Repeat washing foradditional two times.
D) Pour 50 µL of ⑧DT standards or diluted samples. For blank wells, pour 100 µL of distilled water. The typicallayout of microtiter plate is shown in Fig.2
E) Pour 50 µL of ②Primary antibody to all wells expect blank wells. Seal the microtiter plate tightly with ⑨Plate
Seal. Mix gently by shaking the microtiter plate horizontally. Incubate at 4 - 10 °C for overnight. Store all
reagents at 4 °C.
F) Bring all reagents expect working microtiter plate to room temperature.
G) Prepare working washing buffer. Mix 1 vial of ⑦Washing solution with 100 mL of distilled water. Working
washing buffer is stable for one week at 4 °C.
H) Reconstitute working secondary antibody. Add 1 vial of ④Secondary Antibody Buffer to ③Secondary
Antibody, mix gently and stand for 5 minutes at room temperature. Working secondary antibody is stable for
1 week at 4 °C.
I) 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 tapping the plate against clean paper
towel. Add 250 µL of working washing buffer to all well, mix gently by horizontal shaking, and remove thecontents similarly. Repeat washing procedure twice and remove the remaining solution of the well.
J) Pour 100 µL of working secondary antibody to all well. Seal the microtiter plate tightly with ⑨Plate Seal. Mix
gently by shaking the microtiter plate horizontally. Incubate at room temperature (20-24 °C) for 1 hour.
K) Remove the plate seal, and wash the plate as mentioned at STEP I for 3 times. Remove the remaining
solution of the well.
L) Pour 100 µL of ⑤TMB substrate to all well, and incubate at room temperature for 15 minutes in the dark. ⑤
TMB substrate is light sensitive. Avoid room light by covering the working microtiter plate with aluminium foil.
M) Add 100 µL of ⑥Stop Solution to all well, mix gently, wait for 3 minutes, and measure the absorbance at 450nm.

[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.
123
456789101112A
Blank wells(n=3)
X
X
X
X
X
X
X
X
X
⑧Standard-A
Sample-1
Sample-7
Sample-13
B
Sample-2
Sample-8
Sample-14⑧Standard-B
C
Sample-3
Sample-9
Sample-15⑧Standard-C
D
Sample-4
Sample-10
Sample-16⑧Standard-D
E
Sample-5
Sample-11
Sample-17⑧Standard-E
F
Sample-6
Sample-12
Sample-18⑧Standard-F
G
H
X
X
X
X
X
X
X
X
X
X
X
X

6. Procedure flow chart.

Wash plate

NOTE: Pour samples/STD first,
then pour primary antibody.

(250µL stand for 3 minutesｘ3 times）

Samples / ⑧DT standards

Working washing buffer⑦Washing solution: 1 vial
Distilled water: 100mL

50µL②Primary antibody

50µL

Primary antibody
reaction4 - 10°C Overnight
Working washing buffer

Working secondaryantibody

Wash(250µL ｘ3 times)③Secondary antibody: 1 vial
④Secondary antibody
solution: 1 vial

100µL

⑦Washing solution: 1 vial
Distilled water: 100ｍL

Secondary antibody
reaction
Room temperature 1 hour

Wash
(250µL ｘ 3 times)⑤TMB substrate

100µL

Chromogen reaction
Room temperature for 15 minutes at dark place.
CAUTION: Light sensitive. Avoid light using aluminium foil.

⑥Stop solution

100µL

Absorbance at 450nm

Draw standard curve, andestimate DTconcentration

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.

8. Reference.
Kato Y, Wu X, Naito M, Nomura H, Kitamoto N, Osawa T.: Immunochemical detection of protein dityrosine
in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody. Biochem Biophys ResCommun. 275(1), p11-15 (2000).

9. Technical support & troubleshootingA)
B)

Unstableness of incubation temperature may result in unstable results.
Residual water drop in wells may cause unstable results. Please remove water drop completely by tapping microtiterplate to clean and dry paper towel. Please take care not to touch inside wells.
C) Accuracy in pipetting volume for samples and reagents may affect the quality of assay. Please note that samples,standards and primary antibody must be poured accurately 50 µL.
D) Temperature for secondary antibody reaction and chromogen reaction may affect absorbance. Please try to extend or
shorten chromogen reaction time depending on room temperature.
E) TMB substrate is light sensitive. Room light may cause spontaneous / non-specific coloring. Avoid sun light and room
light by covering TMB substrate vial and microtiter plate with aluminium foil.
F) Proteins, high concentration of salts and high / low pH in samples may affect ELISA. Please remove proteins byultrafiltration, dilute samples using saline, adjust pH to 8.0±0.5.
G) For sample dilution, washing solution, working washing buffer or phosphate buffered saline (pH7.4) can’t be used.
Please use saline (0.9% NaCl in distilled water)

Please visit our web site http://www.jaica.com/e/or FAX: +81-538-49-1267(Supported languages: Japanese and English)

Japan Institute for the Control of Aging (JaICA),
Nikken SEIL co., Ltd.
Haruoka 710-1, Fukuroi, Shizuoka 437-0122, Japan
TEL: +81-538-49-0125 FAX: +81-538-49-1267
www.jaica.com/e/ E-mail: biotech@jaica.com