Mouse MDA ELISA Kit

Gronudwork Biotechnology Diagnosticate Ltd.
1.GBD®INTENDED USE
This GBD®MDA ELISA kit is intended Laboratory for Research use only and is not for use in
diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow
and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to
measure the concentration of MDA in the sample, this MDA ELISA Kit includes a set of
calibration standards. The calibration standards are assayed at the same time as the samples and
allow the operator to produce a standard curve of Optical Density versus MDA concentration.
The concentration of MDA in the samples is then determined by comparing the O.D. of the
samples to the standard curve.
2.GBD® REAGENTS PROVIDED
All reagents provided are stored at 2-8°C. Refer to the expiration date on the label.
1. GBD® -586 MICROTITER PLATE 96 wells
2. GBD® -506 BIOLOGICAL 1.0ml 1 vial
3. GBD® -516 ENZYME CONJUGATE 6.0 mL 1 vial
4. GBD® -819 STANDARD.I 0 pg/ml 1.0 mL 1 vial
5. GBD® -829 STANDARD.II 5.0 pg/ml 1.0 mL 1 vial
6. GBD® -839 STANDARD.III 10 pg/ml 1.0 mL 1 vial
7. GBD® -849 STANDARD.IV 25 pg/ml 1.0 mL 1 vial
8. GBD® -859 STANDARD.V 50 pg/ml 1.0 mL 1 vial
9. GBD® -869 STANDARD.VI 100pg/ml 1.0 mL 1 vial
10. GBD® -526 SUBSTRATE A 6.0 mL 1 vial
11. GBD® -536 SUBSTRATE B 6.0 mL 1 vial
12. GBD® -546 STOP SOLUTION 6.0 mL 1 vial
13. GBD® -556 WASH SOLUTION x100 10 mL 1 vial

3.GBD® MATERIALS REQUIRED BUT NOT SUPPLIED
1. Microplate reader capable of measuring absorbance at 450 nm.
2. Precision pipettes to deliver 2 ml to 1 ml volumes.
2. Single or multi-channel precision pipettes with disposable tips: 10-100 μL and 50-200 μL for
running the assay.
3. Adjustable 10ml-100ml pipettes for reagent preparation.
4. 100 ml and 1 liter graduated cylinders.
5. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. （A manifold
multi-channel pipette is desirable for large assays.）
6. Absorbent paper.
7. 37°C incubator.
8. Distilled or deionized water.
9. Data analysis and graphing software. Graph paper: linear（Cartesian）, log-log or semi-log, or
log-logit as desired.
10. Tubes to prepare standard or sample dilutions.
4.GBD® PRECAUTIONS
1. Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates
are matched for optimal performance. Use only the reagents supplied by manufacturer.
2. Allow kit reagents and materials to reach room temperature （20-25°C） before use. Do not use water baths
to thaw samples or reagents.
3. Do not use kit components beyond their expiration date.
4. Use only deionized or distilled water to dilute reagents.
5. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C
in their pouch with the desiccant provided.
6. Use fresh disposable pipette tips for each transfer to avoid contamination.
7. Do not mix acid and sodium hypochlorite solutions.

8. Serum and plasma should be handled as potentially hazardous and capable of transmitting disease.
Disposable gloves must be worn during the assay procedure, since no known test method can offer
complete assurance that products derived from human blood will not transmit infectious agents. Therefore,
all blood derivatives should be considered potentially infectious and good laboratory practices should be
followed.
9. All samples should be disposed of in a manner that will inactivate viruses.
10. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to
stand for a minimum of 30 minutes to inactivate the viruses before disposal.
11. Substrate Solution is easily contaminated. If bluish prior to use, do not use.
12. Substrate B contains 20% acetone, keep this reagent away from sources of heat or flame.
13. Remove all kit reagents from refrigerator and allow them to reach room temperature （ 20-25°C）.
5.GBD® ASSAY PROCEDURE
1. Prepare all Standards before starting assay procedure （see Preparation Reagents）. It is recommended that
all Standards and Samples be added in duplicate to the Microtiter Plate.
2. First, secure the desired number of coated wells in the holder, then add 50 μL of Standards or Samples to
he appropriate well of the antibody pre-coated Microtiter Plate.
3. Add 10 μL of Biological to samples well.（DO not add Biological to standards well）.
4. Add 50 μL of Enzyme Conjugate to each well. Mix well. Complete mixing in this step is important. Cover
and incubate for 1 hours at 37°C.
5. Prepare Substrate Solution no more than15 minutes before end of incubation （see Preparation of Reagents）.
6. Wash the Microtiter Plate using one of the specified methods indicated below:
7. Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste
container. Using a squirt bottle, fill each well compley with distilled or de-ionized water, then aspirate
contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total
of FIVE washes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper
towels until no moisture appears. Note: Hold the sides of the plate frame firmly hen washing the plate to
assure that all strips remain securely in frame.

8. Automated Washing: Aspirate all wells, then wash plate FIVE times using distilled or de-ionized water.
Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 μL/well/wash
（range: 350-400 μL）. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or
paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10
seconds or shaking time of 5 seconds between washes.
9. Add 50 μL Substrate A&B to each well. Cover and incubate for 15 minutes at 20-25°C.
10. Add 50 μL of Stop Solution to each well. Mix well.
11. Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.
6.GBD® CALCULATION OF RESULTS
1. This standard curve is used to determine the amount in an unkown sample.The standard curve isgenerated by
plotting the average O.D.（450nm） obtained for each of the six standard concentrations on the certical（Y）
axis versus the corresponding concentration on the horizontal（X） axis.
2. First,calculate the mean O.D.value for each standard and sample ,all O.D.values,are subtracted by the mean
value of the zero standard before result interpretation.construct the standard curve using graph paper or
statistical software.
3.To determine the amount in each sample ,first locate the O.D.value on the Y-axis and extend a horizontal line
to the standard curve ,at the point of intersection,draw a vertical line to the x-axis and read the corresponding
concentration.
4. Any variation in operator,pipetting and washing technique,incubation time or temperature ,and kit age can
cause variation in result ,each user should obtain their own standard curve.
5. The sensitivity by this assay is 0.1 pg/ml
6. Standard curve match