Human Arginase (Arg) ELISA Kit | MBS454916

Human Arginase (Arg) ELISA Kit

The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of Arg in human serum, plasma,
tissue homogenates, cell lysates, cell culture supernates or other biological fluids.

MATERIALS REQUIRED BUT NOT SUPPLIED

1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. Deionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution.

STORAGE OF THE KITS

1. For unopened kits: All the reagents should be kept according to labels on the vials. The TMB Substrate, Wash
Buffer (30X concentrate) and the Stop Solution should be stored at 4
oC upon receipt while the others should
be at -20oC.
2. For opened kits: Once the kit is opened, the remaining reagents still need to be stored according to the above
storage conditions. In addition, return the unused wells to the foil pouch containing the desiccant pack and reseal
along entire edge of zip-seal. 

Note:
For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this expiration
date.
It is highly recommended to use the remaining reagents within 1 month of opening.

SAMPLE COLLECTION AND STORAGE

 Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at
4
oC before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or
store samples in aliquots at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
 Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at
1000×g at 2-8oC within 30 minutes of collection. Remove plasma and assay immediately or store samples in
aliquots at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
 Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this
assay, tissues should be rinsed in ice-cold PBS (0.01mol/L,pH 7.0-7.2) to remove excess blood thoroughly and
weighed before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with
a glass homogenizer on ice (Micro Tissue Grinders also work). The resulting suspension should be sonicated
with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After
that, the homogenates are centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately
or aliquot and store at ≤-20oC.
 Cell Lysates - Cells must be lysed before assaying according to the following directions.
1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be
collected by centrifugation directly).
2. Wash cells three times in cold PBS.
3. Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4 times (or Freeze cells at ≤-20oC. Thaw
cells with gentle mixing. Repeat the freeze/thaw cycle 3 times.).
4. Centrifuge at 1500×g for 10 minutes at 2-8oC to remove cellular debris.
 Cell culture supernatant or other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove
particulates and assay immediately or store samples in aliquots at -20oC or -80oC. Avoid repeated freeze/thaw
cycles. 

Note:
1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC (≤1 month)
or -80oC (≤2 months) to avoid loss of bioactivity and/or contamination.
2. Sample hemolysis will influence the result, and hemolytic specimen can not be detected.
3. When performing the assay, bring samples to room temperature.

REAGENT PREPARATION

1. Bring all kit components and samples to room temperature (18-25oC) before use.
2. Standard - Reconstitute the Standard with 2.0mL of Diluent Buffer, keep for 10 minutes at room temperature,
shake gently (not to foam). The concentration of the standard in the stock solution is 200ng/mL. Prepare 7 tubes
containing 0.5mL Diluent Buffer and use the diluted standard to produce a double dilution series according to the
picture shown below. Mix each tube thoroughly before the next transfer. Prepare a dilution series with 7 points; for
example: 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.12ng/mL, and the last EP tube with
Diluent Buffer is the blank at 0ng/mL.

Note:
1. Do not perform a serial dilution directly in the wells.
2. Prepare standard within 15 minutes of performing the assay. Do not dissolve the reagents at 37oC directly.
3. Detection Reagent A and B are sticky solutions, therefore slowly pipette them to reduce the volume errors.
4. Carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, avoid
foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting,
use small volumes and ensure that pipettors are calibrated. It is recommended to pipette more than 10μL at a
time to ensure accuracy.
5. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
6. If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently until
the crystals are completely dissolved.
7. Any contaminated water or container used during reagent preparation will influence the detection result.
SAMPLE PREPARATION
1. We are only responsible for the kit itself, not for the samples consumed during the assay. The user should
calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in
advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the standard
curve, users must determine the optimal sample dilutions for their particular experiments. Samples should be
diluted by 0.01mol/L PBS (pH=7.0-7.2).
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is
necessary.
4. Tissue or cell extraction samples prepared using a chemical lysis buffer may cause unexpected ELISA results
due to the impacts from certain chemicals.
5. Due to the possibility of mismatching between antigens from other origin and antibodies used in our kits (e.g.,
antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from
other manufacturers may not be recognized by our products.
6. Samples from cell culture supernatant may not be detected by the kit due to influence from factors such as cellviability, cell number and/or sampling time.
7. Fresh samples that have not been stored for extended periods of time are recommended for the test. Otherwise,
protein degradation and denaturalization may occur in those samples and give inaccurate or incorrect results.