FOR IN VITRO USE AND RESEARCH USE ONLY
NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
[ INTENDED USE ]
The Chemiluminescent Immunoassay kit is designed for the in vitro sensitive quantitative measurement of ACTH in human serum, plasma, tissue homogenates, cell culture supernatesand other biological fluids.
[ REAGENTS AND MATERIALS PROVIDED ]
| Reagents | Quantity | Reagents | Quantity |
| Pre-coated, ready to use 96-well strip plate | 1 | Plate sealer for 96 wells | 4 |
| Standard（lyophilized） | 2 | Standard Diluent | 1×20mL |
| Detection Reagent A （green） | 1×120μL | Assay Diluent A （2 × concentrate） | 1×6mL |
| Detection Reagent B （red） | 1×120μL | Assay Diluent B （2 × concentrate） | 1×6mL |
| Substrate A | 1×10mL | Substrate B | 1×2mL |
| Wash Buffer （30 × concentrate） | 1×20mL | Instruction manual | 1 |
[ MATERIALS REQUIRED BUT NOT SUPPLIED ]
1. Luminometer capable of reading 96-well microplates with the following parameters:
lag time 30.0secs; read time 1.0 sec/well .
2. Precision single or multi-channel pipettes and pipette tips with 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 kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while the others should be at 4 oC.
2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
Note:
It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
[ SAMPLE COLLECTION AND STORAGE ]
Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4oC before centrifugation for 20 minutes at approximay 1000×g. Assay freshly prepared serum immediay or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.
Plasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8oC within 30 minutes of collection. Remove plasma and assay immediay or store samples in aliquot 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 were rinsed in ice-cold PBS（0.02mol/L,pH 7.0-7.2） to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice（Micro Tissue Grinders woks, too）. The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediay or aliquot and store at ≤-20oC.
Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediay or store samples in aliquot at -20oC or -80oC for later use. 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 contamination.
2. Sample hemolysis will influence the result, so 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 1.0mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently（not to foam）. The concentration of the standard in the stock solution is 1,000pg/mL. Please prepare 5 tubes containing 0.6mL Standard Diluent and produce a triple dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted standard such as 1,000pg/mL, 333.33pg/mL, 111.11pg/mL, 37.04pg/mL, 12.35pg/mL, and the last EP tubes with Standard Diluent is the blank as 0pg/mL.
| Tube | 1 | 2 | 3 | 4 | 5 | 6 |
| pg/mL | 1,000 | 333.33 | 111.11 | 37.04 | 12.35 | 0 |
3. Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate（2×） with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B. （In fact, more than 6mL Assay Diluent A and Assay Diluent B are contained in the bottles. Therefore, in every test, please precisely pipette required amount of Diluent and make double dilution in a new container. The prepared working dilution can't be frozen.）
4. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively （1:100）.
5. Wash Solution - Dilute 20mL of Wash Solution concentrate （30×） with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution （1×）.
6. Substrate working Solution -Mix the substrate A and B by the ratio of 99:1 to make the substrate working solution. Mix thoroughly. For example, prepare 1,000μL Substrate working Solution with 990μL Substrate A + 10μL Substrate B.
Note:
1. Making serial dilution in the wells directly is not permitted.
2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37oC directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are compley dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting.
4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. Prepare Substrate working Solution within 15 minutes before assay.
6. If crystals have formed in the Wash Solution concentrate （30×）, warm to room temperature and mix gently until the crystals are compley dissolved.
7. Contaminated water or container for reagent preparation will influence the detection result.
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