1
Human Activin A （ACV Human Activin A （ACVHuman Activin A （ACV Human Activin A （ACVHuman Activin A （ACV Human Activin A （ACV -A）
ELISA Kit ELISA Kit
（96 T）
? This immunoassay kit allows for the in vitro quantitative determination of human ACV-A concentrations in serum, plasma, tissue homogenates.
? Expiration date six months from tmonths from t months from t months from t months from t months from t months from the date of manufacturehe date of manufacture he date of manufacture he date of manufacture he date of manufacture he date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY PRINCIPLE OF THE ASSAY PRINCIPLE OF THE ASSAY PRINCIPLE OF THE ASSAY PRINCIPLE OF THE ASSAY PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an antibody specific to ACV-A. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase （HRP）-conjugated antibody preparation specific for ACV-A and incubated. Then substrate solution A and B are added to each well. Only those wells that contain ACV-A, HRP-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of ACV-A in the samples is then determined by comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
66. 7 pg/ml pg/mlpg/ml -2000 pg/ml pg/ml . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for . The standard curve concentrations used for the ELISA’s were the ELISA’s were the ELISA’s were the ELISA’s were the ELISA’s were the ELISA’s were the ELISA’s were 2000 pg/ml pg/mlpg/ml , 1000 pg/ml pg/ml , 500 pg/ml pg/ml , 166. 7 pg/ml pg/mlpg/ml , 66. 7pg/ml .
SPECIFICITY
This assay recogni This assay recogniThis assay recogni This assay recogni This assay recogni This assay recogni This assay recognizes human human ACV-A. No significant . No significant . No significant . No significant cross cross -reactivity or interference was observed. reactivity or interference was observed. reactivity or interference was observed. reactivity or interference was observed. reactivity or interference was observed. reactivity or interference was observed. reactivity or interference was observed. reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of The minimum detectable dose of human ACV-A is typically less is typically less is typically less is typically less is typically less is typically less is typically less is typically less than than 16.7 pg/ml pg/ml .
The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was The sensitivity of this assay, or Lower Limit Detection （LLD） was defined as the low defined as the low defined as the low defined as the low defined as the low defined as the low est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated est concentration that could be differentiated from zero. from zero. from zero. from zero.
MATERIALS PROVIDED MATERIALS PROVIDED MATERIALS PROVIDED MATERIALS PROVIDED MATERIALS PROVIDED
Reagent Reagent Reagent Reagent
Quantity Quantity
Assay plate Assay plate Assay plate Assay plate
1
StandardStandardStandard Standard Standard （S 0-S5）
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HRP-conjugate
1 x 6 ml 1 x 6 ml 1 x 6 ml
Wash Buffer Wash Buffer Wash Buffer Wash Buffer Wash Buffer Wash Buffer Wash Buffer Wash Buffer
1 x 15 ml 1 x 15 ml
（20×concentrate） （20×concentrate） （20×concentrate） （20×concentrate） （20×concentrate）
Substrate A Substrate A Substrate A Substrate A
1 x 7 ml 1 x 7 ml 1 x 7 ml
Substrate B Substrate B Substrate B
1 x 7 ml 1 x 7 ml 1 x 7 ml
Stop SolutionStop Solution Stop Solution Stop Solution Stop Solution
1 x 7 ml 1 x 7 ml 1 x 7 ml
StandardStandardStandard Standard Standard
S0
S1
S2
S3
S4
S5
Concentration Concentration Concentration Concentration （pg/ml） （pg/ml） （pg/ml）
0
66. 7
166. 7
500
1000
2000
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STORAGE STORAGE STORAGE
1. Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2 Unopened test kits should be stored at 2-8?C upon receipt and C upon receipt and C upon receipt and C upon receipt and C upon receipt and C upon receipt and C upon receipt and C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the e may be used throughout the emay be used throughout the e may be used throughout the emay be used throughout the e may be used throughout the emay be used throughout the e may be used throughout the e may be used throughout the e may be used throughout the emay be used throughout the e may be used throughout the expiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it xpiration date of the kit, provided it is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for is stored as prescribed above. Refer to the package label for expiration date. expiration date. expiration date. expiration date. expiration date.
2. Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2Opened test plate should be stored at 2Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2 Opened test plate should be stored at 2-8?C in the aluminum foil C in the aluminum foil C in the aluminum foil C in the aluminum foil C in the aluminum foil C in the aluminum foil C in the aluminum foil C in the aluminum foil C in the aluminum foil C in the aluminum foil bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits bag with desiccants to minimize exposure damp air. The kits will r will remain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is emain stable until the expiring date shown, provided it is stored as prescribed above. stored as prescribed above. stored as prescribed above. stored as prescribed above. stored as prescribed above. stored as prescribed above. stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10A microtiter plate reader with a bandwidth of 10A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 A microtiter plate reader with a bandwidth of 10 nm or less and nm or less and nm or less and nm or less and nm or less and nm or less and nm or less and an optical density range of 0 an optical density range of 0an optical density range of 0 an optical density range of 0 an optical density range of 0 an optical density range of 0 an optical density range of 0 an optical density range of 0 an optical density range of 0-3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm 3 OD or greater at 450nm wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance mwavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m wavelength is acceptable for use in absorbance m easurement. easurement. easurement. easurement.
REAGENT PREPARATION REAGENT PREPARATION REAGENT PREPARATIONREAGENT PREPARATION REAGENT PREPARATION REAGENT PREPARATION
1. Bring all reagents and plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8°C and avoid sunlight.
2. Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have compley dissolved. Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer.
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OTHER SUPPLIES REQUIRED OTHER SUPPLIES REQUIRED OTHER SUPPLIES REQUIRED OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, Microplate reader capable of measuring absorbance at 450 nm, with t with t he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm.he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm.he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm. he correction wavelength set at 540 nm or 570 nm.
? Pipettes and pipette tips. Pipettes and pipette tips. Pipettes and pipette tips. Pipettes and pipette tips. Pipettes and pipette tips.Pipettes and pipette tips. Pipettes and pipette tips. Pipettes and pipette tips.
? Deionized or distilled water. Deionized or distilled water. Deionized or distilled water. Deionized or distilled water. Deionized or distilled water. Deionized or distilled water. Deionized or distilled water. Deionized or distilled water.Deionized or distilled water.
? Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate Squirt bottle, manifold dispenser, or automated microplate washer. washer. washer.washer.
? An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up An incubator which can provide stable incubation conditions up to 37°C±0.5°C. to 37°C±0.5°C. to 37°C±0.5°C.to 37°C±0.5°C. to 37°C±0.5°C. to 37°C±0.5°C.to 37°C±0.5°C.
SAMP LE COLLECTION AND STORAGELE COLLECTION AND STORAGE LE COLLECTION AND STORAGE LE COLLECTION AND STORAGE LE COLLECTION AND STORAGE LE COLLECTION AND STORAGE LE COLLECTION AND STORAGE
? Serum Use a serum separator tube （SST） and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediay or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
? Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediay or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
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? Tissue Homogenates 100mg tissue was rinsed with 1X PBS, homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g, 2 - 8°C. The supernate was assayed and removed immediay. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE ASSAY PROCEDUREASSAY PROCEDUREASSAY PROCEDURE ASSAY PROCEDURE ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is recommended that all samples and standards be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well.
1. Standard Reconstitute the Standards（S0-S5） with 0.5 ml of ddH2O, respectively. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to use.
2. Add 50μl of Standard or Sample per well. Standard need test in duplicate.
3. Add 50μl of HRP -conjugate conjugate to each well. Mix well and then incubate for 1 hour at 37°C.
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4. Complete remove the liquid. Then fill each well with Wash Buffer （about 200μl）, stay for 10 seconds and Spinning. Repeat the process for a total of three washes. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 50μl of Substrate A and 50μl of Substrate B to each well, mix well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark.
6. Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
7. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.
CALCULATION OF RESULTS CALCULATION OF RESULTS CALCULATION OF RESULTS CALCULATION OF RESULTS CALCULATION OF RESULTS CALCULATION OF RESULTS Using the professional soft "Curve Expert 1.3" to make a standard curve is recommended, which can be downloaded from our web.
Average the duplicate readings for each standardAverage the duplicate readings for each standard Average the duplicate readings for each standard Average the duplicate readings for each standard Average the duplicate readings for each standardAverage the duplicate readings for each standardAverage the duplicate readings for each standard Average the duplicate readings for each standardAverage the duplicate readings for each standard Average the duplicate readings for each standard Average the duplicate readings for each standard Average the duplicate readings for each standardAverage the duplicate readings for each standard Average the duplicate readings for each standard Average the duplicate readings for each standard Average the duplicate readings for each standard Average the duplicate readings for each standardAverage the duplicate readings for each standard Average the duplicate readings for each standard and sample and sample , and subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software standard curve by reducing the data using computer software
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capable of ge capable of gecapable of ge capable of ge capable of ge nerating a four parameter logistic （4 nerating a four parameter logistic （4 nerating a four parameter logistic （4 nerating a four parameter logistic （4 nerating a four parameter logistic （4nerating a four parameter logistic （4 nerating a four parameter logistic （4 nerating a four parameter logistic （4nerating a four parameter logistic （4 nerating a four parameter logistic （4 nerating a four parameter logistic （4 nerating a four parameter logistic （4 nerating a four parameter logistic （4 -PL） curve PL） curve PL） curve -fit. As fit. As fit. As an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean an alternative, construct a standard curve by plotting the mean absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the absorbance for each standard on the x-axis against axis againstaxis against axis against axis against the concentration on the concentration on the concentration on the concentration on the concentration on the concentration on the concentration on the concentration on the concentration on the y-axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the axis and draw a best fit curve through the points on the g points on the g points on the gpoints on the g points on the g points on the g points on the gpoints on the graph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log raph. The data may be linearized by plotting the log of the of the of the ACV-A concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and concentrations versus the log of O.D. and best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. procedure will produce an adequate but less precise fit of the data. If samples If samples If samples If samples have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from have been diluted, the concentration read from standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor.standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor.standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor. standard curve must be multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE LIMITATIONS OF THE PROCEDURELIMITATIONS OF THE PROCEDURELIMITATIONS OF THE PROCEDURE LIMITATIONS OF THE PROCEDURE LIMITATIONS OF THE PROCEDURE LIMITATIONS OF THE PROCEDURE LIMITATIONS OF THE PROCEDURE
? The kit should not be used beyond the expiration date on the kit label.
? Do not mix or substitute reagents with those from other lots or sources.
? If samples generate values higher than the highest standard, dilute the samples with the appropriate solution and repeat the assay.
? Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.
9
? This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
TECHNICAL HINTS TECHNICAL HINTS TECHNICAL HINTS
? Centrifuge vials before opening to collect contents.
? When mixing or reconstituting protein solutions, always avoid foaming.
? To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
? When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.
? To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
10
? Substrate Solution should remain colorless or light blue until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless or light blue to gradations of blue.
? Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

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