FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Human oxyhemoglobinELISA Kit

Cat.No: MBS3802708

Storage：2-8°°C.

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validity：six months.

The sensitivity by this assay is 0.1ng/mL

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Standard concentration was followed by: 20,10,5,2.5,1.25,0ng/mL
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Both intra-assay CV and inter-assay CV is less than 15%.
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Intended use
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This Human oxyhemoglobin 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
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intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the
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concentration of Human oxyhemoglobin in the sample, this Human oxyhemoglobin Kit includes a set of
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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 Human oxyhemoglobin

concentration. The concentration of Human oxyhemoglobin in the samples is then determined by

comparing the O.D. of the samples to the standard curve.

Sample collection and storages

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

 Plasma Cell culture Serum
supernate and other
biological fluids
Use a serum separator
tube and allow samples to
Collect plasma using
Remove particulates by clot for 30 minutes before
EDTA or heparin as an
centrifugation and assay centrifugation for 10
anticoagulant.
immediately or aliquot and minutes at approximately
Centrifuge samples for 30 store samples at -20°C or 3000×g. Remove serum
minutes at 3000×g at -80°C. and assay immediately or
2-8°C within 30 minutes of aliquot and store samples
Avoid repeated

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collection. Store samples at -20°C or -80°C.
freeze-thaw cycles.
Note: The samples should be centrifuged adequately and no hemolysis or granule was

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allowed.

Materials required but not supplied

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1. Standard microplate reader (450nm).

2. Precision pipettes and Disposable pipette tips.

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3. 37 °C incubator.
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Precautions
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1. Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates are matchedfor

optimal performance. Useonly thereagentssuppliedby manufacturer.

2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin

their pouchwith the desiccantprovided.

3. Mix all reagents before using.

Remove allkit reagentsfrom refrigerator and allow them to reachroom temperature(20-25°C)

Materials supplied

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

Name 96determinations 48 determinations

Microelisa stripplate 12*8strips 12*4strips

Standard 0.3ml*6 vials 0.3ml*6 vials

Sample diluent 6.0ml*1vial 3.0ml*1vial

HRP-Conjugate reagent 10.0ml*1vial 5.0ml*1vial

20X Wash solution 25ml*1vial 15ml*1vial

Chromogen Solution A 6.0ml*1vial 3.0ml*1vial

Chromogen Solution B 6.0ml*1vial 3.0ml*1vial

Stop Solution 6.0ml*1vial 3.0ml*1vial

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Closure plate membrane 2 2

User manual 1 1

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Sealed bags 1
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Reagent preparation
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20×wash solution: Dilute with Distilled or deionized water 1:20.

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Assay procedure

1. Prepare allr e a g e n t sbeforestartingassayprocedure. ItisrecommendedthatallStandardsand

Samplesbe addedin duplicateto the MicroelisaStripplate.

2. Add standard: Set Standard wells, testing sample wells. Add standard 50µl to standard well.

3. Add Sample: Add testing sample 10µl Then add sample diluent 40µl to testing sample well; Blank

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

well doesn’t add anything.

4. Add100µlofHRP-conjugate reagent to each well, cover with an adhesive stripandincubatefor 60

minutes at37°C.

5. Aspirate each well and wash, repeating the process four times for a total of five washes.Wash by

filling each well with Wash Solution (400µl) using a squirt bottle, manifolddispenseror auto washer.

Complete removal of liquid at each step is essential to good performance. After the last wash, remove

any remaining Wash Solution by aspirating ordecanting. Invert the plate and blot it against clean paper

towels.

6. Add chromogen solution A 50µl and chromogen solution B 50µl to each well. Gently mix and

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incubate for 15 minutes at 37°C. Protect from light.

7. Add 50µl Stop Solution to each well. The color in the wells should change from blue toyellow. If the

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color in the wells is green or the color change does not appear uniform,gently tap the plate to ensure

thorough mixing.
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8. ReadtheOpticalDensity(O.D.)at450nmusinga microtiterplatereaderwithin15minutes.

Calculation of results
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1. This standard curve is used to determine the amount in an unknown sample. The standard curve is
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generated by plotting the average O.D. (450 nm) obtained for each of the six standard

concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X)
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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.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

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. Standard curve

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FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC

APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE
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BEGINNING!

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES