Dexamethasone ELISA test Kit

Catalog No. LSY-10042

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Dexamethasone in the sample.Thecoupling antigenis pre-coated on the micro-well stripes. TheDexamethasone in the sampleand thecoupling antigen pre-coated on the micro-well stripes compete for the anti-Dexamethasoneantibody. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with theDexamethasone in it. This value is compared to the standard curve and theDexamethasone concentration issubsequently obtained.

 

2. Technical specifications

Sensitivity: 0.1ppb

Incubation Temperature:  25℃

Incubation Time:  30min ～15min

Detection limit:

Tissue0.1ppb

Feed, milk, honey, urine0.2ppb

Milk powder0.3ppb

Cross-reaction rate:

Dexamethasone100%

Recovery rate

Tissue, feed90±25%

Milk powder85±25%

Urine, milk, honey…………………………………………………………. 95±25%

 

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	6× standard solution (1mL each)	0ppb	0.1ppb
		0.3ppb	0.9ppb
		2.7ppb	8.1ppb
3	Enzyme conjugate	7ml	red cap
4	Antibody working solution	7ml	blue cap
5	Substrate A	7ml	white cap
6	SubstrateB	7ml	black cap
7	Stop solution	7ml	yellow cap
8	20× concentrated washing buffer	40ml	white cap
9	5× concentrated redissolving solution	50ml	transparent cap

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4. Materials required but not provided

1) Equipments:microplate reader,homogenizer, nitrogen-drying device,vortex, centrifuge, measuring pipets, andbalance( asensibility reciprocalof0.01 g), Incubator, printer.

2) Micropipettors: single-channel 20-200 µL, 100-1000 µL, andmulti-channel 30～300 μl.

3) Reagents: Ethyl acetate, Acetonitrile (CH3CN), N-hexane.

 

5. Sample pre-treatment 

Instructions

Thefollowing pointsmust be dealt with beforethe pre-treatment ofany kind ofsample:

1)  Only thedisposable tips can be used for the experiments and the tips must be changed when used forabsorbingdifferent reagents;

2)  Before the experiment, eachexperimentalequipment must be checked to beclean and should be re-cleaned ifnecessary,in order to avoid thecontamination whichinterfereswith the experimental results.

Solution preparationbefore sample pre-treatment 

1) Acetonitrile and Ethyl acetate mixing solution: 1 partAcetonitrile + 1 part Ethyl acetate

2) Sample redissolving solution: the 5×concentrated redissolvingsolution is diluted withdeionized water at 1:4.

5.1 Tissue

1. Take 2.0± 0.05 g of the homogenized sample into a 50ml centrifuge tube; Add 2mlsample redissolving solution, then add 6mlEthyl acetate, shake for 3min, centrifuge at above4000 r/minat 15℃ for 10 min.

2. Take 3mL clear organic phase into a dry container, blow to dry bynitrogen or air in50-60℃.

3. Dissolve thedry residues in1 mLN-hexane, add 1 mL ofthesample redissolvingsolution, mix for 30 seconds; centrifugeat above4000 r/min at 15℃ for 10 min, remove the up-layer N-hexane phase.

4. Take50 µL of the down-layer for analysis.

Fold of dilution of the sample:1

5.2 Feed

1.  Weigh 1.0±0.05g homogenized feed sample into 50ml centrifuge tube, add 6mlethyl acetate, shake for 3 min, centrifugeat above4000 r/min at 15℃ for 10 min.

2.  Take 3 ml clearorganic phase into a dry container, blow to dry with nitrogenor airat 50-60℃.

3. Dissolve dry residues in 1 mL N-hexane, add1mL of thesample redissolving solution,mix for 30 seconds,centrifuge at above 4000 r/min at 15℃ for10min,removeup-layerN-hexane phase.

4. Take 50 µLdown-layer liquidfor further analysis.

Fold of dilution of the sample:2

5.3 Milk powder

1. Weigh 1.0 ± 0.05 g milk powder into a 50ml centrifuge tube, add 2mlsample redissolving solution, then add 6mlAcetonitrile and Ethyl acetate mixing solution, shake for 3min, centrifugeat above4000 r/min at 15℃ for 10 min.

2. Take 2ml clearorganic phase into a dry container, blow todry withnitrogen or air at 50-60 ℃.

3. Dissolve dry residues in2ml N-hexane, add1ml sample redissolving solution,mix for 20 seconds,centrifuge at above 4000 r/min at 15℃ for10min,removeup-layerN-hexane phase.

4. Take50 µL down-layer liquid for further analysis.

Fold of dilution of the sample:3

5.4 Honey

1. Weigh 1.0±0.05 g honey into a 50ml centrifuge tube, add 2mlsample redissolving solution, then add 6ml Ethyl acetate, shake for 3min. Centrifugeat above4000 r/min at 15℃ for 10 min.

2. Take 3ml clearorganic phase into a dry container, blow todry withnitrogen or air at 50-60 ℃.

3. Dissolve dry residues in1ml N-hexane, add1ml sample redissolving solution,mix for 30 seconds,centrifuge at above 4000 r/min at 15℃ for10min,removeup-layerN-hexane phase.

4. Take50 µL down-layer liquid for further analysis.

Fold of dilution of the sample: 2

5.5 Urine

1. Take 1.0ml urine sample into a 5ml centrifuge tube, add 2ml Ethyl acetate, shake for 1min, centrifugeat above4000 r/min at 15℃ for 10 min.  

2. Take 1ml clearorganic phase into a dry container, blow todry withnitrogen or air at 50-60 ℃.

3. Dissolve dry residues in1ml N-hexane, add1ml sample redissolving solution,mix for 30 seconds,centrifuge at above 4000 r/min at 15℃ for10min,removeup-layerN-hexane phase.

4. Take50 µL down-layer liquid for further analysis.

Fold of dilution of the sample:2

5.6 Milk

1.  Take 1.0ml milk sample into a 5ml centrifuge tube, add 2mlAcetonitrile and Ethyl acetate mixing solution, shake for 1min, centrifugeat above4000 r/min at 15℃ for 10 min.

2. Take 1ml clearorganic phase into a dry container, blow todry withnitrogen or air at 50-60 ℃.

3. Dissolve dry residues in1ml N-hexane, add1ml sample redissolving solution,mix for 30 seconds,centrifuge at above 4000 r/min at 15℃ for10min,removeup-layerN-hexane phase.

4. Take50 µL down-layer liquid for further analysis.

Fold of dilution of the sample:2

 

6. ELISA procedures

6.1Instructions 

1 Bring all reagents and micro-wellstrips to the room temperature (20-25 ℃) before use;

2 Return all reagentsto2-8 ℃ immediately after use;

3 The reproducibility of theELISA analysis, to alargedegree, depends on the consistencyof platewashing.The correct operationof platewashing is the keypoint inELISA theprocedures;

4 For theincubationatconstant temperatures, all the samples and reagents mustavoid light exposure, andeach microplate should be sealed by the covermembrane. 

6.2Operation procedures

1. Take out all thenecessary reagents from the kit and placeattheroom temperature (20-25 ℃) for at least 30 min. Note that eachreagent must be shaken to mix evenly beforeuse.

2. Take the required micro-well strips and plateframes. Re-sealed the unused microplate,store at 2-8℃,not frozen.

3. Solution preparation:dilute 15 mL of theconcentratedwashing buffer (20 × concentrated) with thedeionized water at 1:19 (1 part of 20Xconcentratedwashing buffer + 19 parts of deionized water), or prepare asquantity needed.

4. Numbering:number themicro-wells according tosamples andstandardsolution; each sample andstandardsolution should be performed in duplicate,record their positions.

5. Add 50 µL of thesampleorstandardsolution toseparate duplicate wells; and addenzyme conjugate,50ul/well, thenaddantibody working solution, 50µl/well.Mix gently by shaking the plate manually, seal the microplate with the cover membrane, andincubate at25 ℃ for 30 min.

6. Pour the liquid, wash the microplate with thewashing buffer at 250 µL/well for 4-5 times. Each time soak the well with thewashing buffer for 15-30 sec,flap to dry withabsorbent paper(if there are thebubbles after flapping, cut them with the clean tips).

7. Coloration: add50 µL of thesubstrate Asolution and then50 µL of theB solution into each well.Mix gently by shaking the plate manually, andincubate at 25 ℃ for 15minatdark for coloration.

8. Determination: add50 µL of thestop solution into each well.Mix gently by shaking the plate manually.Set thewavelength ofmicroplatereader at 450 nm to determine the OD value. (recommend to read the OD value at thedual-wavelength 450/630 nm within 5 min).

 

7. Result judgment

There are two methods to judgethe results; the first one is therough judgment, while the second is thequantitative determination.Note that the OD value of the samplehas anegative correlation with the content of Dexamethasone.

7.1 Qualitative determination

Theconcentration range (ng/mL) can be obtained from the comparison theaverageOD value of the sample with that of the standard solution.Assumingthat the OD value ofthe sampleⅠ is0.3, and that of thesampleⅡ is 1.0, while those of thestandard solutions are as thefollowings: 2.243 for0 ppb,1.816 for0.1ppb, 1.415for0.3ppb,0.74 for 0.9ppb,0.313 for 2.7ppb and 0.155 for 8.1ppb, accordingly theconcentration range of thesampleⅠis2.7 to 8.1ppb, and that of thesampleⅡ is 0.3 to 0.9ppb.

7.2 Quantitative determination

The mean values of the absorbance values obtained for the average OD value (B) of the sample and the standard solutiondivided by the OD value (B0) of thefirststandardsolution(0 standard) andsubsequentlymultiplied by 100%, that is,

 

B—the average OD value of the sample or the standard solution

B0—the average OD value of the 0ng/mL standardsolution

Draw thestandard curvewith theabsorption percentages of the standard solution and thesemilogarithm values of theDexamethasone standardsolution(ng/mL) asY-and X-axis, respectively.Read thecorrespondingconcentration of the sample from thestandard curve byincorporating itsabsorption percentageinto the standard curve.The resulting value issubsequently multiplied bythe corresponding dilution fold, thusfinally obtaining  theDexamethasone concentration in the sample.

Using theprofessionalanalyzingsoftware of thiskit will bemore convenient for theaccurate and rapid analysisof a large amount ofsamples. (Please contact us for thissoftware).

 

8. Precautions

1. Theroom temperature below 25℃ orthetemperature of the reagents andthe samples being notreturned to the room temperature (20-25 ℃) will lead to a lowerstandard OD value.

2. Dryness of the microplate in the washingprocess will be accompaniedbythe situationsincluding the non-linearstandardcurves and theundesirable reproducibility.

3. Mix every reagent and reaction mixture evenlyand wash the microplate thoroughly,otherwise there willbe theundesirable reproducibility.

4. The stop solution is the2 M sulfuric acid solution,avoid contacting with the skin;

5. Put the unused microplate into anauto-sealing bag to re-seal it.Thestandard substance andthecolourless color former is light sensitive, and thus they cannot be directly exposed to the light.

6. Do not usethe kit exceeding itsexpiry date.The use of diluted or adulteratedreagents from the kits will lead to the changes inthesensitivity and thedetecting OD values. Do not exchangethereagents from the kits ofdifferent lot numbers touse.

7. Discard thecolouration solution with anycolor that indicates thedegeneration of this solution.The detecting value of thestandard solution 1 (0 ppb) of less than 0.5 indicates itsdegeneration.

8. The optimum reaction temperatureis25 ℃,and too high or too low temperatures will result inthe changes in thedetectingsensitivity andOD values.

 

9. Storage andexpiry date

Storage: store at2-8 ℃, not frozen.

Expiry date: 12 months; date of production is on the box.

Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.