Chloramphenicol ELISA test Kit

Catalog No. LSY-10007

1. Principle

This test kit is based on theindirect competitive enzyme immunoassay for the detection of Chloramphenicol in the sample.Thecoupling antigenis pre-coated on the micro-well stripes. TheChloramphenicol in the sampleand thecoupling antigen pre-coated on the micro-well stripes compete for the anti-Chloramphenicolantibody. 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 theChloramphenicol in it. This value is compared to the standard curve and theChloramphenicol concentration issubsequently obtained.

2. Technical specifications

Sensitivity: 15 ppt

Incubation Temperature:  25℃

Incubation Time:  30min～15min 

Detection limit:

Tissue(method 1)7.5ppt

Tissue(method 2) 15ppt

Egg 15ppt

Feed 0.2ppb

Recovery rate

Tissue, egg95±25%

Feed90±30%

Cross-reaction rate:

Chloramphenicol100%

Thiamphenicol< 0.1%

Florfeniol< 0.1%

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	7× standard solution (1mL each)	0ppt	15ppt
		45ppt	135ppt
		405ppt	1215ppt  10ppb
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	2×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.

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

3) Reagents: Ethyl acetate,N-hexane, NaOH, K2HPO4·3H2O, HCl.

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) Sample redissolving solution: the2×concentrated redissolvingsolution is diluted withdeionized water at 1:1.

5.1 Tissue (Chicken, duck, pork, fish, shrimp, beef, lamb) Method 1

1. Take 3± 0.05 g of the homogenized sample into a 50ml centrifuge tube. Firstly add 3 mL deionized water, then add 6mLethyl acetate, shake properly for 1 min, centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take 4mL of the supernatant, blow to dry bynitrogen in50-60℃.

3. Dissolve thedry residues in1 mL N-hexane, add 1 mL ofthesample redissolvingsolution, mix for 30 seconds; centrifugeat above4000 r/min at room temperature (20-25℃) for 10 min, remove the up-layer organic phase.

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

Fold of dilution of the sample:0.5

(If cannot take 4ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 9ml ethyl acetate then take 6ml supernatant blow to dry, the dilution factor is same)

5.2Tissue (Chicken, duck, pork, fish, shrimp, beef, lamb) Method 2

1. Take2± 0.05 g of the homogenized sample into a 50ml centrifuge tube. Firstly add 3 mL deionized water, then add 6mLethyl acetate, shake properly for 1 min, centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take3mL of the supernatant, blow to dry bynitrogen in50-60℃.

3. Dissolve thedry residues in1 mLN-hexane, add 1 mL ofthesample redissolvingsolution, mix for 30 seconds; centrifugeat above4000 r/min at room temperature (20-25℃) for5 min, remove the up-layer organic phase.

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

Fold of dilution of the sample: 1

(If cannot take 3ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 10ml ethyl acetate then take 5ml supernatant blow to dry, the dilution factor is same)

5.3 Other tissue samples with high fat content (kidney, liver, intestine, skin, heart, pork belly, etc.) 

1. Take2± 0.05 g of the homogenized sample into a 50ml centrifuge tube.Add10mL N-hexane, shake for3 min, centrifuge at above4000 r/minat room temperature (20-25℃) for5 min, discardN-hexane.

2. Add 6mLethyl acetate, shake for 1min,centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

3. Take3mL of the supernatant, blow to dry bynitrogen in50-60℃.

4. Dissolve thedry residues in1 mLN-hexane, add 1 mL ofthesample redissolvingsolution, mix for 30 seconds; centrifugeat above4000 r/min at room temperature (20-25℃) for5 min, remove the up-layer organic phase.

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

Fold of dilution of the sample: 1

5.4 Egg

1. Take2± 0.05 g of the homogenized sample into a 50ml centrifuge tube.Add 6mLethyl acetate, shake properly for 1 min, centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take3mL of the supernatant, blow to dry bynitrogen in50-60℃.

3. Dissolve thedry residues in1 mLN-hexane, add 1 mL ofthesample redissolvingsolution, mix for 30 seconds; centrifugeat above4000 r/min at room temperature (20-25℃) for5 min, remove the up-layer organic phase.

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

Fold of dilution of the sample: 1

(If cannot take 3ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 10ml ethyl acetate then take 5ml supernatant blow to dry, the dilution factor is same)

5.5 Feed

1. Take 1±0.05g of the homogenized sample into a 50ml centrifuge tube, firstly add9 mL deionized water, then add 5mLN-hexane, shake properly for 3 min;

2. Centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

3.Take5mL middle-layer water phase into another50mL clean centrifuge tube, add10mLEthyl acetate,fully shake for60s;

4.Transfer5mL oftheethyl acetate layer into a newcentrifugal tubeand evaporate to dryness by nitrogen or air at 50℃.

5.Dissolve the dry residues in2mL N-hexane, add 1mL of the diluted redissolving solution, mix properly for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min; Remove up-layerN-hexane phase;

6. Take 50 µL down-layer clear liquid for analysis.

Fold of dilution of the sample: 4

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 40 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;then add 50 µL enzyme conjugate into each well, at last add50 µL of antibody working solution into each 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 the dilutedwashing 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 Chloramphenicol.

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 for0ppt,1.816 for15ppt, 1.415for 45ppt,0.74 for 135ppt,0.313 for405ppt and 0.155 for 1215ppt, accordingly theconcentration range of thesampleⅠis 405 to1215ppt, and that of thesampleⅡ is45 to135ppt.

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 theChloramphenicol 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  theChloramphenicol 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.