Neomycin ELISA test Kit

Catalog no.: LSY-10005

1. Principle

This test kit is based on the indirect competitive enzyme immunoassay for the detection of Neomycin in the sample.Thecoupling antigenis pre-coated on the micro-well stripes. TheNeomycin residues in the sampleand thecoupling antigen pre-coated on the micro-well stripes compete for the anti-Neomycinantibody. 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 theNeomycin in it. This value is compared to the standard curve and theNeomycin concentration issubsequently obtained.

2. Technical specifications

Sensitivity: 0.1ppb

Incubation Temperature: 25℃

Incubation Time: 30min—15min

Detection limit

Tissue, milk, milk powder, feed4ppb

Duck8ppb

Honey7ppb

Egg3ppb

Pig urine1ppb

 Recovery rate

 Tissue95±25%

    Chicken liver, Pig liver80±25%

Milk,milk powder, honey, egg, feed, pig urine90±30%

Cross-reaction rate

Neomycin100%

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	6× standard solution (1 mL 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	15ml	white cap
9	2× concentrated redissolving solution	50ml*2	transparent cap

4. Materials required but not provided

1) Equipment:microplate reader, printer,homogenizer,vortex, centrifuge, measuring pipets, andbalance( asensibility reciprocalof0.01g), incubator.

2) Micropipette: single-channel 20-200µL, 100-1000µL, andmulti-channel30～300µL.

3) Reagents: Trichloroacetic acid(TCA), NaOH, HCl, deionized water.

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 thatinterfereswith the experimental results.

Solution preparationbefore sample pre-treatment 

1) 3% Trichloroacetic acid:dissolve 3gTrichloroacetic acid in thedeionized water to100mL.

2) 1 M NaOH solution:dissolve4gNaOHin thedeionized water to100mL

3) 1 M HCl solution:take 8.6mlHCl, add deionized water to100mL

4) Sample dilution buffer: the2×concentrated redissolvingsolution is mixed withdeionized water at 1:1 (1 part ofconcentrated redissolving solution + 1 part of deionized water). 

5) Sample extracting buffer: diluteSample dilution buffer withdeionized water at 1:9 (or directly dilute the2×concentrated redissolvingsolution withdeionized water at 1:19).

5.1  Tissue(Chicken, pork,duck,shrimp, fish), honey, egg sample 

1. Take 2±0.05g of the homogenizedsample into 50mL centrifuge tube, add 8mL 3% Trichloroacetic acid, shake for4min, centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take 100µl up-layer clear liquid to another tube,add700µl of theSample dilution buffer, shake thoroughly to mix it evenly.

3. Take50µL for analysis.

Fold of dilution of the sample: 40

5.2 Pig Liver sample 

1. Take1±0.05g of the homogenized pig liversample into 50mL centrifuge tube, add5mLof theSample extracting buffer, shake for3min,put it at56℃water bath for 25min,vortex for 30s(or shake by hand for 30s),centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take 100µl up-layer clear liquid to another tube,add 300µl of theSample dilution buffer, shake thoroughly to mix it evenly.

3. Take50µL for analysis.

Fold of dilution of the sample: 20

5.3 Feed sample 

1. Take1±0.05g of thecrushed feedsample into 50mL centrifuge tube, add9mLof theSample extracting buffer and1ml 1M NaOH solution, shake for3min, centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take 100µl up-layer clear liquid to another tube,add 400µl of theSample dilution buffer, shake thoroughly to mix it evenly.

3. Take50µL for analysis.

Fold of dilution of the sample: 50

5.4 Pig urine sample 

1. Take1ml±0.05ml of the pig urinesample into 50mL centrifuge tube, add5mLof the deionized water, shake for2min to mix it evenly.

2. Take 100µl of above liquid,add 300µl of theSample dilution buffer, shake thoroughly to mix it evenly.

3. Take50µL for analysis.

Fold of dilution of the sample: 20

5.5 Milk sample 

1. Take1ml±0.05ml of the milksample into10mL centrifuge tube, add 50µl 1M HCl solution, shake for1min,centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take50µl up-layer clear liquid to another tube,add 950µl of theSample dilution buffer, shake thoroughly to mix it evenly.

3. Take50µL for analysis.

Fold of dilution of the sample: 20

5.6 Milk powdersample 

1. Take1g±0.05g of the milk powdersample into50mL centrifuge tube, add7ml deionized water, shake for3min,centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

2. Take1ml up-layer liquid to another 10ml centrifuge tube,add50µl 1M HCl solution, shake for 1min,centrifuge at above4000 r/minat room temperature (20-25℃) for5 min.

3. Take 100µl up-layer clear liquid to another tube, add 900µl of theSample dilution buffer, shake thoroughly to mix it evenly.

4. Take50µL for analysis.

Fold of dilution of the sample: 80

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 the kit from the refrigerated environment.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 15mL of the20× concentratedwashing buffer with thedeionized waterat 1:19(1 partof 20× concentratedwashing buffer + 19 parts of deionized water) for use, or prepare as quantity needed.

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

5. Add 50µL of thesample or standardsolution toseparate duplicate wells; Thenadd50µL enzyme conjugate, then add 50µL of the antibody working solution into each well.Mix gently by shaking the plate manually, seal the microplate with the cover membrane, andincubate at25 ℃ for 30min.

6. Pour the liquid, wash the microplate with thewashing buffer at 250µL/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: add50µL of thesubstrate A and then50µL of the substrateB into each well.Mix gently by shaking the plate manually, andincubate at 25 ℃ for 15minatdark for coloration.

8. Determination: add50µL of thestop solution into each well.Mix gently by shaking the plate manually.Set thewavelength ofmicroplatereader at 450nm to determine the OD value. (recommend to read the OD value at thedual-wavelength 450/630nm 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 Neomycin.

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 for0ppb,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.Multiplying by its corresponding dilution factor is the actual concentration of neomycin in the sample.

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 0 ng/mL standardsolution

Draw thestandard curvewith theabsorption percentages of the standard solution and thesemilogarithm values of theNeomycin 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 theNeomycin concentration in the sample.

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 andthecolorless 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 thecoloration 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 ℃, If 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.