Colistin ELISA Test Kit

Catalog No. LSY-10056

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection ofColistin in the sample. The couplingantigens are pre-coated on the micro-well stripes. TheColistin in the sample andthecoupling antigens pre-coated on the micro-well stripescompetefor theanti-Colistin antibodies. 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 theColistin in it. This value is compared to the standard curve and theColistin concentrationis subsequently obtained.

 

2. Technical specifications

Sensitivity: 1.5ppb

Detection limit

Tissueabout50ppb

Raw milkabout60ppb

Finished milk about45ppb

Feed about150ppb

Note: ppb= ng/ml or ng/g

Cross-reaction rate

Colistin100%

Recovery rate

90±30%

 

3. Components

1） Micro-well strips: 12 strips with 8 removable wells each

2） 6× standard solution (1mL each): 0ppb,1.5ppb,4.5ppb,13.5ppb,40.5ppb

3） 11X ConcentratedEnzyme conjugate (0.7 mL)

4） Enzyme conjugate dilution (7 mL)

5） Substrate A (7 mL)

6） Substrate B (7 mL)

7） Stop solution (7 mL)

8） 20× concentrated washing buffer (30 mL)

9） Redissolving solution (50 mL)  

 

4. Materials required but not provided

1） Equipments:microplate reader (450nm, 630nm), printer, homogenizer, nitrogen-drying device, vortex, shaker, centrifuge (4000g and above), measuring pipets, balance (a reciprocal sensibility of 0.01 g), incubator(25℃), timer;

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

3） Reagents (AR): Deionized water, HCl, H2SO4, Methanol,NaCl.

 

5. Sample pre-treatment

Instructions

The following points must be dealt with before the pre-treatment of any kind of sample:

1） Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

2） Before the experiment, each experimental equipment must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.

Solution preparation before sample pre-treatment:

1) Tissue sampleextracting solution:Take 5ml HCl, add into container with 115ml deionized water, then add 7.5g NaCl, dissolve and mix them completely.

2) Milk sampleextracting solution:Take 2.8ml 98.3%H2SO4, add into 250ml deionized water, mix them evenly.

3) Washing buffer: 1 part20× concentrated washing buffer + 19 parts deionized water.

5.1 Samples preparation

a) Tissue (Chicken,duck)

1)Weigh1.0g±0.05g of the homogenized sample (tissue) into 50ml plastic centrifuge tube; Add1mlTissue sampleextracting solution and 2mlMethanol, use vortex for2min, Centrifuge at above 3000 g at room temperature for 5min(Note: Vortex immediately after adding extracting solution and methanol to avoid sample agglomeration);

2) Take50ul up-layer liquid, add 450ulRedissolving solution,use vortex for20s;

3) Take 50ul liquid to test.

Fold of dilution of the sample:40

b) Raw milk, finished milk

1)Weigh1ml milk sample into4ml plastic centrifuge tube; Add1mlMilk sampleextracting solution and 1mlMethanol, use vortex for1min;

2)Centrifuge at above 3000 g at room temperature for 5min;

3) Avoid up-layer suspension,take50ul up-layer clear liquid, add 450ulRedissolving solution,use vortex for20s;

3) Take 50ul liquid to test.

Fold of dilution of the sample:30

c) Feed

1)Weigh1g feed sample into10ml plastic centrifuge tube; Add2mlMilk sampleextracting solution and 2mlMethanol, use vortex for1min;

2)Centrifuge at above 3000 g at room temperature for 5min;

3)Take50ul up-layer clear liquid, add 450ulRedissolving solution,use vortex for20s;

3) Take 50ul liquid to test.

      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 all the necessary reagents from the kit and place at the room temperature (20 to 25 ℃) for at least 30 minutes. Note that each liquid reagent must be shaken to mix evenly before use.

2. Enzyme conjugate preparation: take 1 part11X ConcentratedEnzyme conjugate, add 10 partsEnzyme conjugate dilution, dilute at 1:10.

3. Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2-8℃, not frozen.

4. Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate; record their positions.

5. Add 50µL of the sample or standard solution to separate duplicate wells,then add enzyme conjugate, 50µL each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, andincubate at25 ℃ at darkfor30 minutes.

6. Pour liquid out of microwell, add 250 µL/well of washing buffer for 15-30 seconds, repeatthreeto four times, then flap to dry (if there are bubbles after flapping, cut them with the clean tips).

7. Coloration: add100 µLmixture ofthe substrate A andsubstrateB into each well (Note: mix Substrate A and Substrate B at 1:1, the mixture should be used in 10min, never use metal container or metal to stir the solution, otherwise the substrate may be invalid.). Mix gently by shaking the plate manually, andincubate at25 ℃ for15-20 minutes at dark for coloration.

8. Determination: add 50 µL of the stop solution into each well (The substrate color from blue to yellow, it means the stop succeeds). Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450 nm to determine the OD value (Recommend to read the OD value at the dual-wavelength 450/630 nm within 5 minutes).

 

7. Result judgment

There are two methods to judge the results: the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with theColistin concentration.

7.1 Qualitative determination

The concentration range (ng/mL)of Colistin can be obtained from comparing the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sampleⅠ is 0.3, and that of the sampleⅡ is 1.0, the OD value of standard solutions is:2.243 for 0ppb, 1.816 for 0.5ppb, 1.415 for1.5ppb, 0.74 for4.5ppb, 0.313 for13.5ppb, 0.155 for40.5ppb, accordingly the concentration range of the sampleⅠ is13.5 to40.5ppb, and that of the sampleⅡ is0.5 to4.5ppb.

7.2 Quantitative determination

The mean values of the absorbance valuesisobtained for the average OD value (B) of the sample and the standard solution divided by the OD value (B0) of the first standard solution (0 standard) and subsequently multiplied 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 standard solution

Draw the standard curve with the absorption percentages of the standard solution and the semilogarithm values of theColistin standard solution (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, finally obtaining theColistin concentration in the sample.

 

8.Precautions

1) Read the manual carefully before use;

2) Return all reagents in the kit to room temperature(25±2℃) (about 1 hour) before use;

3) Shake the reagent evenly before use, avoid bubbles when mixing;

4) The tips are disposable, to avoid cross-pollution, do not repeat use the tips;

5) Do not use test kits out of date, do not mix use reagents from different batch;

6) Test the sample immediately after sample preparation, otherwise it may affect results;

7) The substrate A and substrate B are both colorless and transparent liquids. If they become blue before use, or turn blue immediately after mixing, the reagents are contaminated or deteriorated.

8) Sampling process must be rapid on the premise of ensuring accuracy, so as to avoid the influence of reaction time difference on the test results.

9) The stop solution contains sulfuric acid. If you accidentally splash on your skin or clothing, rinse immediately with plenty of water. If you accidentally get into the eyes, please go to the hospital for examination after thorough cleaning.

 

9. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.

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