LSY-10058 Kanamycin ELISA Test Kit for milk, pork, chicken

Kanamycin ELISA Test Kit

Catalog No. LSY-10058

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Kanamycin in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Kanamycin in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Kanamycin 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 the Kanamycin in it. This value is compared to the standard curve and the Kanamycin concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 0.2ppb

Incubator temperature: 25℃

Incubator time: 20min～10min

Detection limit

Liquid milk (method 2) about 10ppb

Pork, Chicken about 6ppb

Liquid milk (method 1), Honey, Vaccine about 5ppb

Note: ppb= ng/ml or ng/g

Cross-reaction rate

Kanamycin 100%

Spectinomycin,Streptomycin,Tilmicosin <0.1%

Recovery rate

90±30%

3. Components

• Micro-well strips: 12 strips with 8 removable wells each
• 6× standard solution (1mL each): 0ppb, 0.2ppb, 0.6ppb, 1.8ppb, 5.4ppb, 16.2ppb
• Enzyme conjugate (7 mL)
• Antibody working solution (7 mL)
• Substrate A (7 mL)
• Substrate B (7 mL)
• Stop solution (7 mL)
• 20X concentrated washing buffer (30 mL)
• 20X concentrated tissue redissolving solution (10 mL)
• 10X concentrated honey redissolving solution (10 mL) (Optional)

4. Materials required but not provided

• 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;
• Micropipettors: single-channel 20-200 µL, 100-1000 µL, and eight-channel 30～300 µl;
• Reagents (AR): NaOH, NaCl, KCl, KH₂PO₄, Na₂HPO₄▪12H₂O(The above reagents are of analytical grade), Deionized water.

5. Sample pre-treatment

Instructions

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

• Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;
• 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 redissolving solution: 1 part of 20X concentrated tissue redissolving solution, add 19 parts of deionized water, mix it evenly.

2) Honey redissolving solution: 1 part of 10X concentrated honey redissolving solution, add 9 parts of deionized water, mix it evenly.

• Buffer: 0.1M PBS solution(PH=10-11): weigh 13.4g Na₂HPO₄▪12H₂O, 20g NaCl, 0.32g NaOH, 0.5g KH₂PO₄, 0.5gKCl, add deionized water to 500ml.
• 1M HCl: take 1ml HCl, add 11ml deionized water, mix it evenly.
• Washing buffer: 1 part 20× concentrated washing buffer + 19 parts deionized water.

5.1 Milk (Method 1)

1) Take the collected raw milk , thaw and return to room temperature for above 30min;

2) Put tips into down-layer of raw milk, take out 1ml sample into 2ml centrifuge tube(Note: do not take up-layer cream);

3) Add 50ul 1M HCl, shake strongly for 1min (or vortex for 30s);

4) Centrifuge at above 4000 g at room temperature for 10min;

5) Take 50ul up-layer clear liquid into another centrifuge tube (do not take up-layer cream), then add 450ul Tissue Redissolving solution,shake strongly for 1min (or vortex for 30s);

6) Take 50ul liquid to test.

Fold of dilution of the sample: 10

5.2 Milk (Method 2)

1) Take 50ul liquid milk sample, add 1950ul Tissue Redissolving solution, vortex for 1min, mix it evenly;

2) Take 50 µL for analysis.

Fold of dilution of the sample: 20

5.3 Tissue (Chicken, Pork)

1) Take 1.0±0.05g homogenized tissue sample into 50ml Polystyrene centrifuge tube;

2) Add 3ml Buffer: 0.1M PBS solution(PH=10-11), shake strongly for 5min, then put it at 60℃ water bath for 60min, then take out and centrifuge at above 4000 r/min at room temperature (20 - 25 ℃) for 10min;

3) Take 100ul up-layer clear liquid, add 400ul Tissue redissolving solution, vortex for 20S;

5) Take 50 µL for analysis.

Fold of dilution of the sample: 15

5.4 Honey

1) Take 1.0±0.05g honey sample into 50ml Polystyrene centrifuge tube;

2) Add 2ml deionized water, shake strongly for 2minl(or vortex for 1min)

3) Take 100ul up-layer clear liquid, add 400ul Honey redissolving solution, vortex for 30S;

4) Take 50 µL for analysis.

Fold of dilution of the sample: 10