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1. INTENDED

Streptomycin is a bacteriostatic antimicrobial. Because streptomycin is cheap, it was often added to feed to prevent and control several bacterial and protozoan infections of animal. However, due to resistance and safety concerns, streptomycin has been limited in animal feeding.This elisa kit is a competitive enzyme immunoassay for the quantitative analysis of streptomycin in chicken, fish, shrimps, honey, milk and milk powder.

2. PRINCIPLE

The basis of the test is the antigen-antibody reaction. The microtiter wells are coated with coating antigen. Streptomycin standards or sample solution and anti-Streptomycin antibodies are added. If streptomycin is present in the sample, it will compete for the antibody, thereby preventing the antibody from binding to the coating antigen attached to the well. The secondary antibody, tagged with a peroxidase enzyme, targets the primary antibody that is complex to the drug coated on the plate wells. Substrate/chromogen is added to the wells and incubated. Bound enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm; the absorption is inversely proportional to the Streptomycin concentration in the sample. Chicken, fish,shrimps ..... 5 ppb, milk.......6 ppb

Milk powder....... 2oppb ,oney...5ppb.

3. Composition of the kit

Each kit contains sufficient materials for 96 measurements (including standard analyses). Each test kit contains:

Microtiter plate with 96 wells coated with streptomycin antigen Streptomycin standard solutions：0ppb, 0.15ppb, 0.45ppb, 1.35ppb, 4.05ppb, 8.1ppb)

10×Streptomycin Antibody..................................................................................... 1 ml

10×HRP-Conjugated Antibody.............................................................................1.6 ml

TMB Buffer A............................................................................................................. 7 ml

TMB Buffer B ............................................................................................................ 7 ml

Stop Buffer.................................................................................................................. 7 ml

10×Washing Buffer.................................................................................................50 ml

4.Required Materials Not Provided With the Kit

- Microtiter plate reader (450 nm)

- Balance

- Rotary evaporator or Nitrogen-blow Concentrator

- Vortex mixer

- 10, 20, 100 and 1000 μl pipettes

- Multi-channel pipette: 20-300μl

- Incubator

- Centrifuge Tube: 15ml, 50ml

- Volumetric Flask: 100ml, 1000ml

- Tween 20

- Hexane

5.Solution preparation

- Redissolved buffer: Mix 5.2g Na₂HPO₄.12H₂O, 0.8g NaH₂PO₄.2H₂O, 9g NaCl, 1.095g tween20 and water to 1000ml.

- Washing buffer: Mix 1 volume 10×Washing Buffer with 9 volume of distilled water.

- Antibody working buffer: Mix 1 volume 10×Streptomycin antibody with 9 volume of wash buffer.

- HRP-Conjugated Antibody working buffer: Mix 1 volume 10×HRP-Conjugated Antibody with 9 volume of wash buffer.

6.Sample Preparation

Fish, meat and shrimps

1. Homogenize the sample

2. Use 2±0.05g of the homogenized sample and add 8 ml redissolved buffer. Mix for 5min. Add 3ml hexane. Mix for 10min. Stand for 1h.

3. Centrifuge: 10 min / 3000 g / room temperature.

4. Use 1ml lower layer (water layer) and add 1ml hexane.

5. Centrifuge: 5 min / 3000 g / room temperature.

6. Use 100μl lower layer liquid (water layer), and add 400μl redissolved buffer.

7. Use 20μl in the test.

Note: Dilution factor: 25.

Milk

Use 1ml fresh milk. Centrifuge: 10 min / 3000 g / room temperature. Discard upper layer (fat layer). Use 20μl lower layer and add 780μl redissolved buffer. Mix. Use 20μl in the test.

Note: Dilution factor:40.

Milk powder

Use 2g milk powder, add 10ml distilled water. Mix. Centrifuge: 10 min / 3000 g / room temperature. Discard upper layer (fat layer). Use 20μl lower layer and add 180μl redissolved buffer. Mix. Use 20μl in the test.

Note: Dilution factor:50.

Honey

Use 1g honey, add 40ml redissolved buffer. Mix. Use 0.5g the sample (honey solution). Centrifuge: 10 min / 3000 g / room temperature. Use 20μl clear liquid in the test.

Note: Dilution factor:50.

7.ELISA Testing protocol

1. Insert a sufficient number of wells into the microwell holder for all standards and samples to be run in duplicate. Record standard and sample positions.

2. Add 20 μl of each standard solution or prepared sample to separate duplicate wells. Use a new pipette tip for each standard or sample.

3. Add 80 μl of Antibody working buffer to the bottom of each well. Mix gently by shaking the plate manually and incubate for 30 min at room temperature (37℃).

4. Pour the liquid out of the wells and tap the microwell holder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all the wells with 250 μl washing buffer and pour out the liquid again. Repeat three more times.

5. Add 100 μl of HRP-Conjugated Antibody working buffer to the bottom of each well. Incubate for 30 min at 37℃.

6. Pour the liquid out of the wells and tap the microwell holder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all the wells with 250 μl washing buffer and pour out the liquid again. Repeat three more times.

7. Add 100 μl of TMB Buffer (mix TMB buffer A 1:1 with TMB buffer B) to the bottom of each well. Incubate for 15 min at 37℃ in the dark.

8. Add 50 μl of the stop solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450 nm. Read within 30 minutes after addition of stop solution.

8. INTERPRETATION OF RESULTS

A standard curve can be constructed by plotting the mean relative absorbance (%) obtained from each refernce standard against its concentration in ng/ml on a logarithmic curve.

Use the mean relative absorbance values for each sample to determine the corresponding concentration of the tested drug in ng/ml from the standard curve. A special program with Excel functionality, Triphil ELISA test results. Please contact your local distributor or triphil@126.com for further information.

In order to obtain the Streptomycin concentration in ppb actually contained in a sample, the concentration read from the calibration curve must be further multiplied by the corresponding dilution factor.

9.PERFORMANCE CHARACTERISTICS

（1）Recovery rate:

Chicken, fish, shrimps ............................................................................................80±15%

Honey....................................................................................................80±15%

Milk .................................................................................................80±15%

Milk powder....................................................................................................85±15%

（2）Specificity:

The specificity of the ELISA Kit test was determined by analyzing the cross-reactivities to corresponding substances.

Streptomycin sulfate.............................................................................................. 100.00%

Dihydrostreptomycin................................................................................................<0.1 %

Gentamicin.............................................................................................................<0.1 %

Neomycin ......................................................... ....................................................<0.1 %

Kanamycin...........................................................................................................<0.1 %

10.LIMITATION

1. Make sure plates and reagents are brought up to room temperature. Keep the kit components out of the kit box for a least 1hour before starting the assay.

2. Shake up the reagent before using it

3. Carefully label each reagent to make sure the reagents are not intermixed. Kits with different expiration dates might generate different range of OD readings.

4. In general, a value of less than 0.6 in zero standard reading may indicate certain degrees of deterioration of reagents.

5. Incubate at 37℃, too high or too low temperature may generate different range of OD readings. And avoid sunlight beaming directly.

6. The stop buffer is 1M H₂SO₄, Please caution.

7. The TMB buffer becoming blue before use is indicating the deterioration of reagent

8. Always refrigerate plates in sealed bags at drying condition without light.

9. tore the kit at 2-8℃. The shelf life is 12 months when the kit is properly stored.