1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Streptomycin in the sample. The coupling antigen is pre-coated on the micro-well stripes. The Streptomycin in the sample and the coupling antigens pre-coated on the micro-well stripes compete for anti- Streptomycin antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the testing sample has a negative correlation with the Streptomycin concentration in the sample. This value is compared to the standard curve and the Streptomycin concentration is subsequently obtained.

2. Technical specifications

Sensitivity : 0.1 ppb

Detection limit:

Chicken, Chicken liver, Milk............................................................... 4 ppb

Honey, Royal jelly............................................................................ 2 ppb

Recovery rate:

Milk.............................................................................................. 95±15%

Chicken........................................................................................ 85±10%

Honey, Royal jelly......................................................................... 75±15%

Cross-reaction rate:

Streptomycin.................................................................................... 100%

Dihydrostreptomycin........................................................................ 108%

Kalamycin.......................................................................................... <1%

Gentamycin....................................................................................... <1%

3. Components

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

2）   6× standard solution (1 mL each): 0 ppb, 0.1 ppb, 0.4 ppb, 1.6 ppb, 6.4 ppb, 25.6 ppb

3）   Enzyme conjugate (12 mL)............................................................. red cap

4）   Antibody working solution (7 mL).................................................. blue cap

5）   Substrate A solution (7 mL).......................................................... white cap

6）   Substrate B solution (7 mL)......................................................... black cap

7）   Stop solution (7 mL).................................................................. yellow cap

8）   20× concentrated washing buffer (40 mL)..................................... white cap

9）   10× concentrated redissolving solution (50 mL)................... transparent cap

4. Materials required but not provided

1)        Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance( a reciprocal sensibility of 0.01 g).

2)        Micropipettors: single-channel 20~200 µL, 200~1000 µL; and multi-channel 250 µL;

3)  Reagents:  H₃PO₄, NaOH(for honey sample), Acetonitrile(CH₃CN), N-hexane

5. Sample pre-treatment

Instructions (The following points must be dealt with before the pre-treatment )

1)  Only the disposable tips can be used for the experiments and the tips must be changed when used for 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)  The 10×concentrated redissolving solution is diluted with deionized water at 1:9 (1 mL concentrated redissolving solution + 9 mL deionized water)

2)  0.5% Trichloroacetic acid(for chicken and liver sample): dissolve 0.5 g Trichloroacetic acid in the deionized water to 100 mL.

3)  0.1 M H₃PO₄ (for honey sample): dissolve 680µL H₃PO₄ in the deionized water to 100 mL.

4)  1 M NaOH(for honey sample): dissolve 4 g NaOH in the deionized water to 100 mL.

5.1 milk

1  Take 1 mL milk sample, diluted at 1:39 (1950 µL diluted redissolving solution+ 50 µL milk). Mix for 30 seconds.

2  Take 50 µL for analysis

Fold of dilution of sample :40

5.2 meat, liver( chicken)

1          Take 2±0.05 g homogenized sample(remove fat), add 6 mL 0.5% Trichloroacetic acid and 2 mL Acetonitrile(CH₃CN). Mix for 5 min.

2          Centrifuge at above 4000 r/min at room temperature for 10 min.

3          Transfer 2 mL supernatant into a new vessel, then add 2 mL N-hexane. Mix and then still for 3 min. Take 0.5 mL clear solution on bottom layer. Centrifuge at above 4000 r/min at room temperature for 5 min.

4          Take 50 µL clear solution on bottom layer(if the layer is clear, should remove the upper layer), then add 450 µL the diluted redissolving solution, mix properly for 30 seconds.

5          Take 50 µL for analysis.

Fold of dilution of sample :40

5.3 honey, Royal jelly

1  Weigh 2±0.05 g honey sample, add 4 mL of 0.1 M H₃PO_4, shake properly for 10 min.

2  Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min, until liquid is clear.

3  Add 900 µL 1 M NaOH, adjust PH to 7-9(For Royal jelly, Transfer the Supernatant to a new vessel).

4..Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min, until liquid is clear.

5  Take 50 µL suppernatant, add 350 µL of the diluted redissolving solution, mix evenly.

6  Take 50 µL for further analysis.

Fold of dilution of sample :20 

6. ELISA procedures

1          Bring test kit to the room temperature (20-25 ℃) for at least 30 min, note that each reagent must be shaken evenly before use; put the required micro-well strips into plate frames. Re-sealed the unused microplate, stored at 2-8 ℃, not frozen.

2          Solution preparation: dilute 40 mL of the concentrated washing buffer (20×concentrated) with the distilled or deionized water to 800 mL (or just to the required volume) for use;

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

4          Add 50 µL of the sample and standard solution to separate duplicate wells, then add 50 µL of antibody working solution to each well, shake properly, seal the microplate with the cover membrane, and incubate at 37 ℃ for 30 min;

5          Pour liquid out of microwell, flap to dry on absorbent paper; add 250 µL/well of washing buffer for 15-30 seconds, then take out and flap to dry with absorbent paper, repeat 5 times.

6          Add 100 µL of enzyme conjugate to each well, shake properly, seal the microplate with the cover membrane, and incubate at 37 ℃ for 30 min; continue as step 5 for washing.

7          Coloration: add 50 µL of the substrate A solution, 50 µL of the B solution into each well. Mix gently by shaking the plate manually, and incubate at 37 ℃ for 15 min in the dark for coloration;

8          Determination: add 50 µL of the stop solution into each well. Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450 nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630nm within 5 min).

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 the content of Streptomycin.

7.1 Qualitative determination

The concentration range (ng/mL) can be obtained from comparing the average OD value of the testing 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 0 ppb, 1.816 for 0.1 ppb, 1.415 for 0.4 ppb, 0.74 for 1.6 ppb, 0.313 for 6.4 ppb, 0.155 for 25.6 ppb, accordingly the concentration range of the sampleⅠ is 6.4 to 25.6 ppb, and that of the sample Ⅱ is 0.4  to 1.6 ppb.

7.2   Quantitative determination

The mean values of the absorbance values is equivalent to the percentage of the average OD value (B) of the testing 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 (double wells) OD value of the testing sample or the standard solution

B₀—the average OD value of the 0ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithmic values of the Streptomycin