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1. Product Description

Sulfamethazine is is a member of the sulfonamide antibiotics family. They are effective antimicrobial drugs for the prevention of infections in cattle, poultry, and swine (prophylaxis), to treat veterinary diseases, and to promote growth. As a result of the extensive use of sulfonamides in the animal industry, residues of these drugs in food samples are a major concern because of their contribution to the development of antibiotic resistant pathogenic bacteria. To minimize this risk, maximum residue limits (MRls) have been established for total or individual sulfonamides at 0.1 mg/kg in milk and meat byUnitedStatesand Europe.

This ELISA KIT is a competitive enzyme immunoassay for the quantitative analysis of Sulfamethazine in muscle, liver, kidney, honey, urine and milk.

2. Test principle

The basis of the test is the antigen-antibody reaction. The microtiter wells are coated with sheep anti mouse IgG. Anti-sulfamethazine antibodies are added to combine with the IgG. Free sulfamethazine and sulfamethazine enzyme conjugate compete for the sulfamethazine antibody binding sites. At the same time, the anti-sulfamethazine antibodies are also bound by the immobilized capture antibodies. Any unbound enzyme conjugate is then removed in a washing step. 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 sulfamethazine concentration in the sample.

3. Kit Contents

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

1 × Microtiter plate with 96 wells coated with sheep anti mouse IgG

Sulfamethazine standard solutions(1ml)：0 ppb (zero standard), 1 ppb, 3 ppb, 9 ppb, 27 ppb, 81 ppb

10 ×Sulfamethazine antibody................................................................................... 1.6 ml

10 × HRP-Conjugated Antigen......................................................................1 ml

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

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

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

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

2×Sample Buffer...............................................................................................50 ml

4. Required Materials Not Provided With the Kit

(1) Equipment:

- Microtiter plate reader (450 nm)

- Rotary evaporator or Nitrogen-blow Concentrator

- Vortex mixer

- 20, 100 and 1000 μl pipettes

- Multi-channel pipette: 50-300μl

- Incubator

- Centrifuge

- Centrifuge Tube: 15ml, 50ml

- Volumetric Flask: 100ml, 1000ml

- Balance

(2) Reagents:

- Chloroform

- K₂HPO₄.12H₂O

- Hexane

- Concentrated hydrochloric acid

- NaOH

- Ethyl acetate

5. Solution Preparation

- 0.1M K₂HPO_4: Mix 22.8 g K₂HPO₄.12H₂O with 1000ml distilled water.

- 1M HCl: Mix 8.6ml concentrated hydrochloric acid with 100ml distilled water.

- 1M NaOH Solution: Mix 4g NaOH with 100ml distilled water.

- Sample working buffer: Mix 1 volume 2× Sample Buffer with 1 volume of distilled water.

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

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

- HRP-Conjugated antigen working buffer: Mix 1 volume 10× HRP-Conjugated antigen with 9

volume of wash buffer.

6. Sample Preparation

(1) Chicken, pork, fish and shrimps

1) Use 1±0.05g of the homogenized sample.

2) Add 10 ml chloroform, shake vigorously for 5 min.

3) Centrifuge: 10 min 3000g at room temperature (20 - 25 °C).

4) Use 5ml of upper layer, evaporate the liquid in 50℃(use rotary evaporator or nitrogen-blow Concentrator).

5) Dissolve the residue in 1 ml n-hexane and mix properly with 2 ml sample working buffer. shake vigorously for 5 min.

6) Centrifuge: 5 min 3000g at room temperature (20 - 25 °C).

7) Use 25 μl of the lower, aqueous phase per well in the assay.

Note: Dilution factor:4.

(2) Honey

1) Use 0.8 g of the honey sample.

2) Add 3.2 ml distilled water, 0.32 ml 1 M NaOH and 4 ml ethyl acetate, shake vigorously for 5 min.

3) Discard 3 ml ethyl acetate (upper layer); add 4 ml 0.1M K₂HPO₄, 0.4 ml 1M HCl, 3ml ethyl acetate, shake vigorously for 5 min.

4) Centrifuge: 10 min 3000g at room temperature (20 - 25 °C).

5) Use 2ml of upper layer; evaporate the liquid in 50℃ (use rotary evaporator or nitrogen-blow Concentrator).

6) Dissolve the residue in 0.8 ml n-hexane and mix properly with 0.8 ml sample working buffer. Shake vigorously for 2 min.

7) Centrifuge: 5 min 3000g at room temperature (20 - 25 °C).

8) Use 25 μl of the lower, aqueous phase per well in the assay.

Note: Dilution factor: 2.

(3) Urine

1) Use 2 ml of the urine sample.

2) Add 8 ml ethyl acetate, shake vigorously for 5 min.

3) Centrifuge: 10 min 3000g at room temperature (20 - 25 °C).

4) Use 4 ml of upper layer; evaporate the liquid in 50℃ (use rotary evaporator or nitrogen-blow Concentrator).

5) Dissolve the residue in 2 ml sample working buffer.

6) Use 25 μl in the assay.

Note: Dilution factor: 2.

(4) Milk

1) Use 1 g of the milk sample.

2) Centrifuge: 10 min 3000g at room temperature (20 - 25 °C / 68 - 77 °F).

3) Use 0.1 ml of upper layer, add 0.4 ml sample working buffer.

4) Centrifuge: 5 min 6000g at room temperature (20 - 25 °C / 68 - 77 °F).

5) Use 25 μl of the upper layer in the assay.

Note: Dilution factor: 5.

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 100 μl of Antibody working buffer to the bottom of each well. Mix gently by shaking the plate manually and incubate for 30 min at 37℃.

(3)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.

(4) Add 25 μl of each standard solution or prepared sample to separate duplicate wells. Use a new pipette tip for each standard or sample.

(5) Add 75 μl of HRP-Conjugated Antigen 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. 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 sulfamethazine concentration in ppb actually contained in a sample, the concentration read from the calibration curve must be further multiplied by the corresponding dilution factor.