Rapid detection of Staphylococcus aureus enterotoxin in fresh milk (ELISA method)

[principle]

Coat the known Staphylococcus aureus enterotoxin antibody on the surface of the solid carrier microwell plate, wash off the unadsorbed antigen, add a certain mixture of the antibody and the extract of the sample (containing antigen) to be tested, and then compete in the microtiter plate.

The reaction carrier is a microplate coated with a specific antibody against Staphylococcus aureus enterotoxin

[Instruments and reagents]

1×96-well microplate; homogenizer or stirrer; centrifuge speed ≥3500r/min; magnetic stirrer; pH meter or pH test paper; vortex oscillator; pipette 100~1000uL; counting plate; 0.

(1) Reagents in the kit

① Bottle 1 Negative Gu Control: Ready-to-use Jiye-1×4mL

②Bottle 2 positive quality control Staphylococcus aureus enterotoxin A-50X-1×0.

③ Bottle 3 washing buffer: 20X-2×60mL

④Bottle 4 Enzyme marker: Staphylococcus aureus enterotoxin antibody and catalase mixture-ready-to-use solution-1×15mL

⑤Bottle 5 Substrate: carbamide peroxide -1×10mL

⑥Bottle 6 color-developing agent: TMB-1×10mL

⑦Bottle 7 Reaction stop solution: H ₂ SO ₄ ready-to-use solution-1×10mL

(2) Laboratory preparation reagents

① Extraction buffer (optional): disodium hydrogen phosphate 35: 6g or potassium dihydrogen phosphate 6.

②pH adjusting agent: NaOH (6mmol/L), HCl (6mmol/L)

③Polyethylene glycol : (Mw>17000) Dissolve 30g of polyethylene glycol in 100mL of distilled water and heat it slightly

④Positive quality control diluent: Dilute the positive quality control with distilled water in a ratio of 1:50 (40uL positive quality control plus 2mL distilled water); mix well

[Operation method]

(1) Column preparation: Take 10 mL of fresh milk directly; adjust the pH to 7.

(2) Determination: Insert the corresponding number of microwell strips into the microwell plate rack: 2 for negative control (1 for negative control for surface culture), 1 for positive quality control, and 1 for positive quality control.

(3) 100uL of positive quality control, negative quality control and samples were added to the marked wells
.
The surface culture microtiter plate is directly added with the mixed solution of the coloring agent and the substrate, the negative control is used, and the cover is covered for culture
.

(4) Incubate with shaking for 30 minutes at room temperature, prepare washing buffer, and refer to reagent preparation
.

(5) Pour out the microporous liquid, add washing buffer to the wells, keep it for 5~10s, pour out the washing liquid, tap on the absorbent paper placed on the table several times (holes down) for full pour Liquid in the pores
.
Repeat the washing 4 times
.

(6) Add 100 pL of enzyme marker to each well, being careful not to touch the edge of the pipette tip
.
Mix well and close the lid
.

(7) Incubate with shaking for 30 minutes at room temperature, and prepare the colorant and substrate mixture before the end of the incubation
.

(8) Pour out the microporous liquid, add washing buffer to the micropores, keep it for 5~10s, pour out the washing liquid, tap on the absorbent paper placed on the table several times (holes down) for full pour Remove the liquid in the micropores
.
Repeat the washing 4 times
.

(9) Add 100 uL of the mixed solution of the color developing agent and the substrate to each well, and discard the remaining mixed solution
.
The color agent and the substrate can also be added to the micro wells without mixing in advance
.
Incubate with shaking for 30 min at room temperature
.

(10) Add 100uL reaction stop solution to each microwell, the same steps as adding the substrate
.
Mix the reaction solution to ensure successful dyeing, and the reaction solution will turn from blue to yellow
.
Measure the absorbance under the condition of 450nm with air as a blank
.

[Result Judgment]

(1) The experiment confirms that the absorbance of the positive control should be at least 0.
50; the absorbance of the negative control should not be as high as 0.
50
.
If the quality control results do not meet the above requirements, the test results are invalid
.

(2) Judgment of results

①Positive result: If the absorbance of the sample is not lower than the threshold, the result is positive
.

②Negative result: If the absorbance of the sample is lower than T-0.
05, the result is negative
.

③False positive result: If the absorbance of the sample is between T-0.
05 and T, the result shows a false positive, and it needs to be re-determined by dialysis
.

[Precautions]

(1) In terms of safety, firstly, in order to avoid contamination of all samples and extracts and human poisoning, especially when using hydrochloric acid and sodium hydroxide , wear gloves and laboratory clothes; secondly, bottle 2 is the positive quality control bottle containing golden yellow grapes.
For coccal enterotoxin A, once the skin and eyes come into contact with the reagent, rinse immediately with plenty of tap water
.

(2) Reagent preparation.
First, take out the kit 1h before use; prepare all reagents and samples in advance; buffers should be diluted in advance or in the incubation stage; but the color reagent and substrate mixture is unstable and cannot be advanced Preparation; in the preparation process, washing is very important
.
Secondly, shake each test tube by hand or a vortex shaker before use; the incubation should be carried out in shaking (approximately 600r/min); the reagent bottle should also be shaken before use, especially not to mix reagents of different batch numbers.
All tests need to be newly prepared for positive quality control; reagents are stored at 2~8℃
.

(3) In terms of result determination, the bacteria died during heating or physical treatment, but enterotoxin still exists in the sample
.
In other words, the sample may be positive for enterotoxin, but may be negative for Staphylococcus aureus; substances with similar properties in the sample may have a cross-influence on the results
.

Related link: Rapid detection of Staphylococcus aureus in dairy products