Ochratoxin A（OTA）ELISA Test Kit

Catalog No. LSY-10034

1.  Principle and usage

Ochratoxin is a secondary metabolite produced by certain species of the genus Aspergillus and Penicillium. Among them, ochratoxin A is the most widely distributed in nature and has the strongest toxicity, and has the greatest impact on humans, animals and plants.

The kit uses indirect competitive ELISA to detect ochratoxin (Ochratoxins A, OTA) in cereals and feed samples such as rice noodles, peanuts, and soybeans. The kit consists of pre-coated antigen plate and enzyme conjugate, antibodies, standards and other supporting reagents. During the test, the standard or sample solution is added, the ochratoxin in the sample and the pre-coated antigen on plate to compete against the ochratoxin antibody, and after adding the enzyme conjugate, via the TMB substrate show color, sample absorbance values of Ochratoxin has a negative correlation with its content, compared with the standard curve and then multiplied by the corresponding the dilution factor, can draw Ochratoxin content in samples.

 

2.  Technical specifications

Sensitivity:   0.1ppb(ng/ml)

Incubation: 37℃, 30min~30min~15min

Detection limit:

Grain ……………………………………………………….…1ppb

Feed ……………………………………………………….…2ppb

Cross-reaction rate

Ochratoxin A …………………………………………………………100%

Recovery rate:  

Grain, feed ……………………………………………………………85±15%

 

3. Components

1	OTA Micro-well strips	12 strips with 8 removable wells each
2	6× standard solution (1 mL each)	0ppb	0.1ppb
		0.3ppb	0.9ppb
		2.7ppb	8.1ppb
3	Enzyme conjugate	11ml	red cap
4	Antibody working solution	5.5ml	blue cap
5	Substrate A	6ml	white cap
6	SubstrateB	6ml	black cap
7	Stop solution	6ml	yellow cap
8	20X concentrated washing buffer	40ml	white cap
9	Instruction	1 piece

 

 

4. Materials required but not provided

1) Equipment: ELISA reader (450nm/630nm), printer, vortex (shaker optional), centrifuge, balance: 0.01g quantity sensitive, graduated pipettes, homogenizer;

2)  Micropipettes: single-channel 20ml ~ 200ml, 100ml ~ 1000ml, multi-channel 300ml

3) Reagents:  Methanol, NaHCO₃ , Deionized water

 

5.  Sample pre-treatment

5.1Solution preparation before sample pre-treatment:

1) 70%Methanol

7 parts of Methanol + 3 parts of deionized water; 

2) 0.1M NaHCO₃ solution

Weigh 4.2g NaHCO₃ , add deionized water to dissolve to 500ml;

3)  Washing buffer

1 part of 20X concentrated washing buffer and dissolve with 19 parts of deionized water to obtain the ready to use washing buffer. 

 

5.2Preparation ofGrain(rice, corn, millet etc.)  

1) Weigh 2g homogeneous sample into a 50ml centrifuge tube, add 10ml 70% Methanol, shake for 5min, Centrifuge at 4000 r/min at room temperature for 10min;

2) Take 1ml up-layer clear liquid, add 1ml 0.1M NaHCO₃ solution, shake to evenly;

3) Take 50ul liquid to test

Dilution factor:10               Detection limit:1ppb

 

5.3Preparation of Feed

1) Weigh 2g homogeneous sample into a 50ml centrifuge tube, add 20ml 70% Methanol, shake for 5min, Centrifuge at 4000 r/min at room temperature for 10min;

2) Take 1ml up-layer clear liquid, add 1ml 0.1M NaHCO₃ solution, shake to evenly;

3) Take 50ul liquid to test

Dilution factor:20               Detection limit:2ppb

 

6.  ELISA procedures

1).  Bring test kit to the room temperature (20-25 ℃) for at least 30 min, the 20X concentrated washing buffer may have crystallization when store in cold, need to return to room temperature to dissolve completely; note that each reagent must be shaken evenly before use; Put the required micro-well strips into plate frames. Re-seal the unused microplate, store at 2-8 ℃,not frozen.

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

3).  Add standard/sample: Add 50 µL of the sample or the standard solution into separate duplicate wells, then add antibody working solution, 50µL/well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, incubate at 37℃ for 30 min in the dark.

4).  Wash microplate: Carefully open the cover membrance, pour liquid out of microwell; add 350 µL/well of washing buffer, wash fully for 5 times, 30s each time, then take out and flap to dry with absorbent paper.(Use unused spear to pierce bubble after dry)

5).  Add Enzyme conjugate: add Enzyme conjugate, 100 µL/well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane,incubate at 37℃ for30 min in the dark.

Wash as step 4).

6).  Coloration: add 50 µL of substrate A then 50 µL substrate B into each well. Mix gently by shaking the plate manually, andincubate at 37℃ for 15 minin the dark for coloration. （If the blue color is too light, the reaction time can be extended appropriately)

7.  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 450nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630nm within 10 min).

 

7. Result judgment

In order to calculate the concentration of samples, a standard curve should be made. Before standard curve is made, the concept of % absorbance should be known.

Calculation of % absorbance:

B—the average OD value of the sample or the standard solution

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

The zero standard is thus made equal to 100 % and the absorbance values are quoted in percentages. The values calculated for the standards are entered in a system of coordinates on semilogarithmic graph paper against the ochratoxin A concentration [μg/kg]. The ochratoxin A concentration in μg/kg (ppb) corresponding to the absorbance of each sample can be read from the calibration curve.

A special software for result analysis of ELISA will facilitate double or multiple determinations. If you need, please call to request.

 

8.  Precautions

1.  The room temperature below 25 ℃ or the temperature of the reagents and the samples being not returned to the room temperature (25 ℃) will lead to a lower standard OD value.

2.  Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility; so continue to next step immediately after washing.

3.  Mix evenly before adding any reagents.

4.  The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.

5.  Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use.

6.  Storage: store at 2-8 ℃, not frozen. Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colourless color former is light sensitive, and thus they cannot be directly exposed to the light.

7.  Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value(450/630nm) of the 0 standard solution (0 ppb) of less than 0.5(（A450nm<0.5）) indicates its degeneration.

9. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.

Expiry date: 12 months; date of production is on box.