1.  General

Ochratoxin A are secondary metabolites of Aspergillus and Penicillium strains, exist widely in grain and grain products, spices and coffee beans and other products. Ochratoxin A has a strong liver and kidney toxicity, and has teratogenic, mutagenic and carcinogenic effects, which is serious harmful to human health.

Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) has been a common method of Ochratoxin A detection, but sample preparation and samples analysis of this method is time-consuming and exhausting. The use of Ochratoxin A ELISA kit to analyze Ochratoxin A residue of samples is fast and accurate.

This kit is a new generation of fungal toxins detection products in application of ELISA technology, to minimize operating errors and work intensity.

2.  Principle

The test is based on a direct competitive enzyme immunoassay method. The microtiter wells are coated with Ochratoxin antigen. Free Ochratoxin in sample solutions and the immobilized antigen compete for Ochratoxin enzyme conjugate.

While Ochratoxin antibody and HRP secondary antibody (HRP material) combined, 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.

3.  Application

This test kit can be used to detect Ochratoxin in Corn and corn products, soybeans, oats and other crops and beer samples qualitatively and quantitatively.

4.  Technical specifications

Sensitivity:   0.1 ppb

Detection limit:

Corn, soybean, whole wheat etc. …………………………………………………1ppb

Beer ………………………………………………………………………………..0.1ppb

Recovery rate:  

Corn …………………………………………………………………………………80±10%

Whole wheat ………………………………………………………………………..90±10%

Cross-reaction rate

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

Ochratoxin B ………………………………………………………………………… 9.3%

Ochratoxin C ………………………………………………………………………… 46%

Coefficient of variation:   <10%

5.  Components

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

2)  5 x Concentrated standard (3x) ……………………………………………………1 ml each

(Remark: Concentrated standard (3x) is 0 ppb (zero standard), 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb，While other components of the solution concentration is also 3-fold, all five standards (including 0 points) must have 3-fold diluted to a concentration of 0 ppb,0.1ppb,0.3ppb,0.9ppb,2.7ppb before use, otherwise can not get the correct result. This standard is currently in use in product mixing, please dilute according to the dosage, don't match much.)

3)  OTA Enzyme conjugate .................................................................................... 12 ml

4)  OTA Antibody working solution ........................................................................... 6ml

5)  Substrate A........................................................................................................... 6ml

6)  Substrate B........................................................................................................... 6ml

7)  Stop solution......................................................................................................... 6ml

8)  Concentrated washing buffer（10×）................................................................40ml

6.  Materials required but not provided

1)  Equipment: microtiter plate spectrophotometer (450 nm/630nm), vortex (oscillator optional), centrifuge, balance: 0.01g quantity sensitive, gaduated pipettes: 10ml, ear wash bath, polystyrene centrifuge tube: 50ml, Micropipettes: single-channel 20ml ~ 200ml, 200ml ~ 1000ml, multi-channel 250ml

2)  Reagents:  Methanol, Petroleum ether, Deionized water

7.  Sample pre-treatment

Solution preparation before sample pre-treatment:

1)  washing buffer

Use 1 part of concentrated washing buffer （10×）and dissolve with 9 parts of deionized water to obtain the ready to use washing buffer. Solution is stable for 1 months when stored at 4 °C.

2)  sample extract buffer (for food crops)

Use 6 part of Methanol and dissolve with 4 parts of Deionized water to obtain the ready to use sample extract.

3)  5 OTA standards

Take five 1.5ml centrifuge tubes, numbered from 1 to 5. Take 5 standards(3x) (0 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb) 100μl accordingly to the 5 tubes, then each tube add 200μl deionized water diluted by 1:2 to obtain 0 ppb,0.1ppb,0.3ppb,0.9ppb,2.7ppb standard solution(see following table). The ready to use standard should be used in 3 days (store at 4℃ in the dark)