Lsy-10029 Total Aflatoxin Elisa Test Kits 96 Wells/Kit

Total Aflatoxins ELISA test kit
Catalog No. LSY-10029
1.  General
Aflatoxins are secondary metabolites of Aspergillus flavus and Aspergillus parasiticus in the genus. These molds come from the humid tropics, large-scale cultivation of food crops will be their pollution. Aflatoxins are nature's most dangerous carcinogens. Aflatoxins is almost always exist as AFB1, AFB2, AFG1 and AFG2. They are mainly found in corn, peanuts, nuts, cottonseed and other foods. The U.S. Food and Drug Administration (FDA) has made provisions for food and feed in the maximum permitted level of aflatoxin, therefore, accurate determination of aflatoxin levels has important implications for the food and feed quality control.
This kit can analyze Aflatoxins content in sample quickly and accurately. It is simple, quick and do not need special equipment, can be used for lab, also for test in Farms, feed mixing workshop.
 
2.  Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Aflatoxins in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Aflatoxins in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti- Aflatoxins antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with the Aflatoxins in it. This value is compared to the standard curve and the Aflatoxins concentration is subsequently obtained.
 
3.  Application
This test kit can be used to detect Total Aflatoxins in corn, rice, wheat, beans, peanut, feed and cooking oil samples qualitatively and quantitatively.
 
4.  Technical specifications
Sensitivity: 0.1 ppb
Detection limit:
Grain 0.8 ppb
Peanut, Cake, Cooking oil 1 ppb
Feed, flour, glue pudding 2.4 ppb
Beer 0.5 ppb
Soy sauce, vinegar 0.8 ppb
Cross-reaction rate:
Aflatoxins B1… ...100%
Aflatoxins B2… ...75%
Aflatoxins G1… .....100%
Aflatoxins G2… .....97%
Aflatoxins M1… .....30%
 
5.  Components
1)  Micro-well strips: 12 strips with 8 removable wells each
2)  6 x standards ............................2 ml each 
0 ppb (zero standard), 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb
3)  Enzyme conjugate..................................................... 7ml
4)  Substrate................................................................ ....12ml
5)  Stop solution.................................................................10ml
6)  10X Concentrated washing buffer ................................50ml
7)  10X Concentrated sample diluent ................................50ml
 
6.  Materials required but not provided
1)  Equipment:  microtiter plate spectrophotometer (450 nm/630nm), vortex (oscillator optional), nitrogen-drying device, centrifuge, grinder, balance: 0.01g quantity sensitive.
2)  Micropipettes: single-channel 20ml ~ 200ml, 200ml ~ 1000ml, eight-channel 300ml
3)  Reagents: methanol (AR), petroleum ether, Acetonitrile, deionized water (or distilled water), CHCl3, NaCl.
 
7.  Sample pre-treatment
Solution preparation before sample pre-treatment:
1) Sample diluent
Dilute the 10x concentrated sample diluent at 1:9 with deionized water to use (1 part 10X concentrated sample diluent + 9 parts deionized water).
2)  Washing buffer
Dilute the 10X concentrated washing buffer at 1:9 with deionized water to use (1 part 10X concentrated washing buffer + 9 parts deionized water).
Grain diluent Dissolve 0.9g NaCl with the diluted sample diluent to 100ml
Beer diluent Take methanol, dilute methanol with the diluted sample diluent at 1:4 (1 part methanol + 4 parts diluted sample diluent).
50% methanol 1 part methanol + 1 part deionized water
80% methanol 4 parts methanol + 1 part deionized water
Samples Preparation
7.1 Preparation of grain (Rice, corn, millet and other low-fat content)
1) Take 1.0 g ground sample, mix with 8ml Grain diluent evenly(Can adjust the quantity of sample and grain diluent according to lad conditions);
2) Vortex for 3min;
3) Centrifuge at 5000r/min for 10 minutes.
4) Take 100 μl of the supernatant for assay.
Dilution factor: 8
7.2 Preparation of cake
1) Take 1.0g ground sample, mix with 4ml 50% methanol evenly;
2) Vortex for 3 min;
3) Centrifuge at 5000r/min for 10 minutes;
4) Take 400ul of the down-layer liquid, add 600ul sample diluent, mix it evenly;
5) Take 100 μl of the diluted liquid for assay.
Dilution factor: 10
7.3 Preparation of peanut, cream cake
1) Take 1.0g ground sample, mix with 4ml petroleum ether evenly;
2) Add 4ml 50% methanol, mix it evenly;
3) Vortex for 3 min;
4) Centrifuge at 5000r/min for 10 minutes;
5) Take 400ul of the down-layer liquid, add 600ul sample diluent, mix it evenly;
6) Take 100 μl of the diluted liquid for assay.
Dilution factor: 10
7.4 Preparation of cooking oil
1) Take 1.0g ground sample, mix with 4ml petroleum ether evenly;
2) Add 4ml 50% methanol, shake for 1 min;
3) Be static for 10min;
4) Take 400ul of the down-layer liquid, add 600ul sample diluent, mix it evenly;
5) Take 100 μl of the diluted liquid for assay.
Dilution factor: 10
7.5 Preparation of feed, flour, glue pudding
1) Take 3.0g ground sample, mix with 9ml 80% methanol evenly(Can adjust the quantity of sample and methanol according to lad conditions);
2) Vortex for 3 min;
3) Centrifuge at 2000r/min for 10 minutes;
4) Take 100ul of the supernatant, add 700ul sample diluent, mix it evenly;
5) Take 100 μl of the diluted liquid for assay.
Dilution factor: 24
7.6 Preparation of beer
1) Take 200ul beer (remove CO2), add 800ul beer diluent(Can adjust the quantity of sample and beer diluent according to lad conditions);
2) Shake for 3 min;
3) Take 100 μl of the diluted liquid for assay.
Dilution factor: 5
7.7 Soy sauce, vinegar, wine
1) Take 0.5ml sample, add 0.5ml distilled water and 4.0ml CHCl3(Can adjust the quantity of sample, water and CHCl3 according to lad conditions);
2) Mix it evenly and shake for 3min, centrifuge at 5000r/min for 10 minutes;
3) Take 1.0ml down-layer liquid, blow to dry by nitrogen at 60 ºC;
4) Add 100ul Acetonitrile to dissolve the dry residue, and add 900ul sample diluent, mix it evenly;
5) Take 100 μl of the diluted liquid for assay.
Dilution factor: 8
 
8. ELISA procedures
8.1 Instructions 
1) Bring all reagents and micro-well strips to the room temperature (20-25 ºC) before use;
2) Return all reagents to 2-8 ºC immediately after use;
3) The reproducibility of the ELISA analysis, to a large degree, depends on the consistency of plate washing. The correct operation of plate washing is the key point in ELISA the procedures;
4) For the incubation at constant temperatures, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane.
8.2 Operation procedures

Bring test kit to the room temperature (20-25 ºC) for at least 30 min, note that each reagent must be shaken to mix evenly before use, Put the required micro-well strips into plate frames. Re-sealed the unused microplate, store at 2-8 ºC, not frozen. Washing buffer also need to return to room temperature before use.Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate, record their positions.Add 50 µL of the sample or standard solution into separate duplicate wells; add 50ul enzyme conjugate into each well, mix gently by shaking the plate manually. Seal the microplate with the cover membrane, and incubate at at room temperature (20~25 ºC) for 15 minutes in dark.Pour liquid out of microwell, flap to dry on absorbent paper, add 300 µL/well of washing buffer to wash microplate for 30 s, then take out and flap to dry with absorbent paper, repeat 5 times;Coloration: add 100 µL of the substrate into each well. Mix gently by shaking the plate manually, then incubate at room temperature (20~25 ºC) for 15 min at dark for coloration. Determination: add 50 µL of the stop solution into each well. Mix gently by shaking the plate manully. Set the wavelength of the microplate reader at 450 nm to determine the OD value of every well. 

 
9.  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 know.
Calculation of % absorbance:B-the average OD value of the sample or the standard solution
B0-the average OD value of the 0 ppb 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 Total Aflatoxins concentration (ppb). The Total Aflatoxins  concentration in μg/kg (ppb) corresponding to the absorbance of each sample can be read from the calibration curve.
 
10.  Precautions
1.  The room temperature below 20 ºC or the temperature of the reagents and the samples being not returned to the room temperature (20-25 ºC) 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 0.5M 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 ºC, 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.
8.  After adding the substrate, the general color rendering time can be 15 ~ 30min. If the color is lighter, extended reaction time to 35min (or longer), do not beyond 40min. On the contrary, shorten reaction time.
9. The optimum reaction temperature is 25 ºC, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.
 
11. Storage and expiry date
Storage: store at 2-8 ºC, not frozen.
Expiry date: 12 months; date of production is on box.