Total Aflatoxins in Animal feed ELISA kit

1 Test principle
The basis of the test is on the competitive enzyme immunoassay for the detection of Total
aflatoxins in Animal feed .
The microtiter wells are coated with anti-aflatoxins antibody. aflatoxins enzyme conjugate
and the aflatoxins standards or sample solution are added. Free aflatoxins antibody and
aflatoxins enzyme conjugate compete for the aflatoxins antibody binding sites (competitive
enzyme immunoassay). Any unbound enzyme conjugate is then removed in a washing step.
Chromogen/substrate is added to the wells, bound enzyme conjugate converts the colorless
chromogen into a blue product. The addition of the stop reagent leads to a color change from
blue to yellow.
The depth of the color shows the magnitude of aflatoxins in the sample. The measurement is
made photometrically at 450nm. According to the absorbance, the concentration of aflatoxins
in the sample can be quantified.
2 Technical specification:
Sensitivity: 0.05 ppb

Detect time: Approx.75 min

Detect limit:
Feed: 2.5 ppb
Cross-reaction rate:
Aflatoxin B1: 115%
Aflatoxin B2: 100%
Aflatoxin G1: 98%
Aflatoxin G2: 95%
Aflatoxin M1: 85%
Aflatoxin M2: 80%

Recovery rate:
Feed: 75 10%
3 Reagents provided
1 Microtiter plate; coated with antibodies. 96 wells
2 Standard solutions
0ppb,0.025ppb,0.075ppb,0.225ppb,0.675ppb,2.025ppb-----black cap
3 Enzyme conjugate ----------------------------------------------- red tube
4 Antibody working solution---------------------------------------brown cap
5 Substrate A---------------------------------------------------------brown cap
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7
8
9

Substrate B --------------------------------------------------------black cap

Stop reagent -------------------------------------------------------yellow cap
Washing solution (20 concentrate)----------------------------black cap
Sample dilution (5 concentrate)-------------------------------white cap
-Materials required but not provided
Equipments: microplate reader (450 nm / 630 nm), homogenizer, oscillator, centrifuge,
balance (a sensibility reciprocal of 0.01 g), measuring pipets, Nitrogen Evaporator;
Micropipettors: single-channel 20 to 200 L, 100 to 1000 L, multi-channel 250 L;
Reagents: deionized water, CH3OH (99.5%) , N-hexane, CHCl3 ,Whatman No.1 paper

4. Sample pre-treatment
-Instructions:
The following points must be dealt with before the pre-treatment of any kind of sample:
1) The samples should be stored in a cool place;
2) Only the disposable tips can be used for the experiments and the tips must be changed
when
used for absorbing different reagents;
3) Before the experiment, each experimental equipment must be clean and should be recleaned if necessary, in order to avoid the contamination that interferes with the
experimental results.
-Solution preparation before sample pre-treatment:
1) The 2 concentrated redissolving solution is diluted with deionized water at 1:1
(10 mL concentrated redissolbing solution + 10 mL deionized water), used for the treated
sample redissolving.
2) Sample dilution (5 concentrate) is diluted with deionized water at 1:4
(10 mL Sample dilution + 40 mL deionized water).

5. Samples preparation
- Feed 1 (Lower fat and protein)
1 Take 30.05 g homogenized sample; add 15 mL Sample dilution, mix for 5 minutes.
2 Filter with Whatman No.1 paper,pick up all liquid.
3 Dilute the supernatant with the sample dilution(50L supernatant + 950L Sample dilution,
mix for 30 s).
4 Take 50 L for analysis.
Fold of dilution of sample :100
- Feed 2 (Higher fat and protein,concentrated feed )
1 Take 30.05 g homogenized sample; add 10 mL Sample dilution, mix for 5 minutes.
2 Filter with Whatman No.1 paper, pick up all liquid.
3 Take 5 ml supernatant, add 5mL CHCl3 , evaporate to dry at 65by nitrogen air.
4 Add 1mL sample dilution to dilute residues.
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5 Take 50L ,add 950L Sample dilution, mix for 30 s.

6 Take 50 L for analysis.
Fold of dilution of sample :40

6. ELISA procedures
- Instructions
1) Bring all reagents and micro-well strips to the room temperature (20-25 ).
2) Return all reagents to 2-8 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 the procedures of
ELISA.
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.
- Operation procedures
1) Take out all the necessary reagents from the kit to the room temperature (20 to 25 ) for
at
least 30 minutes. Note that each liquid reagent must be shaken to mix evenly before use.
2) Take the required micro-well strips and plate frames. Re-sealed the unused microplate,
stored at 2-8 , not frozen.
3) Solution preparation: dilute 50 mL of the Washing solution (20 concentrated) with the
distilled or deionized water to 800 mL (or just to the required volume) for use.
4) Numbering: number the micro-wells according to samples and standard solution; each
sample and standard solution should be performed in duplicate; record their positions.
5) Add 50L of the sample or standard solution to separate duplicate wells, and add 50 L
Antibody working solution into each well. Mix gently by shaking the plate manually, seal
with membrane, and incubate at 25 for 30 minutes.
6) Pour liquid out of microwell, add 250 L/ well o Washing solution for 15-30 seconds, repeat
four to five times, then flap to dry .
(if there are the bubbles after flapping, cut them with the clean tips) .
7) Enzyme conjugate: Add enzyme conjugate, 100 L each well. seal the microplate with
the cover membrane, and incubate at 25 for 30 minutes. Repeat the last step ( step 6) .
8) Coloration: add 50 L of substrate A solution and then 50 L of substrate B solution into
each well.Mix gently by shaking plate manually, and incubate at 25 for 15 minutes at
dark.
9) Determination: add 50 L of the stop reagent into each well. Mix gently by shaking the
plate manually. Set the wavelength of the microplate reader at 450 nm to determine the
OD Value
(Recommend to read the OD value at the dual-wavelength 450/630 nm within 5 minutes).
7. Result judgment
There are two methods to judge the results; the first one is the rough judgment, while the
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second is the quantitative determination. Note that the OD value of the sample has a
negative correlation with the content of aflatoxins M1 in the sample.
The mean values of the absorbance values is equivalent to the percentage of the average OD
value (B) of the sample and the standard solution divided by the OD value (B0) of the first
standard solution (0 standard) and subsequently multiplied by 100%, that is,

Absorbance
Absorbance

standard (or sample) 100 = % Absorbance

zero standard

Bthe average (double wells) OD value of the sample or the standard solution
B0the average OD value of the 0 ng/mL standard solution
Draw the standard curve with the absorption percentages of the standard solutions and the
semilogarithm values of the aflatoxins standard solutions (ng/mL) as Y- and X-axis,
respectively. Read the corresponding concentration of the sample from the standard curve by
incorporating its absorption percentage into the standard curve. The resulting value is
subsequently multiplied by the corresponding dilution fold, thus finally obtaining aflatoxins
concentration in the sample. Using the professional analyzing software of this kit will be more
convenient for the accurate and rapid analysis of a large amount of samples. (Please contact
us for this software).
B/B0 (%)
0.2
1.8

%CV
0.6
5.4

16.2
0.2
5.4

0.6

1.8

16.2

8. Precautions
1) The room temperature below 20 or the temperature of the reagents and the samples
being not returned to the room temperature (20-25 ) 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.
3) Mix every reagent and reaction mixture evenly and wash the microplate Thoroughly,
otherwise there will be the undesirable reproducibility.
4) The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.
5) Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance
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and the colourless color former is light sensitive, and thus they cannot be directly
exposed
to the light.
6) 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.

9. Storage and expiry date

The test kit is stable for 12 months at 2-8 in the dark. Do not freeze any test kit
components.

Expiry date: 12 months.

Unibiotest makes no warranty of any kind, either expressed or implied, except that the
materials from which its products are made are of standard quality. If any materials are
defective, Unibiotest will provide a replacement product. There is no warranty of
merchantability of this product , or of the fitness of the product for any purpose. Unibiotest
shall not be liable for any damages, including special or consequential damage, or expense
arising directly or indirectly from the use of this product.

Wuhan Unibiotest Co.,Ltd

Tel: +86 27 81329156,
Fax: +86 2781329156
E-mail: info@unibiotest.com
Web: www.unibiotest.com
NO1, Guanshan Road, Wuhan city ,China.