Aflatoxin Sop

SOP
HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY

TITTLE: Standard Operating Procedure SOP No: HGL/0020
(SOP) For Aflatoxin Test Version: 1
Date Issued: 20/03/2024 Page 1 of 6 Next Review Date:
1.0 Purpose
The purpose of this standard operating procedure is to explain in details the

procedures for total aflatoxin test using veratox kits.
2.0 Scope/Responsibility
These procedures are initiated and reviewed by the Humane Laboratory

staffs and completed by Laboratory Manager and laboratory consultant who
have been well trained on all information listed under “Prerequisite Training
Materials” and this SOP with the scope/view of carrying out accurate and
efficient aflatoxin test
3.0 Sample Acceptance Criteria
Well labelled sample, uncontaminated
4.0 Rejection Criteria
Incompletely or incorrectly labelled, contaminated sample especially with

another sample
5.0 Turn Around Time
3 hours
SOP

(SOP) For Aflatoxin Version: 1
6.0 Test Principle

VERATOX AFLATOXIN is a competitive direct enzyme-linked immunosorbent
assay which allows exact concentration of aflatoxins in parts per billion (ppb)
as the enzymes linked aflatoxins and allow to compete for antibody binding
sites coated in the well. After a wash step, substrate is added, which reacts
with the bound conjugate and produce blue coloration.
7.0 MATERIALS
7.1 Antibody-coated microwells
7.2 Red-marked mixing wells
7.3 4 yellow-labeled bottles of 0, 5, 15, and 50 ppb Aflatoxin controls
7.4 1 blue-labeled bottle of aflatoxin-HRP conjugate solution
7.5 1 green-labeled bottle of K-Blue Substrate solution
7.6 1 red-labeled bottle of Red Stop solution
7.7 250 mL graduated cylinder
7.8 Container with 125 mL capacity
7.9 Sample collection tubes
7.10 High-speed blender
7.11 Scale capable of weighing balance
7.12 100 μL pipette
7.13 12-channel pipette
7.14 Tips for 12-channel and 100 μL pipette
7.15 Paper towels or equivalent absorbent material
7.16 Microwell holder
7.17 Timer
7.18 Waterproof marker
7.19 Wash bottle
SOP

7.20 2 reagent boats for 12-channel pipette

7.21 Distilled or deionized water
7.22 Microwell reader with a 650 nm filter
7.23 pH meter
7.24 1N Sodium Hydroxide (NaOH) Solution.
7.25 1N Hydrochloric (HCl) Acid Solution.
7.26 70% Methanol
7.27 Pasteur pipette
7.28 Funnel
7.29 Filter paper
7.30 Screw capped jar
8.0 Sample Extraction Method:

8.1 Weigh 50 ± 0.2 grams of grounded sample into a screw capped jar.
8.2 Using a 250 mL graduated cylinder, add 250 mL extraction solvent (70%
Methanol) and screw the cap or lid on the jar.
8.3 Blend for 1 minute in a high-speed blender or shake vigorously for 15
minutes. 8.4 Allow the mixture to settle.
8.5 Filter about 3 mL of the extract through a filter paper.
Note: - For Corn Gluten Meal, DDGS and Wheat Bran, check the pH of the filtered
extract using a pH meter. If the pH is not between 7.0 and 8.0, adjust it with 1N
Sodium Hydroxide (NaOH) Solution and/or 1N Hydrochloric (HCl) Acid Solution using
a disposable Pasteur pipette,
SOP

9.0 Test Procedures

9.1 Allow reagents and antibody wells to reach the room temperature of 22 to
250C prior to running the test.
9.2 Remove 1 red-marked mixing well for each sample to be tested plus 4 red-
marked wells for controls and place in the well holder.
9.3 Remove an equal number of antibody-coated wells. Mark one end of strip
with a “1” and place strip in the well holder with the marked end on the left.
Do not mark the inside or bottom of the wells.
9.4 Mix each reagent by swirling the reagent bottle prior to use.
9.5 Using a 100 μL pipette, place 100 μL of conjugate from the blue-labelled
bottle
in each red-marked mixing well.
9.6 Using a new pipette tip for each, transfer 100 μL of controls and samples to
the red-marked mixing wells.
9.7 Using a 12-channel pipette, mix the liquid in the wells by pipetting it up and
down 3 times. Then transfer 100 μL into the antibody-coated wells.
9.8 Mix by sliding the microwell holder back and forth on a flat surface for 10-20
seconds without splashing the reagents from the wells. Incubate for 2
minutes
at room temperature (220C to 250C). Discard red-marked mixing wells.
9.9 Shake out the contents of the antibody wells. Fill the wells with distilled or
deionized water and dump them out. Repeat this step 5 times, then turn the
wells upside-down and tap out on a paper towel until the remaining water
has
been removed.
9.10 With new tips on the 12-channel pipette, prime and pipette 100 μL of
substrate
into the wells and mix by sliding back and forth on a flat surface for 10-20
seconds. Discard the tips. Incubate for 3 minutes.
SOP

9.11 With new tips on the 12-channel pipette, prime and pipette 100 μL of Red
Stop to each well. Mix by sliding back and forth on a flat surface to make sure
there is no layering. Discard the tips.
9.12 Wipe the bottom of the microwells with a lint free Kim wipe and read on the
plate reader using a 650-nm filter.
Note: - Air bubbles should be eliminated, as they could affect analytical results.
Results should be read within 20 minutes after the addition of Red Stop.
10.1 Precautions.
10.1 Store test kit between 2-8°C (35-46°F) when not in use, do not freeze.
10.2 Do not use kit components beyond expiration date.
10.3 Do not mix reagents from one kit serial with reagents from a different kit
serial.
However, substrate and stopping solution are not lot specific.
10.4 Do not run more than 24 wells per test.
10.5 Follow proper pipetting techniques, including priming pipette tips by filling
and
dispensing solution once before use.
10.6 Use of incubation times other than those specified may give inaccurate
results.
10.7 Bring kits to room temperature (22-25°C) prior to use.
10.8 Avoid prolonged storage of kits at ambient temperatures.
10.9 To avoid cross-contamination, use new pipette tips for each sample, and
thoroughly detoxify and wash all glassware between samples.
SOP

11.0 RESULT:
The test is read in a microwell reader to yield optical densities. The optical densities
of the controls form the standard curve, and the sample optical densities are plotted
against the standard curve to calculate the exact concentration of aflatoxins in the
sample
12.0 Reference
www.neogen.com/veratox for aflatoxin/procedure
13.0 Revision History
Revision Author Revisions Made Effected Date

00 Henry Hosea Carl 20/03/2024
01 Mal. Usman Auwal M. 20/03/2024
02 Dr Mustapha H. H. 20/03/2024
03 Dr Rahila Haruna 20/03/2024
04 Abdullahi suleiman 04/10/2024
05 Salma 04/10/2024

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SOP

HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY

The purpose of this standard operating procedure is to explain in details the

These procedures are initiated and reviewed by the Humane Laboratory

3.0 Sample Acceptance Criteria

Well labelled sample, uncontaminated

4.0 Rejection Criteria

Incompletely or incorrectly labelled, contaminated sample especially with

5.0 Turn Around Time

HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY

6.0 Test Principle

HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY

7.20 2 reagent boats for 12-channel pipette

8.0 Sample Extraction Method:

HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY

9.0 Test Procedures

HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY

HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY

Revision Author Revisions Made Effected Date

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