6.SPECIMEN COLLECTION AND STORAGE.
Specimens may be stored for up to 48 hours at 2-8 °C before testing. Freezing/thawing should be avoided.
7.TEST PROCEDURE
7.1. Reagent Preparation
- All reagents (including unsealed microstrips) should be allowed to reach room temperature (+18 to +25 °C) before use.
- All reagents should be mixed by gentle inversion or vortexing prior to use. Avoid foam formation.
- It is recommended to spin down shortly the tubes with calibrators on low speed centrifuge.
- Prepare washing solution from the concentrate BUF WASH 21X by 21 dilutions in distilled water.
It is recommended that pipetting of all calibrators and samples should be completed within 3 minutes.
See the example of calibration graphic in Quality Control data sheet.
1
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Put the desired number of microstrips into the frame; allocate 10 wells for the calibrators CAL 1 - 5 and two wells for each unknown sample. DO NOT REMOVE ADHESIVE SEALING TAPE FROM UNUSED STRIPS.
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2
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Prepare the specimen as indicated in table M
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3
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Pipet 50 µl of EIA buffer into each well.
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4
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Pipet 50 µl of calibrators CAL 1 - 5 and unknown samples into the wells.
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5
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Incubate 60 minutes at 37°C .
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6
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Prepare washing solution by 21x dilution of washing solution concentrate BUF WASH 21X by distilled water. Minimal quantity of washing solution should be 250 µl per well. Wash strips 3 times
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7
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Wash strips 5 times.
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8
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Dispense 100 µl of CONJ HRP into the wells. Cover the wells by plate adhesive tape.
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9
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Incubate 60 minutes at 37°C .
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10
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Wash the strips 5 times.
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11
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Dispense 100 µl of SUBS TMB into the wells
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12
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Incubate 10-20 minutes at 18-25 °C
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13
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Dispense 100 µl of STOP into the wells.
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14
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Measure OD (optical density) at 450 nm.
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15
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Set photometer blank on first calibrator
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16
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Apply point-by-point method for data reduction.
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Sample preparation procedures are essentially different for lump meat (for which only SUPERFICIAL contamination with pork antigens may be suspected) and processed meat products (which may contain pork antigen inside).
An isotonic buffer solution with neutral pH (e.g., 0.1 M phosphate buffer with 0.15 M NaCl) should be used for sample preparation (extraction buffer). If extracts should be stored for more than 24 hours, it is recommended to add a preservatrive (e.g., sodium azide in 0.1% final concentration). We recommend to use our special Sample Preservative (Cat. # S075Z) which may be ordered separately.
For preparation of some sample types, the following disposables are required:
cotton swabs (e.g., ear swabs)
plastic spatula (e.g., those used for mixing of beverages)
disposable blade or scalpel
plastic tubes with screw caps for 15-50 ml (e.g., Sarstedt, Cat.# 60.732.001)
ATTENTION: for all sample manipulations, only disposable materials should be used. For bulky objects, sampling should be made in disposable gloves which should be changed for EACH OBJECT.
Table M
Sample type
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Sample preparation method
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Measuring units
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Recalculation
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Foodstuff
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Take a 0.5-10 g sample of a foodstuff, weight it on an analytical balance and place into a disposable tube with a tightly closing cap (e.g., with a screw cap). Attention! To prevent cross-contamination, use only disposable instrument to take samples! Solid meat products should be cut up small to obtain a homogeneous mass. If a meat is not wet enough, please, add some volume of water or any neutral buffer. Close (screw) the cap tightly and shake the tube vigorously for 15-30 seconds. Place the tube on a horizontal surface and let the aprticles to sediment. The obtained supernatant should be analysed.
Liquid products (stocks, meat washouts, equipment washouts) may be analysed withouf dilution if their pH lays within 6.0-8.0. Viscous stocks should be diluted 2-10 fold with water or any neutral buffer. If a sample contains suspended particles, clear it by filtering (e.g., through a cloth) or sedimentation. Te obtained sample should be analysed within 24 hrs. For longer storage, we recommend either single freezing or adding 1% of a special preservative (Sample preservative, Cat. # S075Z).
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U/ml (of an arbitrary wash-off)
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No
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Fresh-killed meat, cooled meat, surfaces (cutting board, knife)
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Using one swab, take the smears from 3-4 areas of the object. If the piece of meat is too bulky or local contamination is suspected, take the smears from 10-12 areas with different swabs, each swab to be used to prepare a separate sample. Submerge the swab(s) in extraction buffer (1 ml in a tube), shake the tube gently and wring the swab(s) out by pressing to the inner wall of the tube.
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U/ml (of an arbitrary wash-off)
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No
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Frozen meat
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Thaw the meat sample. Using a pipette, take 100 ml of the juice generated during thawing and transfer it to the vial containing 1 ml of extraction buffer. If a carcass or a bulky piece of meat should be tested, it is recommended to take several samples from different areas.
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U/ml (of an arbitrary wash-off)
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No
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Soft meat products (minced meat, paste, rissoles, boiled sausage, canned meat)
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Using a disposable plastic spatula, take a mixed sample from different locations, put 1.5-2.5 g of it into a pre-weighted sampling tube, weigh the sample and determine net weight of the sample. Add 8 ml of extraction buffer, close the tube with a screw cap and mix thoroughly (either by inverting or by vortexing). When testing bigger industrial samples, it is recommended to take a mixed sample of 15-30 g from different locations, put it into a disposable flask and add 80 ml of extraction buffer. In this case, mixing should be made with disposable spatula.
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U/g
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Obtained value/ Net weight, g
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Smoked sausage
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Using a disposable blade or scalpel, cut out thin slides of the sausage in disposable Petri dish. ATTENTION. Try to take only meat of the sausage with minimal amount of fat. Put the cut sample into a pre-weighted tube, weigh the sample, determine the net weight of the sample and add 8 ml of extraction buffer. Close the tube with screw cap and mix thoroughly (either by inverting or by vortexing).
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U/g
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Obtained value/ Net weight, g
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8.QUALITY CONTROL
It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results.
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable federal, state, and local standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test.
The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications.
9.CALCULATION OF RESULTS
9.1. Calculate the mean absorbance values (OD450) for each pair of calibrators and samples.
9.2. Plot a calibration curve on graph paper: OD versus pork concentration.
9.3. Determine the corresponding concentration of pork in unknown samples from the calibration curve. Manual or computerized data reduction is applicable on this stage. Point-by-point or linear data reduction is recommended due to non-linear shape of curve.
9.4. Below is presented a typical example of a standard curve with the XEMA Co. Not for calculations!
Calibrators
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Value
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Absorbance
Units (450 nm)
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CAL 1
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0 U/ml
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0.06
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CAL 2
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10 U/ml
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0.16
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CAL 3
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30 U/ml
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0.37
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CAL 4
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100 U/ml
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0.97
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CAL 5
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300 U/ml
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2.60
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10.EXPECTED VALUES
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|
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Units
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Units alternative
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U/ml
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U/g
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Upper limit for wash-off samples
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Upper limit for extracts
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All meat types (meat, products)
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20.0
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10.0
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11.PERFORMANCE CHARACTERISTICS
11.1. Analytical specificity / Cross reactivity
A high specificity of the test is provided by monoclonal antibodies to pork skeletal muscles (a specific part of the tropomyosin complex) used. The test does not detect meat antigens of the following animals and poultry: cow, sheep, horse, reindeer, other deers, rodents, hen, turkey, duck, goose, rabbit – as well as trace amounts of human blood. By the date of issuing this instruction (version 005), there is no data regarding possible cross-reactivity to camel and kangaroo. ATTENTION: this test can not be used for detection of trace amounts of cardiac and smooth muscles, fatty tissue and skin of swine – for such samples, test gives positive results only in case of admixture of swine skeletal muscles.
Fish
Chicken
Turkey
Goose
Duck
Ostrich
Frog
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<0.1%
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Cow (beef)
Sheep (mutton)
Deer
Rabbit
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<0.5%
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Elk
Horse (meat)
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<1%
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The limit of quantification, or analytical sensitivity of the test is not more than 5 U/ml for ready extract (see “Sample preparation for different sample types”). According to the results of our preliminary biochemical analysis, this value corresponds to ca. 25 ng/ml of the target antigen. However, we recommend to express results in arbitrary Units, as the correspondence of immunoreactivity to the weight of the target antigen is highly variable
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