Mixer Shaker

Isolation and Identification of Foot and Mouth diseases virus

Antigen Detection ELISA

 

An indirect sandwich enzyme-linked immunosorbent assay (ELISA) (Principle of Antigen Detection ELISA), approved by the World Reference Laboratory (WRL) for FMD, is used to determine the presence of FMDV, or swine vesicular disease (SVD), antigen.
Rabbit antisera specific for the different serotypes of FMDV and SVDV are passively adsorbed to the wells of polystyrene microtitre plates. With the addition of the test sample, antigen (if present) is trapped by the immobilised antibodies. Specific guinea pig anti-FMDV and SVDV detecting antibodies are then added which react with the trapped antigen. The bound guinea pig antibodies are detected by means of rabbit anti-guinea pig immunoglobulin (IgG) conjugated with horseradish peroxidase. 

The choice of vesicular viruses to be detected depends on the geographical area and species involved. For example, detection of SVD virus is only relevant in samples from pigs. Samples from animals showing vesicular lesions with direct or indirect epidemiological connection with the Americas should be examined for vesicular stomatitis.

 

Equipments:

Antigen Detection ELISA

Absorbent towels

Bench centrifuge

Freezer: any type in the range -15°C to -20°C.

Colour key buffer solutions pH 4, 7 and 10

Cryopreservation vials: recommended: polypropylene, flat-bottom screw cap with internal thread and O-ring seal (1-5 ml), and appropriate storage racks

ELISA microtitre plates: 96-well, flat-bottomed. NUNC Immunoplate Maxisorp. (Gibco, cat. no4-39454A) (Substitution may alter diagnostic performance). Plates should be stored between +4º and 15ºC

Small glass vials (volume up to 2 ml)

Freezer
Any type in the range -15ºC / -20ºC to +4ºC

Glassware / Plasticware
A selection of beakers (20-4000 ml), flasks, graduated cylinders (10-2000 ml), graduated pipettes (1-20 ml) with suitable safety bulbs, storage bottles with closures (1-100 ml), dilution tubes (2-4 ml) and suitable racks, wash fluid container with tap (5-10 litre) and tubing for connection of this reservoir to plate washer or, alternatively, wash bottles (250 ml).

Incubator: any type of radiant, warm wall incubator or hot room in the range of +35°C to 39°C

Lids for microtitre plates

Marker pens (water-proof) and Adhesive labels

Orbital shaker

Pestle and mortar: any type with a diameter of approximately 7 cm

pH indicator strips range 5 - 10

pH meter: any type with an accuracy of 0.01 pH units; not essential if pH papers available, but may be of assistance in problem solving.

Photometer: Flow Laboratories, Titertek Multiskan Plus MKII microtitre plate reader (or equivalent) with an interference filter of 492 nm; IBM compatible computer, monitor and printer, ELISA data analysis software (useful but not essential).

Pipettes
Flow Laboratories, Titertek Multichannel (or equivalent) pipettes (8 or 12 channel), variable ranges from 5-50 µl and 50-300 µl, and quality tips. Gilson Pipetman (or equivalent) single channel pipettes, variable ranges from 1-10 µl, 5-40 µl, 40-200 µl and 200-1000 µl, and quality tips.

Reagent troughs
Reagent troughs (reservoirs) suitable for simultaneous, multichannel pipetting of a single reagent, and troughs suitable for simultaneous, multichannel pipetting of eight different reagents.

Refrigerator
Any type in the range +2° and 6°C.

Standard laboratory equipment
Measuring cylinders, 10 ml pipettes, assorted small glass bottles and beakers

Timer
Any type, preferably countdown-type with an audible alarm.

Vortex mixer
Any type with variable speed.

Washer
Flow Laboratories, Titertek Handiwash microplate washer (or equivalent). Alternatively, plastic wash bottles or a wash fluid container connected to suitable tubing may be used.

Water bath
Any type in the range +35°C-39°C: not essential, but useful for rapid warming or thawing of reagents.

Water purification system
Minimum: glass-distilled or de-ionized water.
Optimum: Millipore, Milli-Q (or equivalent), Type 1, 18 megohm, pyrogen-free water.

Weighing balance
Any type with an accuracy of +/- 0.1 g.

Weighing boats

Reagents:

Chemicals
Calcium chloride dihydrate (CaCl2.2H2O)
Citric acid monohydrate (C2H8O2.H2O)
Disodium hydrogen phosphate anhydrous (Na2HPO4)
Magnesium chloride hexahydrate (MgCl2 6H2O)
'Merthiolate'
(sodium ethylmercurithiosalicylate-'Thimerosal' - Sigma T5125)
Phosphate-citrate buffer Tablets (Sigma P4809)
Potassium chloride (KCl)
Potassium dihydrogen phosphate anhydrous (KH2PO4)
Sodium azide (NaN3)
Sodium bicarbonate anhydrous (NaHCO3)
Sodium carbonate anhydrous (Na2CO3)
Sodium chloride (NaCl)
Sodium hydroxide (NaOH)
Sulphuric acid (conc) (H2SO4)
Carbonate/bicarbonate buffer capsules (Sigma C3041)
Gelatine
Glycerol
Orthophenylene diamine dihydrochloride (OPD) or
OPD Tablets 30 mg (Sigma P8412)
Phenol red powder
Sand (must be sterile, washed, acid-treated, fine mesh sifted, laboratory grade)
Skimmed milk powder (Marvel or equivalent)
Tween 20 (Polyoxyethylene sorbitan monolaurate)

Biological Reagents
Coating sera

Rabbit antisera against each FMDV serotype and SVDV.
Each rabbit antiserum can be diluted 1:5 with PBS containing either 0.2M sodium azide solution or 0.02% merthiolate

Detection sera

'Blocked' guinea pig sera against each FMDV serotype
and SVDV mixed with equal volume of bovine serum and
heat inactivated at 56°C for 30 min (WRL, Pirbright, UK).
Each 'blocked' guinea pig antiserum is further diluted 1:5 in
(i.e., 1:10 final dilution) PBS containing either 0.2M sodium
azide solution or 0.02% merthiolate solution. Working
stock antiserum reagent is stored at 4ºC.

Conjugate

Peroxidase conjugated rabbit immunoglobulins to
guinea pig immunoglobulins (P141, Dako Ltd). Conjugate
is blocked with an equal volume of non-immune bovine
serum, diluted 1:5 with PBS and mixed with an equal volume
of sterile glycerol (i.e., 1:10 final dilution), and stored at 4ºC.

Reference control antigens

Aziridine inactivated cell culture supernatant fluids
containing each of seven serotypes of FMDV and SVDV.
Each control antigen is mixed with an equal volume of glycerol
and stored at -20ºC.

Preparation of Reagents:

GENERAL NOTE ON BUFFERS
It is important that buffers and stock solutions described below are within the pH ranges indicated. The pH of each solution should therefore be tested. If the pH does not fall within the specified ranges, steps should be taken to correct the problem. Poor water quality and unclean glassware are most frequently implicated. Should any buffer or stock solution show signs of deterioration (i.e., formation of a precipitate) or contamination (i.e., evidence of bacterial, fungal or algal growth), discard immediately and prepare a fresh batch in clean glassware

Acid stopper solution (1.25 M sulphuric acid)
68 ml of concentrated sulphuric acid is added to 1 litre de-ionized/distilled water (exact volume of acid required will vary with the purity of the preparation used).
[WARNING: ADD SULPHURIC ACID TO WATER - NEVER ADD WATER TO SULPHURIC ACID]

Azide solution (1 M)
NaN3 6.5 g
Add de-ionized/distilled water to 100 ml
[WARNING: HANDLE WITH CARE ]

Chromogen
Ortho-phenylene diamine (OPD) in phosphate-citrate buffer pH 5.0.
Citric acid monohydrate 5.11 g
Na2HPO4 anhydrous 7.3 g

Add de-ionized/distilled water to 1 litre
OPD 400 mg
Alternatively, dissolve one tablet of phosphate-citrate (Sigma P4809) in 100 ml of de-ionized water to obtain a 0.05 M phosphate-citrate buffer, pH 5.0. Dissolve one OPD tablet (Sigma P8412, 30 mg) in 50 ml of 0.05 M phosphate-citrate buffer (pH 5.0).

Store at -20°C in 10, 15, 20 and 25 ml aliquots in the dark. (Stock solution should be colourless, with a pH between 5 and 6, and if coloured should be discarded and a fresh batch prepared). Thaw at room temperature in the dark, and immediately before use activate with a 1/2000 dilution of the substrate hydrogen peroxide solution (30-33% concentration), i.e., 12.5 µl H202 per 25 ml OPD.

[WARNING: Handle OPD with care as it is a potential carcinogen; HARMFUL if swallowed, inhaled or adsorbed through skin. WEAR GLOVES]

Coating buffer
0.05 M Carbonate/bicarbonate, pH 9.6 +/- 0.05

Normal strength solution
contains : x 20 strength stock :
Na2CO3 1.59 g Na2CO3 31.8 g
NaHCO3 2.93 g NaHCO3 58.6 g

per litre Add de-ionized/distilled water to1 litre.

Normal strength coating buffer solution is prepared by dilution of x 20 strength stock solution, i.e., 5 ml of x 20 strength solution plus 95 ml sterile de-ionized water.
Alternatively, dissolve one capsule of 0.05 M carbonate/bicarbonate (Sigma C-3041) per 100 ml sterile de-ionized water.

Label and store coating buffer at +4°C for no longer than 1 week.

Diluent buffer A (for dilution of antigens)
0.01 M phosphate buffered saline (PBS), pH 7.4 +/- 0.20, plus 0.05% Tween 20. (Note that Tween 20 is very viscous and care must be taken to ensure that the delivery pipette is filled with the correct volume of this detergent and properly rinsed with the buffer to which it is being added and that the Tween is fully dispersed).

Label and store at +4°C.

Diluent buffer B (for dilution of detecting antibody and conjugate)
0.01 M PBS, pH 7.4 +/- 0.20 plus 0.05% Tween 20 plus 5% (w/v) Skimmed Milk Powder.

On the day of testing, add skimmed milk powder to Diluent buffer A to make the amount of Diluent buffer B required, i.e., add 5 g skimmed milk powder to 100 ml Diluent buffer A.

Do not store diluent buffer containing skimmed milk powder after completion of the test.

Disinfectant solution 0.2% (w/v) citric acid
Dissolve 2 g of citric acid in 1 litre of water.

ELISA wash fluid
1 part PBS to 4 parts distilled water.

Phenol red solution (0.2%)
Phenol red powder 2.0 g
1 M sodium hydroxide solution 29.2 ml

Grind the powder with the sodium hydroxide solution using a pestle and mortar, make to 1 litre with de-ionized/distilled water, and filter.

Phosphate buffered saline (PBS) (Dulbecco PBS) 0.01 M

NaCl 8.0 g
KCl 0.2 g
MgCl2.6H2O 0.1 g
KH2PO4 0.2 g
Na2HPO4 1.14 g
CaCl2.2H2O 0.1 g

Add de-ionized water to 1 litre, and check pH is 7.3-7.4.

Sample buffer
0.04 M phosphate buffer, pH 7.4 +/-0.05

NaH2PO4.2H2O 6.1 g
KH2PO4 0.78 g
make up to 1 litre with sterile distilled water

Preparation of test samples

Place tissue sample, after removal of as much glycerol/phosphate buffer as possible, in the mortar.

Grind the tissue with a little sterile sand and add sufficient sample buffer to make a 10% (w/v) suspension of epithelium. Transfer ground suspension to a small bottle and centrifuge at approximately 1000g for 10 minutes. Harvest the clarified supernatant fluid into a small glass bottle.

If a cell culture sample is to be tested, clarify the sample by centrifugation at approximately 1000g for 10 minutes to remove cell debris.

NOTE:
Samples may be stored in 0.04 M PBS at -70°C for prolonged periods. Alternatively, they may be stored at -20°C after the addition of an equal volume of sterile glycerol (50% v/v)

Method:

PLATE COATING

Coating of ELISA microtitre plates with coating (trapping) antibody

1. Plates are coated with rabbit antiserum diluted in coating buffer.

2. Prepare working dilutions of the rabbit sera in coating buffer in an 8-well reservoir; a dilution of 1/1000 is used for each rabbit serum, i.e., 1 µl of serum is added to 1 ml of coating buffer (sufficient to coat 12 wells on one microtitre plate).

3. A suitable distribution of antisera to FMDV and SVD serotypes is made as required over a microtitre plate. The WRL ELISA examines for the seven serotypes (O, A, C, SAT1, SAT2, SAT3, ASIA 1) of FMDV, and SVDV, laid out in a particular configuration. Rows A to H on each plate receive, respectively, antisera to serotypes O, A, C, SAT1, SAT2, SAT3, ASIA 1 and SVDV.

4. Each plate well receives a 50 µl volume of the appropriate coating serum, transferred by multichannel pipette, and the plates are tapped to ensure even distribution in the wells.

Check that each tip of the multichannel pipette delivers the correct amount at each stage, i.e., ensure that air bubbles are not present.

Number plates with an indelible marker, and ensure that all the plates are aligned correctly (with the letters on the left hand side) as the ELISA reader will only accept the plates in one position

5. (a) Cover the plates with lids or an empty plate (if plates are stacked, cover only top plate) and, ideally, place on a pre-warmed orbital shaker, set at 100 to 120 revolutions per minute, at 37ºC for 1 hour.

The agitation of the plate shaker should be sufficient to cause thorough mixing without spillage of the contents.

In the absence of a plate shaker, tap each side of the plates intermittently to obtain uniform dispersion of the reagents. If microplates must be stacked, cover only the top plate, and minimise the effects of thermal gradients and evaporation in this situation.

5. (b) Alternatively, plates can be left to coat overnight at +4ºC in a stationary position.

6. Return remainder of the trapping antibody stocks to +4°C.

7. After incubation, empty plates and wash with ELISA wash fluid by filling and emptying wells three times. Empty residual fluid from plates by tapping plate onto several layers of absorbent paper using an abrupt downward hand motion.

Plates are now ready for test assay.

ELISA plates precoated with rabbit antisera

There is considerable advantage in having ELISA plates precoated with rabbit typing antisera, and stored at either +4°C or -20°C. Precoated plates can be removed and immediately used for detecting and typing antigens from field samples, etc.

The sensitivity of precoated plates will remain at maximum levels for several weeks at +4ºC or months at -20ºC. If problems arise after storage at +4°C, discard plates after 1 week and coat fresh plates.

Precoated plates are prepared as described above but, after the incubation step, the coating buffer is retained in the wells during storage. Cover with cellophane seal.

For use, plates are removed as required and, after removal of cellophane seal, emptied and washed three times in ELISA wash fluid immediately before the test assay.

ASSAY

Note: Total volumes of each reagent required for a given number of microtitre plates in a test are tabulated in Required Reagent Volumes.

1. Prepare two dilutions for each antigen in diluent buffer A, representing strong and weak antigen controls for each serotype. The actual dilutions to be used will depend on the batch of antigen.

On test microtitre plate 1, load wells in columns 5 and 6 with 50 µl of diluent buffer A (Plate Layout-Antigen Detection ELISA) using, for example, a multichannel pipette and reagent trough.

To wells 3 and 4 in row A add 50 µl of the weak control antigen type O, using a single channel micropipette, and to wells 1 and 2 add 50 µl of the strong control antigen type O (the same micropipette tip can be used). Using a new micropipette tip, add 50 µl of the weak control antigen type A to wells 3 and 4 of row B, and 50 µl of the strong control antigen type A to wells 1 and 2 of this row. Using new micropipette tips continue in a similar manner with the other control antigens.

SAMPLE ADDITION

1. The remainder of the plate can be loaded with the test sample(s). Add 50 µl of sample 1 to wells 7 and 8 of rows A to H; second and third samples are added to rows A to H in wells 9 and 10, and 11 and 12, respectively (Plate Layout-Antigen Detection ELISA).

If more than three samples are to be run in the same test, other microtitre plates should be used as follows. Antigen controls are not required on these plates, but 50 µl of diluent buffer A should be dispensed to the wells (rows A to H) in columns 5 and 6 as buffer control columns. Five test samples may be added in 50 µl volumes in rows A to H, columns 1,2; 3,4; 7,8; 9,10 and 11,12, respectively.

Note that reference control antigens are required on only one microtitre plate for any given test run, provided that the same batch of plates and working preparations of reagents and buffers are used.

Use a fresh tip for each test sample and the control antigens; tips should be discarded into either disinfectant solution, or a container for eventual boiling.

2. Cover plates with lids and incubate on the orbital shaker at 37°C for 1 hour. Remove sufficient aliquots (Required Reagent Volumes) of OPD chromogen from -20°C and leave to thaw.

3. While plates are incubating, prepare working dilutions of the 'blocked' guinea pig detection sera in the appropriate order in an eight-well reservoir; 1/100 dilutions of each antiserum are used.

4. After incubation, wash plates by flooding with ELISA wash fluid three times, and empty residual fluid. Invert the plates and tap onto absorbent paper towelling to remove residual liquid. Proceed quickly to the next step.

5. Transfer 50 µl volumes of each guinea pig detecting serum dilution to each plate well in the appropriate order, e.g., rows A to H receive antisera to serotypes O, A, C, SAT1, SAT2, SAT3, ASIA 1 and SVDV, respectively.

6. Cover plates with lids, replace on the orbital shaker, and incubate at 37°C for 1 hour.

CONJUGATE ADDITION

1. While plates are incubating prepare the required volume of conjugate (Required Reagent Volumes). This is used at a dilution of 1/200, or that dilution previously determined by titration.

2. After incubation, wash plates by flooding with ELISA wash fluid three times, and empty residual fluid. Blot the plates dry on paper towelling.

3. Load a reagent trough with conjugate and add 50 µl to all wells of all the plates. Tap the sides of the plates to ensure that the conjugate is evenly distributed over the bottom of each well.

4. Cover plates with lids and return to the orbital shaker for 40 min at 37°C.

5. After incubation, wash plates by flooding with ELISA wash fluid three times, and finally flood the plate to eliminate totally the presence of unreacted conjugate.

SUBSTRATE/CHROMOGEN

1. Thaw and pool required aliquots of chromogen, and activate with hydrogen peroxide according to reagent preparation.

2. Remove wash fluid from the plates, and blot dry by inversion on paper towelling; at this stage it is essential that this is done thoroughly.

3. Add 50 µl volumes of substrate/chromogen to every well of each plate IN NUMERICAL ORDER. START TIMING FOR 15 MINUTES AS SUBSTRATE/CHROMOGEN IS ADDED TO THE FIRST WELLS. In addition, add substrate/chromogen to the wells of the first column (1) A to H of a new microtitre plate, to act as a control blank for later calibration of the photometer (plate reader). (This plate can eventually be washed and re-used for future tests.)

4. Cover the plates with lids and leave in the dark for 15 mins at room temperature.

5. After 15 minutes add 50 µl volumes of acid stopper solution to every well of each plate IN NUMERICAL ORDER, not forgetting the control 'blanking' plate.

(After addition of the stopper solution discard tips immediately to prevent corrosion of multichannel pipette.)

6. Read plates visually and record result

READING THE PLATE(S)

The optical density (OD) of each well in the test plate(s) is measured with the photometer (plate reader).

1. Before reading the microtitre plates, ensure that there are no bubbles in any of the wells, as this will cause optical aberrations, and ensure that there are no marks (e.g., fingerprints) or condensation on the bottom of the plates. If necessary, rupture any bubbles with a clean pipette tip and wipe the bottom of the plate clean with a soft dry cloth.

2. Check that there has been colour development (Colour Development in Plates) resulting from each of the inactivated control antigens on plate 1 (rows A to H, columns 1 to 4) to indicate that the plates have been correctly coated with antisera and that the test is valid. Columns 5 and 6 (buffer controls) should show no or very little colour (Plate Layout-Antigen Detection ELISA).

3. Check that the 492 nm filter is installed, and place the control 'blanking' plate in the carriage of the photometer and initiate the calibration sequence. Different photometers may have different blanking procedures so the relevant manual should be consulted.

4. Place each test microtitre plate in turn in the carriage of the photometer and initiate the reading sequence for each.

Results:

1. If colour development (Colour Development in Plates) occurs in the control wells of the microtitre plates (rows A to H, columns 1 to 4 on plate 1, checked before reading of plate) this indicates that the wells have been correctly coated with reagents. Colour reaction in the test wells should be estimated approximately by eye, and can be compared with optical density (OD) values.
2. Calculate the mean background reactions for each plate by adding the the OD values of wells 5 and 6 for each row (serotype) and dividing by two; these values are due to the reagents and not to a specific reaction between antigen and antisera. Subtract each mean background OD for each serotype from the recorded OD for that serotype to obtain the 'corrected' value. For example, for type O row A, add the OD values for wells 5 and 6, and divide by two to give the mean background OD for row A. Subtract this figure from all recorded OD values in row A. Repeat for each row (serotype). A mean of each group of two wells for each test sample can thus be obtained to give a mean 'corrected' OD value for each sample against each antiserum serotype.

   If there are problems (Trouble Shooting) tests may have to be repeated

Controls

Note that SAT antigen control has failed. This test should normally be repeated.

Trouble Shooting:

The ELISA procedure is easy to perform once reagents have been constituted correctly and standardised, but it is important to follow a code of practice for its successful operation.

In general:

Concentrate at each stage to prevent problems - ensure that the right reagent is added at each step and in the correct microtitre plate well.

It is essential that all glassware and pipette tips used are clean and sterile. Use a fresh tip for each test sample and the control antigens. Change tips for multichannel pipettes etc., and use fresh reagent troughs at each reagent stage.

[If pipette tips have to be recycled - Do not recycle pipette tips that have been used for conjugate].

For the antigen detection ELISA, mix reagents carefully in eight-well reservoirs so as not to cross mix serotypes.

Wash reagent troughs thoroughly immediately after use and soak in distilled water.

Wash plates thoroughly after each incubation step. This is especially important after adding conjugate and before adding OPD.

Keep OPD in the dark as far as possible and WEAR GLOVES when handling it. Ensure that OPD is not heated above room temperature and is at this temperature when used, and has been activated by hydrogen peroxide immediately prior to use.

Add OPD and H2SO4 to the plates in numerical order to ensure that reaction times are similar for each plate.

Avoid touching bottom of plates and ensure underneath is dry as finger marks and water can cause optical aberrations.

In particular:

If the results of ELISA assays are not as expected, it is vital to check that the correct type of plate was used, and that the reagents were stored and reconstituted as described.

It is then necessary to retrace the test procedure and check that the correct steps were taken; in particular establish whether correct dilution of all the reagents was used and if these reagents were correctly distributed on the plate.

If colour development is patchy (i.e., there is large variation between triplicates of individual samples), or if colour develops all over the plate, the pipetting, mixing and/or washing have been poor. More care is therefore needed with these procedures.

It is useful to keep a sample of the conjugate and the chromogen/substrate used in a test until the end of that particular test. If problems occur (no colour, slow colour or quick colour in the test) then the reagents can be checked as follows :

Pipette 100 µl of chromogen/substrate solution used for the test (H2O2 + OPD solution at the correct concentration) into a well of a microtitre plate or into a tube.

Add 50 µl of conjugate as prepared for the test (correctly diluted in buffer)

Colour should develop within 30 seconds.

If no colour develops prepare a new chromogen/substrate, but use the original conjugate preparation, and repeat the above steps.

If no colour develops prepare a new conjugate solution (correct dilution in buffer) and repeat the above test using the 'old' and 'new' chromogen/substrate solution.

If the above tests fail to give satisfactory results it may be necessary to obtain new conjugate.

Example of a Typical Photometer Print Out

 

Recorded ODs given under OPTICAL DENSITY
'Corrected' ODs given under WRL TYPING

Date of test =
Filename :
Experiment = EXAMPLE
Plate 1
Lowest OD = 0.00 Highest OD = 2.55 Filter = 492 nm
Background = 0.00 Threshold OD = 0.00 100% value = 0.00

OPTICAL DENSITY

Sample1          Sample2       Sample3

1

2

3

4

5

6

7

8

9

10

11

12

A

1.26

1.25

0.28

0.24

0.04

0.03

0.03

0.02

2.55

2.47

0.03

0.02

B

1.28

1.28

0.27

0.29

0.03

0.03

1.13

1.11

0.00

0.01

0.02

0.02

C

1.04

1.08

0.20

0.24

0.04

0.04

0.04

0.02

0.04

0.04

0.03

0.02

D

1.32

1.32

0.32

0.32

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

E

1.27

1.26

0.24

0.22

0.03

0.02

0.03

0.02

0.03

0.02

0.03

0.03

F

1.26

1.20

0.26

0.20

0.02

0.02

0.06

0.01

0.06

0.05

0.05

0.02

G

1.06

1.04

0.27

0.23

0.02

0.03

0.02

0.01

0.03

0.03

0.03

0.02

H

1.01

1.01

0.31

0.31

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

-----------------------
CONTROLS

Mean of columns 5 and 6 subtracted from all columns in each row

A        B        C        D        E        F        G        H
0.03    0.03    0.04    0.02    0.02    0.02    0.02    0.01

Date of test =
Filename :
Experiment = EXAMPLE
Plate 1
Lowest OD = 0.00 Highest OD = 2.55 Filter = 492 nm
Background = 0.00 Threshold OD = 0.00 100% value = 0.00

WRL TYPING 1
Sample1     Sample2    Sample3

 

1

 

2

 

3

 

4

 

5

 

6

 

7

 

8

 

9

 

10

 

11

 

12

A

1.23

1.22

0.25

0.21

0.01

0.00

0.00

-.01

2.52

2.44

0.00

-.01

B

1.25

1.25

0.24

0.26

0.00

0.00

1.10

1.08

-.03

-.02

-.01

-.01

C

1.01

1.05

0.16

0.20

0.00

0.00

0.00

-.02

0.00

0.00

-.01

-.02

D

1.30

1.30

0.30

0.30

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

E

1.25

1.24

0.22

0.20

0.01

-.00

0.01

0.00

0.01

0.00

0.01

0.01

F

1.24

1.28

0.24

0.18

0.00

0.00

0.04

-.01

0.04

0.03

0.03

0.00

G

1.04

1.02

0.25

0.21

0.00

0.01

0.00

-.01

0.01

0.01

0.01

0.00

H

1.00

1.00

0.30

0.30

-.00

-.00

-.00

-.00

0.00

0.00

0.00

0.00

------------------------
CONTROL ANTIGENS

Mean of columns

1-2

3-4

5-6

7-8

9-10

11-12

Interpretation

O

1.22

0.23

0.00

0.00

2.48

-.01

A

1.03

0.25

0.00

1.09

-.02

-.01

Control antigens: satisfactory

C

1.70

0.18

0.00

-.01

0.00

-.02

Sample 1 : positive FMDV serotype A

SAT 1

1.30

0.30

0.00

0.00

0.00

0.00

Sample 2 : positive FMDV serotype O

SAT 2

1.25

0.21

0.01

0.00

0.01

0.01

Sample 3 : negative

SAT 3

1.26

0.21

0.00

0.02

0.04

0.02

ASIA1

1.03

0.23

0.00

0.00

0.01

0.01

SVD

2.00

0.30

0.00

0.00

0.00

0.00

 

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Perfecting Your Acoustic Set List.

Perfecting an acoustic set list can really open your band up to a number of positive opportunities. A great level of emotion is often sparked from this type of music. Several things must be remembered though. The low tone must be fitting, the percussion must be fitting and the music needs to be performed in the proper type of environment. This type of music should be performed in the right type of way and the same also goes for recording it.

Low tones have always been an important part of layering acoustic music just as in the layering of electric music. Electric bass is just to over whelming for an acoustical piece. There are a few solutions to this problem. The bassist can play an acoustic bass or even do the bass line on a standard six string instrument. In a tight situation the guitarist can play the low tones with his thumb while he performs the rest of the music.

The proper type of percussion will also help set the right type of mood for the acoustic music you are playing. Traditional drum kits do not sometimes fit, unless played softly. Keep in mind there are many types of hand percussion like bongo drums that compliment acoustic arrangements. A tambourine or bean shaker might also keep the beat appropriately.

Playing the proper environment is often overlooked. It is a very important part of the acoustic set however. It helps to accomplish the right type of sound. An environment with good projection and acoustics is very important. The tighter the better. Any audience members should always be moved in as close as possible. A guitar that produces loud clear tones is also preferred. The dreadnought acoustic guitar often works very well for such a thing. The best quality is achieved from this.

Do not forget that to achieve the right sound, acoustic music has a certain way that is should be played by the musician. A musician is typically very relaxed and in some type of seat or chair. The beat to this music is usually much slower. Keep this in mind when performing acoustic renditions of electric work. A pick may be used, however sometimes fingers are preferred. An instrument like the alvarez acoustic guitar was practically made to be picked.

If playing this acoustic music has a certain protocol to it, please realize that recording it is very important as well. The most useful route is an acoustic guitar with an electric out put. This allows electric style recording while maintaining an acoustic tone. The ibanez acoustic electric guitar is one instrument that never fails in this category Boom mics can be placed at the feet of musicians or another type of microphone that attaches directly to the instrument may be used.

It is not hard to see why developing a quality acoustic set can be so very rewarding for a band. Doing so has several great benefits. The most useful one would be to display the versatility of your act.

I would like to make a really good alcoholic drink!?

I have very little experience mixing alcohols, i want to make something good note i do not have a shaker... The Alcohol's i do have are, Triple-sec, vodka, tequila, jagermeister, gin, and White rum... I know i can make long island iced tea or a jersey long island iced tea, but i am not very much into those drinks... I am fairly limited with mixers... any ideas? i have lime and lemon juice, i have tabasco, i may have v8, i have dr. pepper, some diet citrus soda's yuck, honey, agave syrup, pina colada mix that is past the best by date... i have lemon lime salt, and chili lime salt... i have celary salt, and sugar, normal household cooking stuff... any ideas? i do have to use measuring stuff most of the time i am bad at eyeballing and usually it comes out too strong :-/... I like fruity tastes, i have vitamin water haha maybe that could work with something maybe some apple and grape juice too! Thank you for your help!

go buy pineapple juice or orange juice and grenadine (cherry syrup). Add ice to glass, then an ounce and 1/2 (one shot) of tequila, half ounce triple sec, pineapple juice. Stir that then drizzle a small amount of grenadine in. That my friend, is the Tequila Sunrise

Tornado Mixer/Shaker