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Diagnostic Procedure
Required Reagent Volumes
Plate Layout-Antigen
Detection ELISA
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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.
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