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ELISA can only be used as an optional test in addition to IF, PCR or FISH due to a relatively low sensitivity of this test. When DAS ELISA is used enrichment and the use of monoclonal antibodies are compulsory (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). Enrichment of the samples before using ELISA may be useful in order to increase the sensitivity of the test, but it can fail due to competition by other organisms in the sample.
Note: Use an validated source of antibodies to R. solanacearum (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main) It is recommended that the titre is determined for each new batch of antibodies. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 10 5 to 10 6 cells per ml of the homologous strain of R. solanacearum and using appropriate secondary antibody conjugates according to the manufacturer’s recommendations. During testing, the antibodies should be used at a working dilution close to or at the titre of the commercial formulation. U.K.
Determine the titre of the antibodies on a suspension of 10 5 to 10 6 cells per ml of the homologous strain of R. solanacearum .
Include a sample extract that previously tested negative for R. solanacearum and a suspension of a non-cross reacting bacterium in phosphate buffered saline (PBS) as negative controls.
As positive control use aliquots of sample extract, that previously tested negative, mixed with 10 3 to 10 4 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 2 A and B). For comparison of results on each plate use a standard suspension of 10 5 to 10 6 cells per ml in PBS of R. solanacearum . Ensure positive controls are well separated on the microtitre plate from the sample(s) under test.
Standardised positive and negative control materials available for use with this test are listed in Appendix 3 A.
Test control material in an identical manner as the sample(s).
Two ELISA protocols have been validated. U.K.
Use 100 to 200 µl aliquots of sample extract. (Heating at 100 °C for four minutes in a waterbath or heating block may reduce non-specific results in some cases).
Add an equal volume of double strength coating buffer (Appendix 4) and vortex.
Apply 100 µl aliquots to each of at least two wells of a microtitre plate (e.g. Nunc-Polysorp or equivalent) and incubate for one hour at 37 °C or overnight at 4 °C.
Flick out the extracts from the wells. Wash the wells three times with PBS-Tween (Appendix 4), leaving the last washing solution in the wells for at least five minutes.
Prepare the appropriate dilution of antibodies against- R. solanacearum in blocking buffer (Appendix 4). For validated commercial antibodies, use the recommended dilutions (usually twice as concentrated as the titre).
Add 100 µl to each well and incubate for one hour at 37 °C.
Flick out the antibody solution from the wells and wash as before (4).
Prepare the appropriate dilution of secondary antibody-alkaline phosphatase conjugate in blocking buffer. Add 100 µl to each well and incubate for one hour at 37 °C.
Flick out conjugated antibody from wells and wash as before (4).
Add 100 µl alkaline phosphatase substrate solution (Appendix 4) to each well. Incubate in the dark at ambient temperature and read absorbance at 405 nm at regular intervals within 90 minutes.
Prepare the appropriate dilution of anti- R. solanacearum polyclonal immunoglobulins in coating buffer pH 9.6 (Appendix 4). Add 200 µl to each well. Incubate at 37 °C for four to five hours or at 4 °C for 16 hours.
Wash the wells three times with PBS-Tween (Appendix 4).
Add 190 µl of sample extract to at least two wells. Also add positive and negative controls in two wells each per plate. Incubate for 16 hr at 4 °C.
Wash the wells three times with PBS-Tween (Appendix 4).
Prepare an appropriate dilution of R. solanacearum -specific monoclonal antibodies in PBS (Appendix 4) also containing 0,5 % bovine serum albumin (BSA) and add 190 µl to each well. Incubate at 37 °C for two hours.
Wash the wells three times with PBS-Tween (Appendix 4).
Prepare an appropriate dilution of anti-mouse immunoglobulins conjugated with alkaline phosphatase in PBS. Add 190 µl to each well. Incubate at 37 °C for two hours.
Wash the wells three times with PBS-Tween (Appendix 4).
Prepare an alkaline phosphatase substrate solution containing 1 mg p-nitrophenyl phosphate per ml of substrate buffer (Appendix 4). Add 200 µl to each well. Incubate in the dark at ambient temperature and read absorbance at 40 nm at regular intervals within 90 minutes.
The ELISA test is negative if the average optical density (OD) reading from duplicate sample wells is < 2x OD of that in the negative sample extract control well, providing the OD for the positive controls are all above 1,0 (after 90 minutes incubation with the substrate) and are greater than twice the OD obtained for negative sample extracts.
The ELISA test is positive if the average OD readings from duplicate sample wells is > 2x OD in the negative sample extract well provided that OD readings in all negative control wells are < 2x those in the positive control wells.
Negative ELISA readings in positive control wells indicate that the test has not been performed correctly or that it has been inhibited. Positive ELISA readings in negative control wells indicate that cross-contamination or non-specific antibody binding has occurred.]