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The ELISA - Kit Enzyme Linked Immuno Sorbent Assay - a practical test for class room demonstrations The test detects infection of pelargoniums (geraniums) with the Pelargonium Flower Break Virus (PFBV). The virus is widespread but absolutely harmless for humans. The sample material for the assay comes from any garden or balcony with ornamental flowers. The experiment illustrates a modern immunochemical analytical method and serves as a model for many diagnostic parameters. The test is easy to carry out, provides the result quickly (within one lesson) and includes a positive and a negative control to check correct performance of the assay.		ELISA-Kit with transferable Solid Phase (Pin format)
Two versions of the test are available: a. ELISA-Kit with transferable solid phase (pin format) for 2 x 10 analyses b. ELISA-Kit with microwell solid phase for 1 x 14 analyses Both test versions have the same speed and sensitivity. The microwell version is less expensive, but requires filtration of the plant extract. For this purpose, a re-usable accessory package is offered, which need only be purchased once.		ELISA-Kit with Microwell Solid Phase

Manual ELISA with transferable solid phase | Manual ELISA with microwell solid phase | EIBE

PFBV - ELISA with transferable solid phase (2x10 analyses)

Enzyme Linked Immuno Sorbent Assay
for the detection of
Pelargonium flower break virus (PFBV)

Complete test kit for 2 x 10 analyses; Art.-No. 04093E00.CWD. For laboratory use only. The reagents contain Bronidox L as preservative. Toxic when swallowed. Keep away from children.

1. INTRODUCTION
The PFBV ELISA is a sandwich enzyme immunoassay, featuring a unique design: the antibody has been immobilised on the teeth of a special comb rather than in conventional wells. This design facilitates and speeds up the assay procedure greatly (total incubation time: 30 minutes).

The test kit provides the reliable and sensitive detection of PFBV in 2 x 10 samples. To assure the proper performance of the assay, a negative and a positive control have been coated on the 11th and the 12th tooth of the comb (green und red dot, respectively).

The detection limit of the test was established by extracting and diluting infected plant tissue (Chenopodium quinoa) in sample buffer. The virus could be detected up to dilutions of 1:1.000 of a 10 % extract (>100 mOD over healthy control).

This test kit has been developed in a cooperation between BIOREBA AG and STEFFENS GmbH.

2. PRINCIPLE OF THE TEST
The teeth of the comb are coated with a polyclonal antibody which specifically recognises PFBV.
1st reaction: PFBV antigens present in the sample are bound to the immobilised antibody, forming the antigen-antibody-complex.
2nd reaction: a second PFBV antibody, labeled with horseradish peroxidase (enzyme conjugate), binds to the antigen-antibody-complex.
3rd reaction: the enzyme-labeled antigen-antibody-complex converts a substrate into a blue-coloured product. PFBV-infected samples exhibit the blue coloration, whereas non-infected samples remain colourless.

3. CONTENTS OF THE KIT
a. 2 combs, coated with PFBV-antibodies, each one with desiccant and 3 x 12-well-strips, sealed separately in air-tight plastic bags.
b. 20 extraction bags for sample homogenisation.
c. 2 vials with sample buffer (yellow, ready for use, 50 ml each).
d. 24 pipettes, for 2 x 10 samples, 2 x 1 diluted conjugate and 2 x 1 substrate.
e. 2 graduated pipettes for 2 x 1 sample buffer.
f. 2 pipettes with thin aperture for 2 x 1 concentrated conjugate.
g. 2 vials with concentrated conjugate (colourless, 50-250 µl each, depending on the kit lot, specified on the label).
h. 2 vials with conjugate buffer (blue, 2,0 ml each).
i. 2 vials with substrate solution (colourless, ready for use, 2,0 ml each).
j. 1 pipette stand (part of the inner packing).
The reagents of the test kit are stable at 2 - 8°C at least until the expiry date stated on the box label.

4. ACCESSORIES REQUIRED BUT NOT SUPPLIED
a. A homogeniser (can be supplied on request).
b. Cold tap water.
c. Optional (test may also be conducted on a purely visual basis): photometer with 650 nm filter. The optical density can be read directly in a special strip reader or in a conventional microtitre plate photometer for which a frame is required (can be supplied on request).

5. PREPARATION
Unpack the box completely and remove the inner packing. Take out the pipettes from the inner compartment. The test kit is intended for 2 tests of 12 analyses each; therefore, every part is supplied in duplicate. Return one set of components to the box and keep refrigerated (2 - 8°C) until required. By unfolding the lid of the inner compartment, the pipette stand designed to hold the 3 x 12-well strips is formed.
Cut open the plastic bag containing the comb. Remove the 3 strips of 12 wells and insert them into the pipette stand (no special order or orientation is required). Discard the desiccant bag. Return the comb to the plastic bag, avoiding contact with the teeth.

6. ASSAY PROCEDURE
Before starting the assay, all reagents should have reached room temperature (18 - 24°C). All reactions are performed at room temperature.

Sample preparation: Put the plant material, a piece of leaf approximately 15 cm² in size (0,5 g), into the center of the extraction bag. Add 5 ml of sample buffer (yellow) using the graduated pasteur pipette. Be sure not to contaminate the sample buffer with any plant extract.

Place the extraction bag on an even and firm surface. Extraction is achieved conveniently and quickly by means of an homogeniser. As a make-shift substitute, any smooth and solid object may be used, e.g. the handle of a screwdriver. Break the tissue down by moving the homogeniser in circles over the bag, applying moderate pressure, until an homogeneous suspension is formed. The release of chlorophyll (green colour) is a good indication of the degree of extraction.

Dispensing the samples: Dispense the samples with separate pipettes into the first row of wells (3 drops per well). Keep track of their identity: samples numbered 1 - 10 from left to right. Omit wells no. 11 and 12, which remain empty during the initial reaction step.

Incubation with samples: Insert the comb into the first row in correct orientation - the controls (immobilised on the comb, see dots) should be inserted into the empty wells. The first reaction is thus started. An incubation time of 10 min is required.

Conjugate preparation: Transfer the concentrated conjugate into the blue conjugate buffer as entirely as possible using the pipette with thin aperture. Attention: drops can stick to the lid. Mix thoroughly. Discard the pipette after use.

Dispensing the conjugate: While the comb incubates with the samples, dispense 3 drops of the diluted conjugate into each well of the second row, including wells no. 11 and 12. Use a new pipette and discard it afterwards. Avoid any contamination of the third row.

Washing the comb: After completion of the sample incubation, remove the comb and wash it carefully with cold, moderately running tap water for about 15 - 30 seconds. Thorough washing is important for low background staining. Then, shake off any excess water by some fast, flinging movements of the comb.

Incubation with conjugate: Insert the comb into the second row, maintaining its orientation with controls (i.e. dots) on the right hand side. Again, incubation takes 10 min..

Dispensing the substrate: Dispense 3 drops of the substrate solution into each well of the third row (including wells no. 11 and 12), using a new pipette.

Washing the comb: After 10 minutes incubation, remove the comb from the conjugate row and wash it thoroughly with cold tap water, as described above.

Incubation with substrate: Insert the comb into the third row, again ensuring the dots are on the right hand side. Incubate for 10 min. As the substrate is photosensitive, avoid intense light exposure (e.g. direct sunlight) during incubation.

7. EVALUATION
After removal of the comb, an evaluation of the assay is possible either visually or by means of a microtitre photometer, reading the optical density of the wells at 650 nm. This should be carried out immediately after the last incubation step.

The controls are intended to check the assay performance. The assay has been conducted properly if the positive control exhibits an intense blue colour, whilst the negative control remains colourless. Samples with a more pronounced colour development than the negative control are definitely infected. Samples with colour development less than that of the negative control are most probably uninfected. However, it cannot be ruled out that the concentration of virus in the sample may be below the detection limit of the test kit.

Having interpreted the assay, discard the comb and the well strips. Do not try to re-use the well strips; even after thorough washing, false-positive results are possible.

8. WARRANTY AND LIABILITY
STEFFENS BIOTECHNISCHE ANALYSEN GmbH guarantees that the delivered product has been thoroughly tested in order to ensure that its properties specified herein are fulfilled. No further warranties are given. In particular, STEFFENS BIOTECHNISCHE ANALYSEN GmbH is unable to accept liability for any damage which results from inappropriate storage or use of this product.

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PFBV - ELISA with microwell solid phase (1x14 analyses)

Enzyme Linked Immuno Sorbent Assay
for the detection of
Pelargonium flower break virus (PFBV)

Complete test kit for 2 x 14 analyses; Art.-No. 2609BE00.FWD. For laboratory use only.
Toxic when swallowed. Keep away from children.

1. Introduction
The PFBV-ELISA is a sandwich enzyme immunoassay which enables the detection of a PFBV infection of pelargoniums (geraniums). It is easy to perform and provides the result within one hour.

The detection limit of the test was established by extracting infected plant tissue (Chenopodium quinoa) and measuring dilutions of the extract. The virus could still be detected in dilutions of up to 1:1000 of a 10% extract.

The test allows analysis of 14 samples. It contains a positive and a negative control, immobilised on the solid phase on which the immunological reactions take place. These controls check the proper performance of the assay.

2. Precautions, disposal
Sample buffer and conjugate contain Bronidox as a stabiliser. The substrate contains tetramethylbenzidine and hydrogen peroxide.

All reagents are harmful only to such a low degree that no risk- and safety-phrases apply. Nevertheless, the reagents should not be swallowed and should be kept away from children. Avoid skin contact and rinse accidentally wetted spots with water.

After use, the reagents can be disposed of as waste water and poured into a sink. All solid components of the test are disposed of as ordinary garbage.

3. Contents of the kit

Solid phase: This is a plastic strip with 16 wells, arranged in 2 columns. The solid phase together with a desiccant is hermetically packed in an airtight foil laminate pouch.
14 extraction bags for homogenisation of the samples
14 folded filters for clarifying the extracts
Vial with 50 mL sample buffer (yellow, ready to use)
14 pipettes for the samples, 1 labelled pipette for each if the sample buffer, conjugate and substrate. The pipettes are graduated.
Vial with 2,5 mL conjugate (red, ready to use)
Vial with 2,5 mL substrate (colourless, ready to use). The vial is black in order to protect the substrate from light.

The test is stable at least until the expiry date stated on the packaging. It has to be stored at about 4°C.

4. Accessories required but not supplied

1 glazed pestle to homogenise the samples, 14 funnels, 14 reagent tubes and 1 tube rack (Set: Art.-No. 2709BE00.FWD). All components are intended for repeated use.
A pair of scissors, cold tap water, adsorbent paper

5. Principle of the test
The 2 x 8 wells of the solid phase are coated with a polyclonal antibody which specifically recognises PFBV. In addition, a negative (PFBV-free) and a positive (PFBV-containing) sample are immobilised in 2 wells on the edge. These are the control wells; they are marked green and red, respectively.

First reaction: The wells (except the control wells) are loaded with the samples, i.e. extracts from geranium plants. PFBV antigens eventually present in the sample are bound to the immobilised antibody, forming the antigen-antibody complex.

Second reaction: After a washing step which flushes all non-bound sample components from the solid phase, a second PFBV-antibody is added; the control wells are included. This antibody is conjugated with peroxidase ("enzyme conjugate") and binds to the immobilised antigen-antibody complex.

Third reaction: After a further washing step which removes non-bound conjugate from the solid phase, colourless substrate is added; the control wells are included. It is converted into a blue product by the conjugate. PFBV-infected samples exhibit the blue coloration (like the positive control), whereas non-infected samples remain colourless (like the negative control).

6. Preparation
Samples: Cut out 14 pieces of geranium leaves (about 10 cm² each) and put them into the vertically placed extraction bags. Note the designation of the samples on the corresponding bag and numerate according to the solid phase (see table above): B1-H1, B2-H2. Then, using the sample buffer pipette, add about 3 mL (corresponds to the uppermost pipette mark) sample buffer.

Place the extraction bag on an even surface, the opening facing upwards. Homogenise the leaf by rubbing the glazed pestle in circles over the bag, applying moderate pressure. When the extract turns green (release of chlorophyll), the extraction process is completed.

Transfer the extracts one by one, each with a separate sample pipette, into separate folded filters. Collect the filtrates into numbered reagent tubes. These filtrates are the actual test samples. The pipettes will be used again and must not be mixed up!

7. Assay procedure
Before starting the test, all reagents should have reached room temperature (19-29°C). All reactions are performed at room temperature.

Incubation with samples: Cut open the pouch with the solid phase and discard the desiccant bag. Insert the solid phase into its support (tray of the box), the green and red marked control wells facing upwards. Dispense the filtrates with the appropriate sample pipettes into the wells; 3 drops in each. Omit the control wells which remain empty during the initial reaction step! The incubation takes 10 minutes.

Note the identity of the samples:

Washing the solid phase: Take the solid phase out of the support and pour its contents into a sink with a flinging movement. Then, wash the entire solid phase thoroughly (3 times for about 5 seconds) under cold tap water. Finally, empty the solid phase, remove adherent water by shaking and tapping on adsorbent paper. Replace the solid phase into its support.

Incubation with conjugate: Dispense the red conjugate with the conjugate pipette into the wells, including the control wells; 3 drops per well. The incubation takes 10 minutes.

Washing the solid phase: as described above.

Incubation with substrate: Dispense the colourless substrate with the substrate pipette into the wells, including the control wells; 3 drops per well. The incubation takes 10 minutes. As the substrate is photosensitive, avoid intense light exposure (e.g. direct sunlight) during incubation.

8. Evaluation
The assay has been conducted properly if at the end of the substrate incubation the positive control exhibits an intense blue colour, whilst the negative control remains colourless. The result could look something like this:

In this case, samples 1B, 1G and 2D are strongly infected by PFBV, sample 1E is weakly infected and all other samples probably originate from healthy plants.

9. Warranty and liability
Steffens Biotechnische Analysen GmbH guarantees that the delivered product has been thoroughly tested in order to ensure that the specifications are fulfilled and that it conforms to the properties specified here. No further warranties are given. In particular, no liability can be accepted for any damage which results from inappropriate storage or use of this product.

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