ADGEN Phytodiagnostics - Protocol 1

1 ADGEN UNIT = 2 TEST WELLS

DAS-ELISA PROTOCOL (PAb and MAb)

Assay Principle

The double antibody sandwich ELISA (DAS) uses antibodies
(IgG) which are bound to the surface of a microtitre plate to
capture the antigen (A) of interest. A specific antibody-enzyme
conjugate (E) is then used to detect the trapped antigen. The
presence of enzyme (in this case alkaline phosphatase) is detected
by a colorimetric substrate (S) reaction.

Reagents required for DAS ELISA

Coating antibody (codes in the ADGEN catalogue ending in -
01/-02).
Conjugate (ADGEN codes ending in -03/-04)
Coating buffer (ADGEN codes 02-001/02-002)
Phosphate buffered saline + Tween 20 (ADGEN code 02-003)
Extraction buffer (ADGEN codes 02-004 - 02-016 depending on
antigen of interest)
Conjugate buffer (ADGEN codes 02-008/02-009)
pNPP tablets (ADGEN code 03-001/03-002)
Substrate buffer (ADGEN code 02-010/02-011)

Alternatively, for your convenience ADGEN supply a DAS
ELISA buffer pack (02-017/02-018) and prepared liquid
substrates which are stable, convenient and easy to use (03-
003/03-004) and for enhanced detection in your assay choose
ADGEN Blue liquid substrate system (03-005/03-006).

Additionally, positive and negative controls are available from
ADGEN (-11/-12) for a wide variety of pathogens, these allow
you to have increased confidence in the performance of the assay.

To cover all of your ELISA requirements ADGEN supply
ADGEN Total sets which contain all of the antibodies, reagents
and controls that are required for testing. ADGEN Total is
available in 500, 1000, 2500 and 5000 unit sizes.

Protocol (please read before starting the assay)

1. Dilute coating antibody in coating buffer as recommended on
the bottle label and add 100l to the required number of wells
for your test.

2. Wrap the plate tightly in cling film or place in a plastic box
with some damp paper towels and close the box. Incubate the
plate at 37
0
C for 4 hours.

3. Wash the plate three times with phosphate buffered saline +
Tween 20 (0.05%) - PBST. To do this fill the wells of the
plate with PBST and invert to remove the buffer. Repeat
twice, pat the plate dry on paper towels.






4. Extract the samples by grinding 1g of tissue with 10ml of
general extraction buffer in a mortar and pestle (or an
alternative method of grinding). Then filter the sample
through a layer of muslin (or similar fine cotton gauze). If this
is not available then allow the plant material to settle and use
the supernatant in the test. In some cases the recommended
ratio of sample to buffer may have to be reduced to allow a
clear signal to be obtained if the plant material is not highly
infected.

5. Add 100l of each sample, positive and negative control to
the coated wells. ADGEN recommend that all samples and
controls are tested in duplicate.
Remember, 1 ADGEN UNIT = 2 TEST WELLS.
ADGEN positive and negative controls are reconstituted by
adding 2ml of distilled/deionised water and gently shaking.
Any unused reconstituted control may be stored at -
20
0
C.However, the performance of the positive controls may
decrease when stored in this manner.

6. Wrap the plate as described in (2) above and incubate at
4
0
C overnight (at least 16 hours).

7. Wash the plate as described in (3) above.

8. Dilute the antibody-enzyme conjugate as recommended on the
bottle label in conjugate buffer and add 100l to each test
well.

9. Wrap as in (2) above and incubate at 37
0
C for 1 hour.

10. Wash four times as described in (3) above. An extra wash is
included at this stage to ensure that all unbound antibody-
enzyme conjugate is removed from the wells.

11. Prepare the substrate just before use - add pNPP at 1mg/ml to
substrate buffer (one 5mg tablet in 5ml of buffer).
Alternatively, use one of the ADGEN liquid substrates. All of
these substrates may change colour when exposed to light and
should be protected from light to prevent this occurring.

12. Add 100l of prepared substrate to each test well.

13. Wrap the plate as in (2) above and incubate in the dark at
room temperature for 1 hour.

14. Read the absorbance using a spectrophotometer at 405nm (for
pNPP and ADGEN Yellow) or 595 - 650nm (for ADGEN
Blue). Alternatively, positive and negative samples may be
scored visually although this may not be as accurate as using
a spectrophotometer. A positive sample may be determined as
one which gives an absorbance value which is greater than the
absorbance values of the negative control. A negative sample
is one which gives an absorbance value which is the same as,
or less than, the negative control. Visually, a positive sample
will give a darker colour than the negative control and a
negative sample will give a similar, or lighter, colour to the
negative control.

Notes

1. ADGEN antibodies and conjugates are sold in units of 500,
1000 and 5000. When they are diluted as indicated in the
protocol each unit provides sufficient for each test to be
conducted in duplicate wells containing 100l of each
reagent.
That is, 1 ADGEN UNIT = 2 TEST WELLS.

2. ADGEN recommend that, whenever possible, positive and
negative controls be run during all tests to ensure that all of
the test components are working properly.

3. The antibodies/conjugates should be stored at 4 - 6
0
C on
receipt.




Recommended buffers for DAS ELISA


ADGEN Phytodiagnostics - Protocol 1
1 ADGEN UNIT = 2 TEST WELLS

Coating Buffer (Carbonate Buffer)

Sodium carbonate 1.59g
Sodium hydrogen carbonate 2.93g


Make up to 1 litre with dH20. The pH of this buffer is 9.6 and
does not require to be adjusted.


Phosphate buffered saline (PBS)x10

Sodium chloride 80g
Potassium diHydrogen orthophosphate 2g
diSodium Hydrogen orthophosphate 11.5g
Potassium chloride 2g

Make up to 1 litre with dH20. The pH of this solution when
diluted to 1xs is 7.2


Wash buffer (PBS + Tween 20)

Phosphate buffered saline 1 litre
Tween 20 0.5 ml


General Extraction Buffer

Polyvinylpyrrolidone (PVP) 20g
Ovalbumin 2g
Sodium sulphite (anhydrous) 1.3g
Sodium azide 0.2g
Tween 20 0.5ml
Sodium chloride 8g
Potassium diHydrogen orthophosphate 0.2g
diSodium Hydrogen orthophosphate 1.15g
Potassium chloride 0.2g

Make up to 1 litre with distilled/deionised water. This buffer can
be difficult to get into solution and it is easier if the PVP is mixed
into a "paste" with a small volume of water before adding the other
components and the remainder of the water.


Conjugate buffer

Bovine serum albumin 0.2g
PBST 100ml


Substrate buffer (Diethanolamine buffer 1M)

Diethanolamine 90.39g
Diethanolamine-HCl 19.82g
Magnesium chloride 0.1g

Make up to 1 litre with dH20. The pH of this buffer is 9.8 and it
does not require to be adjusted. (The diethanolamine and
diethanolamine-HCl are liquids however, it is easier to weigh them
out than to measure their volumes as they are extremely viscous.)

pNPP is added to the above buffer at 1mg/ml to make up the
substrate for alkaline phosphatase.









Trouble Shooting Guide

A uniform high level of colour appears in all wells.

The plate may not have been washed properly.
The substrate solution may have been contaminated. pNPP
tablets can be contaminated by touching them, using
ADGEN Yellow removes this potential source of
contamination.
The substrate solution may have been exposed to bright
light.
One of the buffers may have been contaminated by
conjugate.
The plate may have been coated with antibody-enzyme
conjugate.
The plate may have been coated with coating antibody which
has been contaminated with antibody-enzyme conjugate.

All wells are coloured but to different degrees

The plate may not have been properly washed.
The plate may have been exposed to bright light.

Colour appears in all of the outer wells of the plate

The outer wells may not have been properly washed.
You may be experiencing 'edge effects'. This is a problem
associated with the cooling procedure employed when the
plates are being moulded. Newer, high quality plates such as
those used at, and supplied by, ADGEN do not cause this
problem.

Some colour appears in a negative control well, while some of
the other wells are clear.

A positive sample may have been added to the negative
control well.
During washing some positive control sample may have
been washed into the negative control well.
Contamination of the negative control well may have
occurred by carry over of a positive sample if the same
pipette tip was used. New tips must be used for every
sample.

All of the wells, including the positive control, are clear.

The antibody-enzyme conjugate may not have been added to
the conjugate buffer.
The positive control may have gone off.
The pNPP substrate tablet may not have been added to the
substrate buffer.

The colour in the positive control wells is very low

One of the buffers that have been used may be too old.
The positive control may be starting to go 'off'.
The substrate buffer may have been diluted with PBST and
not water as recommended. Phosphate in PBST reduces the
amount of colour produced.

If you still have a problem then prompt and
comprehensive technical advice regarding
ADGEN Phytodiagnostics products is always
available from Neogen Europe Ltd.

Tel: ++44 (0) 1292 525275
Fax: ++44 (0) 1292 525477
e-mail: [email protected]
website: www.neogeneurope.com