TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.3 DETECTION/Serology

Section 2.3.3
Purification and
Conjugation of Antiserum

The sensitivity and specificity of the serological techniques for virus

detection such as Double Antibody Sandwich (DAS) and Nitrocellulose
Membrane (NCM) Enzyme-Linked Immunosorbent Assay (ELISA), latex,
micropreci-pitation, etc., require the use of antibodies and specific
conjugated antibodies. Therefore, the methods used for antiserum
purification should be suitable for each particular virus. The aim of this
chapter is to show some alternatives for processing antisera.

Purification of Immunoglobulins (IgG)

1. Take 1 ml crude antiserum, add 9 ml distilled water and pour into a
30-ml centrifuge tube.

2. Stirring carefully, add 10 ml saturated ammonium sulfate (drop-by-

drop), and store for 45 min. at room temperature.

3. Precipitate the proteins by centrifuging at 13,000 g for 15 min. at

4 C.
 4. Decant supernatant and re-suspend the pellet in 1 ml dializing
buffer solution.

5. Dispense the resuspended sample in dialysis tubes and identify

each sample.

6. Dialyze at 4 C with dialyzing buffer solution, making 4 changes of

500 ml each.

7. Label silicone-treated vials with the following information: IgG

(virus), code identifying the animal used in the research, volume,
date of bleeding, and concentration.

8. Dilute the globulin appropriately, and read its absorbance at 280

nm to determine the exact concentration by photometry (1 mg/ml
corresponds to A280 nm=1.4).

9. Make the corresponding calculations and store at 4 C.

Alternative Purification
7* Prepare a column of DEAE Sephacel of 3.5 ml, total volume and
equilibrate with dialyzing buffer.

8* Wash the column several times with dialyzing buffer which is also
used as an elution buffer.

9* Apply the dialyzed fraction and collect fractions of about 0.5 ml:
measure the concentration of the fractions at 280 nm.

Preparation of Chromatographic Columns

DEAE Sephacel is a gel commonly used for the purification of IgG (a 3.5-
ml column of DEAE Sephacel is needed). The procedure for using
Sephacel S-300 is described in the CIP Gugerli conjugation method (25
mm x 200 mm).

In each case, the gel contains a preservative (ethanol or thimerosal),

which must to be removed by washing the gel several times with double
the volume of the eluting buffer. This provides the gel with the required
characteristics for its use (pH, ionic strength, etc.; these requirements
vary according to each situation).

After stabilizing the gel for the particular conditions of use, the gas should
be removed both from the eluting buffer and the stabilized gel to avoid
air-bubble formation. This can be done by shaking the mixture slowly and
using an air pump.

Mounting the column is easy: Take the required volume; verify the height
the column will reach according to the specificity of each case; mark
where necessary, taking care that the column is exactly perpendicular to
the surface of the table to ensure that the sample flows evenly. Fill the
column by closing the lower outlet and pouring the gel, which has been
previously stabilized and degassed to prevent bubbles.

P.V. • Sec 2.3.3 – 99 • Page 2 - INTERNATIONAL POTATO CENTER

 When packing the column, keep it from drying out, because this could
cause breaks that will interfere with the normal flow.

The speed of the flow is specific for each case. Generally, a slow flow
prevents the column from overpacking or from losing its optimum
characteristics. Normally, a flow of 10–20 ml per hour is enough. Once
the column is packed, it is washed several times with eluting buffer to
ensure that the preservative has been completely removed.

To insert the sample, temporarily stop the flow and insert a sample that is
5% of the volume of the column.

The volume of the fractions must be controlled before collecting the

samples. The tubes are numbered and absorbance read at 280 nm to
determine the concentration of proteins (shown by the occurrence of
peaks in the reading).

Preparation of Enzyme Conjugated IgG

1. CIP method

1. Mix 1 ml IgG (1mg/ml) with 1250 units of alkaline phosphatase (in

the appropriate volume).

2. Add 2.6 l glutaraldehyde 25% solution for each ml of IgG. Mix

carefully to avoid bubbles.

3. Allow it to rest at room temperature for 2 hours.

4. Dialyze at 4 C in 1 liter of 10mM Phosphate Buffer Saline (PBS)

pH 7.4. Change the buffer 4 times.

5. Dialyze for the last time with PBS.

6. Pour the contents of the dialysis bag into a vial (previously treated
with silicone and rinsed in distilled water).

7. Add 5 mg BSA (bovine serum albumin globulin-free).

8. Store at 4 C.

2. CIP-Gugerli Method

This method differs from the original in the following:

a) When purifying the IgG, Gugerli proposes skipping the step of passing
through the DEAE-Sephacel column after dialyzing and before
conjugation.

b) Unlike the CIP method for enzymatic conjugation, Gugerli does not
dialyze, so the process takes less time (3 hours).

1. Dilute 3000 units of alkaline phosphatase in 0.6 ml conjugate

buffer.

2. Add 5 l glutaraldehyde 25% (undiluted) and stir vigorously.

P.V. • Sec 2.3.3 – 99 • Page 3 - INTERNATIONAL POTATO CENTER

 3. Incubate at 25 C for 50 min.

4. Add 0.75 ml IgG in a concentration of 1–2 mg/ml and mix

thoroughly.

5. Incubate at 25ºC for 70 min.

6. Add 50 g/l of sucrose, place the mixture on ice, mix well and pour
into a chromatography column in Sephacryl S-300 gel (25 mm x
200 mm, previously buffered) plus Tris/HCl, pH 8.0.

7. Elute with the same Tris/HCl solution, collect 2-ml fractions, and
measure the concentration of protein at 280 nm. The antibody
conjugated with the enzyme will elute as the first fraction, then the
elution of unreacted antibodies and enzymes could be observed.

8. Separate the fractions of eluted conjugate into 15 ml aliquots and

add 5 mg/ml BSA (globulin free) to each ml.

9. Store at 4 C.

Immunoglobulin Titration

The aim of titrating antibodies and antibodies conjugated with enzymes is

to determine the appropriate dilution for use in immunological assays.

The following dilutions should be prepared:

a) IgG (1mg/ml) in coating buffer: 1/500, 1/1000 and 1/1500

b) Conjugate-IgG in conjugate buffer: 1/500, 1/1000 and 1/1500

c) Sap from infected plants in extracting buffer: 1/10 and 1/100

d) Sap from healthy plants in extracting buffer: 1/10 and 1/100

For the distribution of the dilutions in the ELISA plates, see the diagram.

Note: The wells of the edges that are not used should be filled with the
appropriate buffer solution in each step of the test (to prevent nonspecific
reactions).

APPENDIX

1. Preparing the Solutions

a) 10 mM Phosphate Saline Buffer (PBS), pH 7.4

0.2 g KH2PO4
2.9 g Na2HPO4 12 H20
8.0 g NaCl (137 mM)
0.2 g KCl (2.7 mM)
0.2 g NaN3 (3 mM)*

P.V. • Sec 2.3.3 – 99 • Page 4 - INTERNATIONAL POTATO CENTER

 b) 5 mM Dialysis buffer; pH 7.4 (1/2 PBS)

0.1 g KH2PO4
1.45 g Na2HPO4 12 H20
4.0 g NaCl (69 mM)
0.1 g KCl (1.4 mM)
0.1 g NaN3 (1.5 mM)*

*Optional: only if the buffer solution is stored several days.

c) Saturated ammonium sulfate

In 1 liter of hot distilled water, gradually dissolve 750 g

(NH4)2SO4. When ready, pour into a plastic flask and cool to
room temperature. No refrigeration is needed.

IMPORTANT:

The enzyme used to conjugate could come in the form of:

1. Ammonium sulfate solution. In this case, it is necessary to dialyze

before use making 2 changes of the dialyzing buffer prior to its use
for conjugation.

2. NaCl Solution. In this form, the enzyme could be used directly

without dialysis.

Depending on the batch and the provider, the enzyme’s label could
provide the following information (for example):

a) Approximate number of enzyme units per flask: 5000 units

(Glycine, pH 10.4)

b) Volume of the flask: 0.47 ml.

c) Protein concentration in the flask expressed in mg prot./ml: 9.3-mg

prot./ml (Biuret).

d) Units of enzyme per mg of protein for the Diethanolamine, pH 9.8:

2870 units/mg prot. With this information we can get:

1. Total Units in the Flask

For the Glycine system pH 10.4:

1360 units 1 mg prot.

X 9.3-mg prot./ml

X = 12,648 units/ml

There is only 0.47 ml in the flask, so:

1.0 ml 12,648 units

0.47 ml Y
P.V. • Sec 2.3.3 – 99 • Page 5 - INTERNATIONAL POTATO CENTER

Y = 5944.56 units (contained in the flask)

 For the Diethanolamine system pH 9.8

2870 units 1 mg prot.

X 9.3-mg prot./ml

X = 26,691 units/ml

There is only 0.47 ml in the flask, so:

1.0 ml 26,691 units

0.47 ml Y

Y = 12,544.77 units (contained in the flask)

2. Determining the volume needed for the conjugation

In the DAS-ELISA system, Diethanolamine buffer solution pH 9.8 is used.

So we need:

- lgG 1 mg/ml and

- 1250 units of enzyme for conjugation

26,691 unit 1000 µL

1,250 unit X

X= 46.83 µl

2. Reagents

1. Phosphatase alkaline from calf intestine for enzyme

immunoassay. Cat. 567,752. Boehringer Mannheim GmbH or
Phosphatase alkaline from bovine intestinal mucose, TypeVII-NT
Cat. P-0405. Sigma Chemical Company.

2. Glutaraldehyde 25% aqueous solution. Grade 1. Specially

purified. Cat. G-5882. Sigma Chemical Company.

3. Albumin Bovine. 98–99% albumin. Essentially fatty acid-free.

Cat. S-2002. Sigma Chemical Company.

4. Sodium Azide Cat S-2002 Sigma Chemical Company.

P.V. • Sec 2.3.3 – 99 • Page 6 - INTERNATIONAL POTATO CENTER

 Recommended Literature
Clark, M.F. and A.N. Adams. 1977. Characteristics of microplate method
of Enzyme Linked Immunosorbent Assay for the detection of plant
viruses. Journal of General Virology 34:475–483.
Gugerli, P. 1986 in H.U. Bergmeyer. Methods of Enzyme Analysis. vol XI
p. 430–446.
Gugerli, P. 1979. Phytopath. Z. 96:97–107.
Mathews, R.E.F. 1957. Plant virus serology. Cambridge University Press,
Church Army Press, Cowley, Oxford.

P.V. • Sec 2.3.3 – 99 • Page 7 - INTERNATIONAL POTATO CENTER