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20 Production of Antiserum (Hurn and Chantler 1980)

20 Production of Antiserum (Hurn and Chantler 1980)

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Qualitative and Quantitative Estimations of Amino Acids and Proteins


1. Dilute the purified antigen (150 mg) to 0.2 mL in phosphate-buffered saline

(PBS: 100 mg CaCl2, 120 mg MgSO4; 200 mg each of KCl and KH2PO4; 8.0 g

NaCl; 1.15 g Na2HPO4 per litre) and emulsified with 0.8 mL of incomplete

Freund’s adjuvant by thorough vortexing. Draw the mixture inside a syringe.

The syringe is emptied and refilled with the mixture 3–5 times to produce a

uniform, stable emulsion which can then be administered to a rabbit.

2. Prepare the rabbit for immunization by shaving away the hair at one or two

places on the hind thighs. Inject the emulsion in these places subcutaneously

using a 21-gauge needle.

3. Upto three booster shots at 10-day intervals following the initial shot could be

delivered. Each time, 50 mg protein (antigen) emulsified in a total volume of

0.5 mL of incomplete Freund’s adjuvant is used.

4. Eight days after the last booster shot, prepare the animal for bleeding. Rub the

surface of back of an ear with alcohol-moistened tissue to expand the veins.

5. Nick the marginal vein with a scalpel blade and collect the blood in a glass

vessel (about 15 mL can be collected in 10 min). Every minute or so clean the

clot at the puncture wound with alcohol-moistened tissue for continuous

bleeding. Alternately, bleeding is carried out under infrared light. At the end,

thoroughly clean the stained surface of the ear to avoid any infection. A testbleeding may be carried out even before the third booster shot to examine the

antiserum for the immune response and titre.

6. The blood is allowed to clot standing for 3–4 h at room temperature. The serum

is separated from the clot by low speed centrifugation and stored at 4 C in the

presence of 0.1% sodium azide as antibacterial agent. The serum is usually

straw yellow-coloured. If RBCs are partly lysed then the serum is coloured red.

7. Bleeding up to 3 times can be made on successive days. The rabbit is allowed to

rest for 4–6 weeks before further bleeding.

8. The animal can be used for about 6 months to collect blood and then abandoned. If after boostering, the antiserum is not of the desired quality, it is better

to disregard the particular rabbit and look for the other rabbits for the antiserum. If the whole lot is unsatisfactory possibly due to the weak immunogens,

either repeat the immunization or use other animals.

9. The antiserum is then tested by immunodiffusion or immunoelectrophoresis for

its ability to form immunoprecipitate.

10. The antiserum may be stored at À20 C in small aliquots in the presence of

0.1% sodium azide for longer duration.

11. The antiserum may be further processed to isolate IgG or for other



• A good antiserum should possess three important qualities: avidity (measure of

the strength of the interactions of its antibodies with antigen), specificity (ability


Immunodiffusion in Agarose Gels


of the antibody to recognize its antigen from related molecules) and titre (the

concentration of antibodies present, and on their affinities for the antigen).

When large amounts of pure immunogen are available, a high initial immunization dose can be used. However, lower the does of antigen, the greater is the

avidity of the antiserum.

Immunization can be done on any part of the body-skinfold of the neck and of

back, footpad, etc.

A long gap of 3–10 weeks is also normally used between the initial and booster

immunizations in order to a state of tolerance to the antiserum by the animal.

Since the antisera produced by conventional methods consist of mixtures of

different antibody molecules, the properties of antisera collected during the

prolonged period of immunization may change. Hence, each bleeding should

be tested for the antiserum qualities before any use.

Complete Freund’s adjuvant induces antibody production greatly than the

incomplete Freund’s adjuvant. CFA contains killed Mycobacterium cells over

the IFA, which are commercially available.


Immunodiffusion in Agarose Gels (Ouchterlony 1968)

Immunodiffusion in gels is often classified as single or double diffusion. In single

diffusion technique, the antigen (Ag) is usually allowed to diffuse into a gel

containing the gel antiserum/antibody (Ab). On the other hand, in the double

diffusion technique, both the antigen and antibody are allowed to diffuse into the

gel and meet each other. A lot of qualitative and quantitative information on the

antigen can be derived from the diffusion techniques. This is a simple technique to

test for the antibody titre during immunization and to study the antigen–antibody

reactions in gels. The antigen–antibody complex forms an insoluble precipitate

when the reaction is carried out both in solution and gels. Performing such reactions

in agarose gel is advantageous because of higher sensitivity and resolution. The

antigen and antibody react to produce the antigen–antibody complex. At equilibrium, the complex produced is immobile and forms a thin band of (protein)

precipitin. The precipitin is visualized either directly or after protein staining for



1. Agarose

2. 0.05 M Borate buffer (pH 8.0): (a) Dissolve 1.90 g borax (Na2B4O7Á10H2O) in

100 mL water (b) Dissolve 1.24 g boric acid in 100 mL water. Mix 30 mL of

(a) and 70 mL of (b) dilute to 400 mL by adding 300 mL water.

3. Solutions of antigen and antiserum

4. Glass Petri dishes or rectangular plates or microscope slides.

5. Gel punch and template – various sizes



Qualitative and Quantitative Estimations of Amino Acids and Proteins

6. Humidity chamber

7. Physiological saline solution: Dissolve 0.9 g sodium chloride in 100 mL water.

8. Staining solution: 0.1% Coomassie Brilliant Blue R250 in methanol: acetic acid:

water (4:1:5).

9. Destaining solution: methanol: acetic acid: water (4:1:5).


1. Ouchterlony double immunodiffusion

The double diffusion of the antigen and antibody was first described by

Ouchterlony. This technique is most widely used in the qualitative analysis of

antigens and antisera.

(i) Prepare 100 mL of 0.05 M borate buffer (pH 8.0) containing 0.9% NaCl

and 3% polyethylene glycol.

(ii) Dissolve 0.9 g of agarose in the above buffer solution by heating to 90 C

on a water bath with constant stirring or by autoclaving.

(iii) Pour the agarose solution to a depth of 2–3 mm into the sterile Petri dishes

or onto the rectangular plates or on a number of slides placed on a

horizontal level surface. Allow the gel to set for 10–15 min and store in

a humid chamber.

(iv) Place a template of a suitable pattern depending upon the number of

samples to be analysed on top of the gel. The gel punch that is connected

to a water vacuum pump is carefully inserted in to the gel through each

hole of the template to suck out the agarose plugs thus to form wells.

(v) Fill the centre or inner well with the antiserum and the outer or radial wells

with the antigen(s) solutions. Use serially diluted solutions of the same

antigen or the same volume of different antigens. The concentration of

antigen solutions and the dilution of the antiserum to be used have to be

established largely by trial and error by running pilot experiments.

(vi) Keep the gel plate in a humidity chamber at a constant temperature

(between 16 and 20 C) for 1–3 days and examine for the precipitin line


(vii) The precipitin line is visualized directly. The presence of polyethylene

glycol in the gel enhances the visibility of the precipitin. Alternatively, the

gel can be stained for proteins, especially if the precipitin line is very faint.

(viii) Prior to staining remove excess moisture from the gel plate by placing

some weight to a wad of filter paper placed on top of the gel for

15–30 min. Wash out the unreacted antigen and antiserum with several

changes in the physiological saline for 2–3 h. Dry the gel after further

pressing, in a stream of cold air from a hair-dryer.

(ix) Stain the dried gel for 10–15 min with Coomassie Brilliant Blue dyeDestain the plate for appropriate period to visualize the precipitin lines

clearly. The gel may be photographed and dried subsequently for a

permanent record.


Immunodiffusion in Agarose Gels


2. Single radial immunodiffusion

This method is widely used for the quantitative analysis of antigens.

The method followed is essentially as described for the Ouchterlong double

diffusion with the following modifications:

(i) Double amount of agarose (1.8/100 mL) is used.

(ii) The agarose solution is allowed to cool to 50–55 C, and is mixed with an

equal volume of a suitable dilution of the monospecific antiserum also at

55 C; the gel is then poured on the gel plates. The plates are used within a

day; may be stored for a week or so at 4 C.

(iii) The wells of 2 mm diameter are cut in a row on the plate using a gel

known concentration. The other wells are filled with the solution

containing the antigen at unknown concentrations. The volume of

antigens filled should be precisely known and equal in all wells.

(iv) A few wells in the row are filled with standard solutions of antigen of

known concentration. The other wells are filled with the solution

containing the antigen at unknown concentrations. The volume of

antigens filled should be precisely known concentrations. The volume of

antigens filled should be precisely known and equal in all wells.

(v) A disc of precipitin is formed around the antigen well. The disc size

enlarges with progress of period of diffusion.

(vi) The area of each disc is measured in terms of its diameter at every 12 h

until no more increase is noticed. A magnifying lens on suitable oblique

illumination such as colony counter may be used for measurement.

(vii) At the end of diffusion, processed gel may be stained and preserved as

described in the previous section.

(viii) A standard graph is constructed by plotting the diameters of the disc

against the logarithm of the antigen solutions of known concentration. The

concentration of the antigen in the test sample can then be determined

using the standard graph.


Double immunodiffusion

• It is important to run a few pilot experiments to standardize the antigen concentration and the dilution of antiserum for satisfactory results.

• It is preferable to precoat with 0.4% agarose gel prior to formation of the main

gel to prevent the latter from being detached during the staining procedure when

Petri dishes or glass plates are used.

• Microscope slides are considered ideal for rapid microanalysis.

• When the antigen and antiserum wells are closer the sensitivity is greater and the

time required for diffusion is shorter.

• If the antigen used is not highly purified, the antiserum will contain antibodies to

the protein impurities a well. Consequently, there will be many precipitin lines in

the diffusion patterns.




Qualitative and Quantitative Estimations of Amino Acids and Proteins

Enzyme-Linked Immunosorbant Assay

(Wim Gaastra 1984)

The enzyme-linked immunosorbant assay (ELISA) technique is used for a semiquantitative determination of the concentration of certain antigens/antibodies. It is

vividly used in medicine to detect the antigen or antibodies in serum samples. At

present, it has versatile applications in the immunodiagnosis of several infectious



The ELISA technique was first introduced in early 1970s by Engvall and Perlmann.

The principle underlying the double antibody sandwich technique of ELISA is

described below.

The antibodies against the antigen to be measured are adsorbed to a solid

support, in most cases a polystyrene microtiter plate. The support after coating

with antibody is washed. The antigen is now added and binds to the adsorbed

antibodies. Then, an enzyme-linked antibody molecules called the conjugate is

added which also binds to the antigen. A chromogenic substrate for the enzyme

is added and the coloured product generated is measured. The intensity of the colour is

proportional to the bound enzyme and thus to the amount of the bound antigen.

Hence, the intensity of the colour produced by a series of standard antigens

allows the calculation of the amount of antigen in an unknown sample.












Flat-bottomed polystyrene microtitre plate with 96 wells.

Micropipettes 0–250 mL.

Multi-channel Pipette 0–250 mL for pipetting of all reagents.

ELISA reader (Multiscanphotometer).

0.1 M Carbonate buffer (pH 9.6): Prepare 0.1 M Na2CO3 solution and adjust to

pH 9.6 with NaOH. The chemical should be of the highest quality and water

double distilled.

Wash solution: Mix 90 mL Tween 80 with 910 mL water.

BST: 0.2% (w/v) Bovine serum albumin, 0.01% Tween 80 and 0.9% (w/v)

sodium chloride in distilled water.

Substrate solutions: It depends upon the enzyme that is coupled to the conjugate. Two widely used enzymes in ELISA technique are horseradish peroxides

(HRP) and alkaline phosphatase. For HRP, there are two substrate solutions and

are prepared as below:

Solution 1: Dissolve 80 mg 5 amino-salicylic acid (purple-red brown colour) in

100 mL 0.05 M potassium phosphate buffer (pH 6.0) containing 0.001 M

EDTA. Add 20 mL H2O2 (30%) and mix.

Solution 2: Mix 24.3 mL 0.1 M citric acid

• 25.7 mL 0.2 M NaHPO4

• 50 mL H2O


Enzyme-Linked Immunosorbant Assay


• 40 mg ortho-phenylenediamine (yellow colour)

• 40 mL H2O2 (30%)

11. Stop solution

0.3 M NaOH (in the case of solution 1) or 1 M H2SO4 (in the case of solution 2)

is used.

(When alkaline phosphatase is the enzyme coupled, the substrate is then

p-nitrophenyl phosphate and is released as yellow coloured p-nitrophenol).

12. A diluted solution of IgG against the antigen to be measured. The dilution is

usually 1,500–2,500 fold depending on the titre of IgG. Dilutions are made in

0.1 M carbonate buffer.

13. Antigen solutions to be tested and standard antigen solutions.

14. Enzyme (HRP) labelled diluted IgG solution. As a rule the conjugate solution

has to be diluted 500–2,000 times in BST.


The double antibody sandwich technique

1. Pipette 150 mL of the diluted IgG solution to each of the wells of a microtitre

plate manually or using multichannel pipette. Cover the plate and incubate

overnight at room temperature.

2. Wash the plates with wash solution. The wells can be emptied by tapping the

plate over a sink and then beating the plate upside down against a filter paper.

The plates can be stored several months, covered and cooled. An appropriate

volume of wash solution is pipetted into the wells and left for a couple of

minute. The wells are then emptied as described above. Washing is repeated a

few times to ensure good results.

3. After washing add 100 mL BST to each well.

4. Add 100 mL of an antigen solution to be tested to the first well of each row.

Avoid air bubbles or splashing of small drops. Mix carefully and thoroughly.

5. Take 100 mL from the first wells and transfer to the second wells in each row.

Repeat the mixing procedure. Take 100 mL from the second wells and add to

the third wells and so on. By this way a twofold dilution series from wells 1–12

is created. Finally, remove the 100 mL excess from the last wells.

6. Incubate the plate for 2 h at 37 C, to allow the antigen bind to the coated

antiserum, and then wash thoroughly.

7. Add 100 mL of the diluted conjugate solution to each well and incubate for 2 h

at 37 C, then wash the plate thoroughly.

8. Add 100 mL of substrate solution to each well and incubate for 1–2 h at 37 C in

the dark.

9. Stop the reaction by adding 100 mL of stop solution.

10. Read the titre of the antigen solutions by either using an ELISA Reader or

visually by observing the last well that still gives some colour and could be

observed with the naked eyes.



Qualitative and Quantitative Estimations of Amino Acids and Proteins

Preparation of conjugate

A conjugate is the covalent complex of IgG and an enzyme. The coupling of HRP is

described below:

1. Dissolve 5 mg of HRP in 1 mL 0.3 M Na2CO3 (pH 8.1). This solution should be

prepared fresh.

2. Add 0.1 mL of 1% fluorodinitrobenzene in pure ethanol. If the HRP used is not

pure, a precipitate may be formed that must be removed by centrifugation

(10 min, 18,000 rpm).

3. Mix thoroughly and incubate for 1 h at room temperature.

4. Add 1 mL of 0.16 M ethylene glycol, mix, and incubate for another hour at room

temperature. The total volume is now 2.1 mL.

5. Dialyze the mixture against 0.01 M sodium carbonate buffer (pH 9.5) for 25 h.

The buffer should be changed at least 3 times.

6. Add IgG dissolved in 0.01 M sodium carbonate buffer (pH 9.5) to the peroxidase

aldehyde solution in the ratio of one volume of IgG solution to one volume

activated peroxidasealdehyde or 5 mg purified IgG protein to 3 mL peroxidase


7. Mix well and incubate 2–3 h but not longer at room temperature. If any precipitate is formed, clarify by centrifugation (10 min, 10,000 rpm).

8. Dialyze extensively against 0.01 M phosphate buffer (pH 7.2) containing 0.9%

NaCl at 4 C. Store the conjugate in a refrigerator or freezer in small aliquots and

use once only.


1. Purification of IgG fraction from whole serum:

• Mix 100 mL serum with 200 mL of 0.06 M sodium acetate (pH 4.6). The final

pH of the mixture should be 4.8.

• Add 8.2 mL (for rabbit serum) of caprylic acid dropwise at room temperature.

The volume of caprylic acid (6.8–8.2 mL) needed to precipitate IgG varies

from sera to sera depending upon the source.

• Stir for 30 min and remove the precipitate (10,000 rpm, 10 min).

• Dialyze the IgG fraction against 0.9% NaCl solution and store after


The experiment is wrong when many or all wells develop the same amount of

colour. In such case, the problem can be overcome by using freshly prepared




Preparations of S-30 Extract for Protein Synthesis In Vitro


Preparations of S-30 Extract for Protein Synthesis

In Vitro (Roberts and Paterson 1973)

Protein synthesis is a complex biosynthesis reaction involving a large number of

cell components and molecules. The process takes place on ribosomes and involves

polymerization of amino acids at the expense of energy as directed by messenger

ribonucleic acid. The protein synthesis as takes place in vivo could be conducted

in vitro using cell-free extracts. These extracts are prepared from a variety of

sources such as reticulocyte, wheat embryo, wheat germ, etc. These extracts are

very useful tools to study the synthesis of protein products programmed with

exogenous mRNAs. This procedure describes a method to prepare an efficient

cell-free extract from wheat embryos/germs.


The starting material is extracted in a suitable buffer to release that cell content,

centrifuges to get rid off fat and mitochondria and the post-mitochondrial supernatant containing ribosomes and soluble components is used for protein synthesis

in vitro.


Wheat Embryo/Wheat Germ

Standard HEPES buffer (SHB)

20 mM HEPES-KOH (pH 7.6)

120 mM KCl

2 mM Magnesium acetate

6 mM 2-Mercaptoethanol

SHB (pH 6.25)


1. Prechill a mortar and pestle by adding a few millilitre of liquid nitrogen. Add 3 g

of wheat embryo/wheat germ when liquid nitrogen is still there. Grind to a fine

powder before thawing. Add 20 mL of SHB (pH 6.25) in increments and

continue grinding to gel a fine homogenate.

2. Centrifuge the homogenate at 17,000 rpm (30,000 g) for 10 min in a refrigerated


3. Remove the supernatant carefully using a Pasteur pipette avoiding both the

precipitate and the top lipid layer.

4. Repeat the steps 2 and 3.

5. Load the supernatant onto a Sephadex G-25 column (25 Â 1 cm) preequilibrated with SHB (pH 7.6) and elute with the same buffer.



Qualitative and Quantitative Estimations of Amino Acids and Proteins

6. Collect the eluant in fraction. Combine the most turbid fractions. Immediately,

dilute 20 mL of the combined fraction to 2 mL with water and measure the

absorbance. The A260/A280 should be above 1.6.

7. Freeze immediately and store the combined fractions in small aliquots

(0.2–0.5 mL) in microfuge tubes under liquid nitrogen (À190 C) or at À70 C.


1. Embryos can be collected from freshly harvested wheat grains. Final purification

of embryos is carried out briefly by floating in a mixture of organic solvents –

cyclohexane: carbon tetrachloride (1:4) – change the solvent ratio slightly to

float the embryos on the surface. Collect the floating embryos quickly and dry on

filter paper. Embryos can be stored in sealed vials in freezers for a few weeks

before extraction.

2. The translation efficiency of the extract will be lower if A260/A280 is below 1.6.

Carefully combine only peak turbid fractions.

3. Storing of the extract at À20 C leads up to 70% activity loss in the 3 weeks.

4. The thawed extract should be used once.


In Vitro Translation Assay (Marcus et al. 1974)

In vitro translation study is an excellent procedure to study protein synthesis and to

characterize the product encoded by mRNAs. The cell-free protein synthesizing

system efficiently translates mRNAs from exogenous source under optimal

conditions. The assay is usually carried out in a small volume of 20–25 mL,

which could be scaled-up for preparative purposes.


The cell-free (S-30) extract containing the necessary protein synthesis machinery

components translates the genetic message in the mRNAs into protein when

provided with energy source under proper ionic conditions. The hot TCA perceptible radioactivity due to the labelled amino acid incorporation is measured.


1. Cell-free extract (see preceding experiment)

2. Salt mix (10Â)

200 mM HEPES-KOH (pH 7.6)

750 mM Potassium chloride

25 mM Magnesium acetate

20 mM Dithiothreitol

6 mM Spermidine (optional)

Store in aliquots at À20 C

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20 Production of Antiserum (Hurn and Chantler 1980)

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