Preparation of Immunogens and Production of Antibodies

Summary

The quality of reagents greatly affects the interpretation of serological tests. Methods used in conventional viral purification and molecular cloning and expression of target viral proteins to obtain antigens for immunization are presented. Immunization of rabbits, mice and chickens and isolation of immunoglobulin from immunized animals also are described.

1 Introduction

Immunology, a more commonly used term than serology, has had an important role in the advancement of biological science. Interest in diagnosing causal agents has provided the greatest impact to the development of immunodiagnostic techniques. In plant virology the primary concern is the detection of viral agents that causes plant diseases. The basis of the technique lies in the specific interaction of antigen (viruses) with immunoglobulins (antibodies).

When a foreign substance is introduced into an animal it elicits an immune response resulting in humoral antibody production. Plant viruses and viral proteins are immunogenic when used as immunizing agents (immunogen).

Numerous immunization protocols have been used successfully to generate antibodies to a variety of plant viral antigens. No single protocol proves to be the best (1). Many of the variables including the choice of animals, the doses of antigen, route of immunization, frequency of injection and choice of adjuvants need to be considered. They all, however, share common features that include a primary immunization followed by a 1- to 2-week resting period before subsequent one or more booster injections, and collection of immune sera about 1–2 weeks after the last injection.

Among the most important considerations for production of antibodies is the selection of a species or strain of animal. Polyclonal antibodies (antisera) have been prepared in numerous animal species. If a large volume of diagnostic reagent or extensive research utilizing the antibody reagent is the primary objective, goats or horses would be the better option. On the other hand, if antibodies are to be used for analysis in a few experiments, the use of mice which produce an adequate amount of antibody-rich ascitic fluids is an appropriate choice. The rabbit is, however, the most common species for polyclonal antibody production. Chicken antibodies (IgYs) are gaining more and more attention in basic research but are not fully utilized in the study of plant viruses (2).

Immunization of animals with highly purified antigens produces antiserum that does not contain significant amount of antibodies to host-plant proteins. The numbers of injections of a given antigen will, however, affect the specificity of an antiserum produced. Antibodies to major antigenic determinants can be produced by just one or two injections of purified virus antigens. The result is a highly specific antiserum. Too many injections of animals with a viral preparation may produce higher titer antisera that contain antibodies to both major and minor antigenic determinants resulting in an antiserum with broad-range reactivity. In addition, virus preparation considered pure may otherwise contain contaminants that also elicit immune responses when injected repeatedly into animals. Such sera react generously with plant tissue antigens and require extensive clean-up before use.

When hybridoma technology producing monoclonal antibodies was introduced, many thought that polyclonal antibody reagents were things of the past. Progresses made in molecular biology have extended the application of polyclonal antibodies into areas previously thought to be exclusive to monoclonal antibodies. It is now possible to produce mono-specific polyclonal antibodies to short, define peptide segments (3).

In this chapter we shall describe preparation of antigens, immunization of animals and isolation of immunoglobulins.

2 Materials

2.1 Preparation of Antigens

2.1.1 Virus Purification

Purified viruses, and recently the isolated capsid proteins, are routinely prepared from infected plants for production of diagnostic polyclonal antibodies. An excellent resource reference is available. Readers are recommended to consult Plant Virology Protocols in the Methods in Molecular Biology series (4). We will describe a procedure using Watermelon silver motel virus (WSMoV) as an example for purification of nucleocapsids of tospoviruses (5). The same procedure has been successfully used in the isolation of nucleocapsids of other members in the Tospovirus genus.

• 1. Infected plant tissue: Tospoviruses are readily transmitted by manual inoculation and virions attain high concentration in the infected Chenopodium quinoa leaf tissues when chlorotic spots developed. The inoculum is prepared in 0.1 M potassium phosphate, pH 7.0, containing 0.01 M sodium sulfite. C. quinoa plants are inoculated at 8–10 leaf stage and kept in a greenhouse at 25–30°C (see Note 1 ).

• 2. TB extraction buffer: 0.01 M Tris–HCl, pH 8.0, containing 0.01 M sodium sulfite and 0.1% cysteine.

• 3. Cheese cloth or Miracloth.

• 4. TBG buffer: TB buffer containing 0.01 M glycine.

• 5. 35% Cesium sulfate in TBG buffer.

• 6. Triton X-100.

• 7. Beckman high speed JA41, ultracentrifuge SW 41, 35 Ti and 60 Ti rotors.

• 8. Protein dissociation buffer.

• 9. 0.25 M KCl.

2.1.2 E. coli Expression

Traditionally, virions purified from infected tissue are used as immunogen to prepare diagnostic polyclonal antiserum. However, not every virus is easily purified from infected tissue. Some viruses accumulate very low concentration in infected tissues, and others are unstable when extracted from plant tissues. Such preparations after purification procedures are unsatisfactory to incite immune responses in injected animals. We have successfully used E. coli expressed viral coat proteins as immunogen to produce diagnostic antibodies (6, 7). We will describe a procedure using Chrysanthemum virus B (CVB) as an example to illustrate the preparation of E. coli expressed CVB coat protein for antiserum production (8). The same protocol has been successfully applied to prepare antisera against many ornamental plant viruses (9–11).

• 1. Culture of CVB maintained in chrysanthemum plants.

• 2. pCRII-TOPO vector and cloning kit (Invitrogen, Carlsbad, CA).

• 3. Sequence analysis ScanDNASIS program (Hitachi Software Engineering America, Ltd, CA, USA).

• 4. Protein expression vector plasmid pET28b(+) (Novogen, Inc., Madison, WI, USA).

• 5. Restriction enzymes NcoI and XhoI.

• 6. E. coli strain Rosetta(DE3).

• 7. Bacteria culturing M9 medium containing 50 μg/ml kanamicin and 34 μg/ml chloramphenicol.

• 8. 1 mM of isopropyl β-d-thiogalactopyranoside (IPTG).

• 9. VCX 600 sonicator (Sonics & Materials Inc., CT, USA).

• 10. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) system.

• 11. Primers T7 promoter (5′-TAATACgACTCACTATAGGG-3′) and T7 terminator (5′-gCTAgTTATTgCTCAgCgg-3′).

• 12. Protein denaturing solution (0.25 M Tris–HCl, pH 6.8, containing 2% (w/v) of SDS, 4% (v/v) of 2-mercaptoethanol and 10% (w/v) of sucrose).

2.1.3 ZYMV Expression

Recent success of plant viral vectors provide a fast and convenient technique for obtaining various proteins in eukaryotic plant cells (12, 13). In this section, we will illustrate a method that uses a ZYMV vector to express the nonstructural NSs protein of WSMoV in zucchini squash plants (3). The NSs open reading frame (ORF) was inserted in between the P1 and HC-Pro cistrons of ZYMV following a hexahistidine tag and an additional NIa protease cleavage sequence for purification and processing of the expressed protein, respectively. The expressed NSs protein was purified from squash tissues for production of antibodies.

• 1. WSMoV maintained in Nicotiana benthamiana plants.

• 2. ZYMV vector: p35SZYMVGFPhis plasmid.

• 3. TOPO TA cloning kit (Invitrogen; Carlsbad, CA).

• 4. Ultraspec RNA isolation system (Biotex Laboratories; Houston, TX).

• 5. E. coli strain DH5α.

• 6. Oligonucleotide primers.

• 7. Restriction enzymes SphI and KpnI.

• 8. T4 DNA ligase.

• 9. Agarose and DNA gel equipment.

• 10. Ampicillin.

• 11. TE buffer: 10 mM Tris–HCl and 1 mM EDTA, pH 8.0.

• 12. Miracloth (Calbiochem, La Jolla, CA).

• 13. Buffer A: 50 mM Tris–HCl, pH 8.0, 15 mM MgCl₂, 10 mM KCl, 20% glycerol, 0.05% β-mercaptoethanol (β-Me), and 0.1 mM phenylmethylsulphonyl fluoride (PMSF).

• 14. Buffer B: 50 mM Tris–HCl, pH 8.2, 15 mM MgCl₂, 20% glycerol, 0.05% β-Me, and 0.1 mM PMSF.

• 15. 0.45-μm Filters (Millipore, Billerica, MA).

• 16. Ni-NTA SUPERFLOW (Qiagen; Germany).

• 17. Wash buffer: buffer B containing 5 mM imidazole.

• 18. Elution buffer: buffer B containing 250 mM imidazole.

• 19. Monoclonal antibody (MAb) against the histidine tag (MAb-His, Amersham Pharmacia Biotech; Buckinghamshire, England).

• 20. SDS–PAGE equipment.

• 21. AlphaInnotech IS2000 image system (Alpha Innotech Corporation, San Leandro, CA).

2.2 Immunization and Isolation of Immunoglobulins (IgG/IgY)

2.2.1 Production and Isolation of Rabbit IgG

• 1. Two to three New Zealand White rabbits.

• 2. Adjuvant: Complete Freund’s adjuvant (CFA), a water-in-oil emulsifying agent with heat-killed Mycobacterium tuberculosis or incomplete Freund’s adjuvant (IFA) in which the bacterium is omitted.

• 3. PBS: Phosphate buffered saline solution, 0.01 M disodium phosphate, pH 7.5, 0.85% sodium chloride.

• 4. 50-ml Centrifuge tubes, serum vials.

• 5. Preservative stock solutions: 1% Merthiolate, 10% sodium azide.

• 6. Ammonium sulfate solution, saturated at room temperature, adjusted to pH 7.8 prior to precipitation of gamma globulin. 2N sodium hydroxide.

• 7. 10% Barium chloride.

2.2.2 Production and Isolation of Mouse IgG

• 1. Three to five Balb/c mice.

• 2. Pristane (Sigma-Aldrich, St. Louis).

• 3. PBS: Phosphate buffered saline solution, 0.01 M disodium phosphate, pH 7.5, 0.85% sodium chloride.

• 4. Adjuvant: Complete Freund’s adjuvant (CFA).

• 5. Mouse myeloma cells.

• 6. 70% Ethanol.

• 7. Protein-A Sehparose.

• 8. 0.02 M Disodium phosphate, pH 7.3.

• 9. 0.1 M Glycine, pH 3.0.

• 10. 2.0 M Tris–HCl, pH 8.3.

2.2.3 Production and Isolation of Chicken IgY

• 1. Two to three egg-laying hens.

• 2. Adjuvant: Complete Freund’s adjuvant (CFA).

• 3. PBS: Phosphate buffered saline solution, 0.01 M disodium phosphate, pH 7.5, 0.85% sodium chloride.

• 4. Polyethylene glycol 8000 (PEG, formally PEG 6000).

3 Methods

3.1 Preparation of Antigens

3.1.1 Purification of WSMoV Nucleocapsids

All purification steps should be carried out in a 0–4°C cold room with pre-cooled buffer, equipment, and rotors.

• 1. Harvest infected C. quinoa leaves 4–6 days after inoculation.

• 2. Homogenize the tissue with a Waring blender in TB extraction buffer (300 ml/100 g tissue).

• 3. Filter through four layers of cheesecloth.

• 4. Centrifuge the filtrate at 15,000 × g in a Beckman JA 14 rotor for 10 min to remove cell debris.

• 5. Carefully decant the supernatant into a beaker and discard pellets.

• 6. Add to supernate 1 to 2% Triton X-100 (final concentration) and stir for 5 min in the cold room.

• 7. Centrifuge at 80,000 × g in a Beckman 35 Ti rotor for 120 min through a 20% sucrose cushion.

• 8. Resuspend pellets in TBG buffer and further centrifuge in 35% cesium sulfate at 84,000 × g in a Beckman SW41 rotor for 16–18 h.

• 9. Collect opalescent zones containing virus nucleocapsids and centrifuge for 75 min at 160,000 × g in a Beckman 60 Ti.

• 10. Pellets resuspended in TBG buffer (3 ml/100 g initial weight of tissue) constitute the purified nucleocapsids. If not used immediately nucleocapsid preparation may be stored at −20°C. Nucleocapsid proteins are isolated by the following gel electrophoresis procedure.

• 11. Proteins are dissociated by the addition of 1/3 volume of 4× protein dissociation buffer.

• 12. The mixture is heated in boiling water for 3 min.

• 13. Centrifuge at 2,500 × g for 5 min to remove insoluble materials.

• 14. Supernatant is loaded onto stacking gel and electrophoresed at 80 V for 12 h. The NP proteins are visualized by soaking the gel in cold 0.25 M potassium chloride and eluted from the gel using an ISCO electrophoretic concentrator. Purified proteins are measured by absorbance at 280 nm, and store at −20°C.

3.1.2 Coat Protein Expression by E. coli

A protocol developed for the preparation of E. coli expressed CVB coat protein (9) is outlined as follows:

3.1.2.1 Construction of CVB Coat Protein Expression Vector

• 1. Total RNA was extracted from CVB-infected chrysanthemum tissue by the use of QIAamp total plant RNA mini kit (Qiagen, Hilden, Germary).

• 2. Coat protein gene of CVB was amplified using primers CVB-dw (5′-ATCTTCACAATGACATCCAT-3′) and CVB-up (5′-TAGGTTGTGGAGTGGTTACA-3′) by reverse transcription-polymerase chain reaction (RT-PCR) using 3 μl of total RNA extracted from CVB-infected chrysanthemum as template.

• 3. A PCR product about 1,000 bp was consistently amplified and it was cloned into pCRII-TOPO vector for sequencing studies.

• 4. The revealed 5′- and 3′-terminal sequences of CVB coat protein open reading frame were used to design PCR primers for the cloning of complete coat protein sequence.

• 5. Two restriction enzymes, NcoI and XhoI digestion sites (underlined) were created at the 5′-end of the upstream primer, CVB-up1 (5′-AGTCACCATGGCTCCCAAA-3′) and downstream primer, CVB-dw1 (5′-ACACTCGAGCACCACCACCACCACCACTGA-3′), respectively, to facilitate subsequent directional cloning of the coat protein into expression vector plasmid pET28b(+).

• 6. Using the CVB coat protein inserted pCRII-TOPO vector as template, the complete CVB coat protein open reading frame was amplified by PCR with CVB-up1 and CVB-dw1 as primers.

• 7. The amplified PCR product was digested with NcoI and XhoI and then ligated with a NcoI–XhoI cleaved pET28b(+).

• 8. The recombinant pET28b(+) vector was subsequently transformed into E. coli strain Rosetta(DE3) for protein expression.

• 9. Bacterial clones containing recombinant pET DNA were identified by PCR using primer pairs T7 promoter (5′-TAATACgACTCACTATAGGG-3′) and T7 terminator (5′-gCTAgTTATTgCTCAgCgg-3′).

• 10. Bacteria clones identified to have CVB CP inserted pET28b(+) were grown overnight at 37°C in M9 medium containing 50 μg/ml kanamicin and 34 μg/ml chloramphenicol.

• 11. When optical density (OD600) of bacteria culture filtrate reached 0.6–0.7, 1 mM of isopropyl β-d-thiogalactopyranoside (IPTG) was added to the medium to induce protein expression.

• 12. Four hours after IPTG induction, bacteria culture was subjected to centrifugation at 8,000 rpm for 10 min. Bacteria cells were resuspended in TE buffer (10 mM Tris–HCl, pH 8.0, 0.5 mM EDTA) and frozen at −20°C overnight.

• 13. After thawing , the bacteria cells were further disrupted by a VCX 600 sonicator (Sonics & Materials Inc., CT, USA) followed by centrifugation at 3,000 rpm for 10 min to remove cell debris.

• 14. Supernatants were treated with an equal volume of protein denaturing solution (0.25 M Tris–HCl, pH 6.8, containing 2% (w/v) of SDS, 4% (v/v) of 2-mercaptoethanol and 10% (w/v) of sucrose) and heated in boiling water bath for 3 min. The sample was then analyzed in SDS–PAGE (14). Size and expression level of viral CP was identified by western blotting analysis with a CVB antiserum purchased from Agdia Inc. (Elkhart, IN, USA).

• 15. Bacteria clones with satisfactory CP expression ability were selected and grown in a 1,000-ml flask of M9 medium containing 50 μg/ml kanamicin and 34 μg/ml chloramphenicol. Protein expression was induced and processed similarly as in steps 11–14. Bacteria expressed CP was further purified by a preparative SDS–PAGE protocol as described previously (7, 9).

• 16. Purified protein was always adjusted to a concentration of 1.0 OD₂₈₀ per ml, divided into 1.0 ml aliquot and preserved at −80 C for subsequent immunization.

3.1.3 NSs Protein Expression by ZYMV

An affinity chromatography (13, 15) was modified for purification of the ZYMV-expressed NSs protein from infected zucchini squash plants (3).

3.1.3.1 Construction of ZYMV Vector

• 1. The full-length NSs ORF was amplified from WSMoV S RNA using primers WNSs67KS (5′-GGGTACCGCATGCATGTCTACTGCAAAGAATGCTGCT-3′) and WNSs1383cK (5′-GGGTACCTTCTGCTTTCACAACAAAGTGCTG-3′) by RT-PCR.

• 2. The PCR product was cloned into pCR2.1-TOPO by TOPO TA cloning kit (Invitrogen, Carlsbad, CA) to generate pTOPO-WNSs.

• 3. The DNA fragment corresponding to NSs ORF was released from pTOPO-WNSs using restriction enzymes SphI and KpnI, and then ligated with the SphI/KpnI-digested ZYMV vector p35SZYMVGFPhis.

• 4. The plasmid of the ZYMV recombinant carrying NSs ORF was isolated by the mini-prep method, dissolved in TE buffer, and mechanically introduced with a glass spatula on C. quinoa leaves (10 μg in 10 μl per leaf) dusted with 600 mesh carborundum.

• 5. Local lesions developed were individually transferred to cotyledons of single zucchini squash plants.

• 6. Total RNAs extracted from symptomatic squash leaves using the Ultraspec RNA isolation system and primers WNSs67KS and WNSs1383cK were used to check the presence of the insert in the recombinant by RT-PCR.

• 7. PCR products were analyzed in 1.0% agarose gels by electrophoresis.

3.1.3.2 Purification of ZYMV-Expressed NSs Protein

• 1. 50 g Infected zucchini squash leaves were ground in 100 ml buffer A with a blender.

• 2. Extracts were clarified by centrifugation at 3,000 × g for 10 min, and supernatants were filtered through Miracloth.

• 3. The filtrates were treated with 1% Triton X-100 at 4°C for 30 min and then centrifuged at 30,000 × g for 30 min.

• 4. The supernatants were filtered through 0.45-μm filters.

• 5. Approximately 1 ml of Ni²⁺-NTA resins, pre-equilibrated in buffer B, was added.

• 6. The mixtures were gently shaken for 1 h at 4°C and loaded onto a column.

• 7. After allowing the resins to settle, the unbound materials were discarded and the resins were washed with twofold bed volume of wash buffer.

• 8. The proteins bound to the resins were eluted with 10 ml elution buffer.

• 9. The ZYMV-expressed NSs protein was further purified by gel electrophoresis method.

• 10. Each fraction of the purification steps was monitored by western blot using MAb-His.

• 11. The amount of purified NSs protein was estimated with standardized histidine-tagged GFP using MAb-His in western blotting or by comparison with bovine serum albumin (BSA) in SDS–PAGE, and estimated by the software Spot Density of AlphaInnotech IS2000.

3.2 Immunization and Immunoglobulin Isolation

Although marking experimental animals is of primary importance in laboratory studies, it is generally not required for plant virology investigations. Unless a mass production of antisera for a variety of viruses is a major responsibility of the facility, temporary cage marking for identification is sufficient since it is less stressful to the animals through the period of production. Procedures minimizing stress and pain and proper handling and care of animals should be observed all the time.

Both ammonium sulfate precipitation and Protein A-Sepharose chromatography are preferred methods for isolation of mammalian immunoglobulin (IgG) from immune sera and ascitic fluids. Protein A chromatography is not applicable to isolation of chicken immunoglobulin (IgY) since IgY does not bind with mammalian Fc-receptors nor with protein A or G. Isolation of chicken antibodies employs polyethylene glycol fractionation and precipitation.

3.2.1 Rabbits

3.2.1.1 Immunization

• 1. Day 0: Primary, intramuscular injection with 0.5–1.0 mg protein in 0.5 ml Tris–acetate buffer (containing 0.002 M EDTA) emulsified with 0.5 ml CFA equally divided into two hind legs (see Notes 2 and 3 ).

• 2. Day 7: Booster injection with 0.5–1.0 mg protein in a 0.5 ml Tris–acetate buffer (containing 0.002 M EDTA) emulsified with 0.5 ml ICA subcutaneously behind the neck or equally divided into two hind legs (see Note 3 ).

• 3. Day 14: Repeat step 2.

• 4. Day 21: Repeat step 2.

• 5. Day 28: Repeat step 2.

• 6. Day 35 and weekly thereafter: Serum collection (see Note 4 ).

• 7. Allow the blood to clot at room temperature for 2–4 h.

• 8. Dislodge the clot by rimming the tube with a wood applicator.

• 9. Centrifuge at 10,000 × g for 5 min and carefully transfer the serum to a new tube.

• 10. Sterile immune serum can be held at 4°C for a long time. If in doubt, add merthiolate (0.01% final concentration) or sodium azide (0.1% final concentration). Freezing at −20°C or lower and lyophilization will preserve sera for a long time, however.

3.2.1.2 Isolation of Rabbit IgG

Rabbit serum volume 50 ml or less is convenient and easy to manipulate. Using a 50 ml serum sample, ammonium sulfate precipitation procedure is presented.

• 1. At room temperature, while stirring, add, drop-by-drop, 25 ml saturated ammonium sulfate (adjust to pH 7.8 prior to use) to 50 ml serum sample in a 150-ml beaker. Continue stirring the suspension for an additional 2–3 h.

• 2. Centrifuge at 1,400 × g for 30 min in a clinical centrifuge.

• 3. Discard supernatant, and dissolve pellets in PBS to restore the original serum volume.

• 4. Repeat steps 1–3.

• 5. Repeat steps 1 and 2.

• 6. Dissolve precipitate in one half or less (25 ml or less) volume of PBS.

• 7. From hereon all steps are performed at 4°C. Dialyze against several changes of PBS overnight at 4°C and test the presence or absence of sulfate ions by adding a few drops pf 10% barium chloride to a small volume of dialysate.

• 8. Remove the solution from the dialysis tubing and centrifuge the solution at 4°C for 30 min at 1,400 × g.

• 9. Determine the immunoglobulin concentration by measuring the absorbance at 280 nm.

• 10. Purified immunoglobulin can be stored frozen.

3.2.2 Mice (Balb/c)

3.2.2.1 Immunization

When limited amounts of antigens are available for immunization, mice are excellent substitutes for larger animals. Antibody rich ascitic fluids can be induced in immunized mice by injection of FCA or implantation of mouse myeloma cells in the peritoneal cavity. Rouse sarcoma virus and the virus transformed tumor cell lines although serve the same purpose, are not recommended for plant pathologists who are not trained to handle animal pathogens.

• 1. Mice are primed with an intraperitoneal injection of Pristane (0.5 ml/mouse) 1–2 weeks before immunization (see Note 5 ).

• 2. Day 0: Intraperitoneal injection of 10–50 μg proteins in PBS (see Notes 3 and 5 ).

• 3. Day 7: Intraperitoneal injection of 10–50 μg proteins in PBS with CFA.

• 4. Day 21: Repeat step 2 and allow 3–5 weeks for development of ascites. Alternatively inject peritoneally (10⁶) myeloma cells in PBS, and allow 2–3 weeks for ascitic fluids to develop.

• 5. Tap mice when abdomen becomes distended. Surfaces sterilize the abdomen with 70% ethanol lightly and, with a finger, insert an 18 gauge needle. The fluid will drip out. Gentle massaging of the abdomen also increases the yield of ascites during tapping. Tapping can be repeated two to three times whenever abdomen becomes distended (see Note 6 ).

3.2.2.2 Isolation of Mouse IgG

Mouse immunoglobulins are generally purified by the method employing Protein A or Protein G column chromatography because of its small volume. The details of the isolation procedure can be found with accompanied literature from Protein A suppliers. Ammonium sulfate precipitation technique outlined in the rabbit immunoglobulin purification also can be applied in isolation of murine immunoglobulins.

• 1. Rehydrate 0.5 or 1 g Protein A-Sepharose in 0.02 M sodium phosphate, pH 7.3. Pack into a small column by running several bed volumes of buffer through column no faster than 1.0 ml/min.

• 2. Load sample on top and continue adding buffer. If the sample is serum, then dilute it with an equal volume of buffer before loading. Continue running buffer through until absorbance returns to zero.

• 3. Elute antibody off column with 0.1 M glycine, pH 3.0. Collect 1.0 ml fractions and immediately neutralize with 40 μl 2.0 M Tris–HCl, pH 8.5.

• 4. Concentrate antibody by ammonium sulfate precipitation (using 0.9 volumes saturated ammonium sulfate) and followed by dialysis against several changes of PBS.

• 5. Stabilize column with 0.02 M sodium phosphate, pH 7.3. Add a drop of sodium azide solution and store at 4°C.

• 6. For the next use, warm the column and all buffers to room temperature. De-gas the buffers before running through the column to eliminate air bubbles.

3.2.3 Chicken

Use of chickens for antibody production has been reported but not fully utilized in plant virology (1, 16). The concentration of chicken immunoglobulin Y (IgY, which is equivalent to mammalian IgG) in the yolk is essentially similar to (in some cases much higher than) the concentration of IgY in the serum. IgY is transferred from plasma through egg follicle to the yolk. Daily collection of chicken egg eliminates bleeding steps that every animal worker tries to avoid. A single chicken egg may yield as much as 100–200 mg of IgY.

3.2.3.1 Immunization

• 1. Day 0: Primary intramuscular injection with 0.1–0.5 mg protein in CFA.

• 2. Day 14: Booster injection with similar amount of protein in CFA.

• 3. Start collecting eggs 2–3 weeks after the beginning of immunization.

3.2.3.2 Isolation of Chicken IgY

• 1. Separate yolk and egg white.

• 2. Measure the volume of the yolk.

• 3. Add to 1 part egg yolk + 2 parts PBS, while stirring, PEG 8000 to 3.5% (w/v). Incubate at room temperature for 30 min.

• 4. Centrifuge the mixture at 14,000 × g for 10 min.

• 5. Carefully decant the liquid phase onto absorbent cotton in a funnel to remove fatty materials.

• 6. Add PEG 8000 to a final concentration of 12% (remember there was 3.5% before) while stirring and incubate at room temperature for 30 min.

• 7. Centrifuge at 14,000 × g for 10 min.

• 8. Dissolve the precipitate in PBS to the original yolk volume.

• 9. Add PEG 8000 to 12% (w/v) as in step 6 and sit at room temperature for 30 min.

• 10. Centrifuge at 14,000 × g for 10 min.

• 11. Remove as much as possible the supernatant containing PEG.

• 12. Dissolve the precipitate in one half the original yolk volume.

• 13. Aliquot the IgY preparation and store at −20°C.

4 Notes

• 1. Other susceptible species Nicotiana benthamiana and N. tabacum, etc., have been used as propagation hosts for tospovirus purification. Virion concentration, however, decreases before leaf necrosis occurs in these hosts.

• 2. Hyperimmunized animals may become very sensitive and thus susceptible to anaphylaxis. It is suggested that lower dose of antigen be used.

• 3. For most soluble antigens prepared with adjuvant, subcutaneous and intramuscular injections are preferred in rabbits, and intraperitoneal is more practical for mice. Interdermal and footpad injections are not recommended since injection via this route often causes animals discomfort due to local swelling, inflammation, and infection.

• 4. For a successful bleeding, ear vein should be dilated by rubbing the ear with your hand/or a gauze soaked in hot water (that your hand can tolerate). Coat the ear with a thin layer of Vaseline. With a quick stroke of a brand new razor nick the vein diagonally. Allow the blood to drip into a centrifuge tube. Care must be taken not to cut through the vein.

• 5. Injections should be made in the lower abdomen. To prevent the injected materials leak out of the abdomen. Injections work best when the needle first pierces the skin followed by inserting needle, parallel to and under the skin, about one cm before puncturing the abdominal wall.

• 6. Lipids in the fluid can be removed by passing through glass wool placed in a funnel.