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An improved chromatographic method for the separation of egg yolk IgG into subpopulations utilizing immobilized metal ion (Fe3+) affinity chromatography
Journal of Immunological Methods 209 Ž1997. 155–164
An improved chromatographic method for the separation of egg yolk IgG into subpopulations utilizing immobilized metal ion žFe 3q / affinity chromatography Cam R. Greene ) , Peter S. Holt U.S. Department of Agriculture, Agricultural Research SerÕice, Southeast Poultry Research Laboratory, 934 College Station Road, Athens, GA 30605, USA Received 19 November 1996; revised 2 September 1997; accepted 5 September 1997
Abstract An improved methodology is described for the separation of yolk IgG into subpopulations using immobilized metal ion ŽFe 3q . affinity chromatography. The yolk IgG was first extracted using a prechilled, pre-acidified method. After extraction, the yolk IgG was then fractionated using an Fe 3q column. Using an ascending pH gradient, four IgG containing peaks were well resolved based upon the elution pH, specific activity and the relative avidity index. q 1997 Elsevier Science B.V. Keywords: Immobilized metal ion affinity chromatography; Yolk IgG; Subpopulations
1. Introduction Various animal species possess IgG subclasses with the exception of the chicken and the rabbit. Many IgG subclasses can be separated using protein
Abbreviations: AI, avidity index; ELISA, enzyme-linked immunosorbent assay; HEPES, N-w2-hydroxethylxpiperazine-N-w2ethanesulfonic acidx; IDA, imino-diacetic acid epoxy-activated Sepharose 6B; IMAC, immobilized metal ion affinity chromatography; IEP, immunoelectrophoresis; MES, 2-w N-morpholinex ethane sulfonic acid; PB, phosphate buffer; PBST, phosphate buffered salineqTween-20; RID, radial immunodiffusion; SE, Salmonella enteritidis; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis ) Corresponding author. Tel.: q1-706-5463441; fax: q1-7065463161.
A or DEAE ion-exchange chromatography. Because of its failure to bind to protein A, egg yolk IgG has been separated into subpopulations using DEAE ion-exchange ŽMcCannel and Nakai, 1990.. This was accomplished by maintaining a constant pH while increasing the phosphate ion concentration. Immobilized metal ion affinity chromatography ŽIMAC. is a protein purification technique based upon the affinity that proteins have for certain metals. This metal-protein binding is mediated through the presence of the amino acids histidine, tryptophan and cysteine being located on the surface of the protein molecule ŽPorath and Olin, 1983.. Elution of bound proteins from an IMAC column is usually brought about by lowering the pH, or increasing the salt concentration, or by both. The most commonly used metals in IMAC chromatography include Cu2q,
0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 1 5 5 - 5
156
C.R. Greene, P.S. Holt r Journal of Immunological Methods 209 (1997) 155–164
Ni 2q, Co 2q and Zn2q, while the Fe 3q cation occupies a special niche in IMAC chromatography. The Fe 3q ion has been shown to have a very strong affinity for phosphorylated amino acids and proteins ŽAndersson and Porath, 1986.. Using an Fe 3q column, Sulkowski Ž1988. found that Fe 3q can act as a carboxymethyl ŽCM. exchanger and that model proteins elute in order of their isoelectric points. Using this special property of Fe 3q, we were able to separate yolk IgG into four well resolved subpopulations based upon the elution pH and the relative avidity index.
2. Materials and methods 2.1. Immunizations Ten specific-pathogen-free White Leghorn laying hens Ž5 birds per experiment. were each immunized, subcutaneously, with an acetone-killed preparation of Salmonella enteritidis ŽSE. which was mixed with an equal volume of an incomplete adjuvant composed of mineral oil, Arlacel A and Tween-80. Each hen received a booster injection six weeks after the initial immunization. Eggs were collected three weeks after the booster injection and were stored at 48C until used. All animals used in this study were treated in a manner consistent with USDA animal welfare guidelines. 2.2. Bacterial growth conditions Stock cultures of SE, phage type 13A, were maintained at y208C in 15% Žvrv. glycerol-tryptic soy broth ŽDifco Laboratories, Detroit, MI.. For the flagella extraction, a stock culture was thawed and streaked on a nutrient agar plate ŽDifco. to obtain well isolated colonies. After growth at 378C for 18–24 h, individual colonies were transferred to U-shaped glass tubes ŽFisher Scientific, Atlanta, GA. containing GI motility medium ŽDifco. and were passaged five times to obtain highly motile SE which were then used to inoculate 6 = 500 ml of the chemically defined minimal medium of Ibrahim et al. Ž1985.. These cultures were then incubated at 358C
in an orbital shaker incubator ŽPrecision Scientific, Chicago, IL. at 80 rpm for 16 h. 2.3. Flagella extraction and purification Flagella were extracted by the method of Ibrahim et al. Ž1985.. Briefly, Salmonella cells were harvested by centrifugation and resuspended in a saline solution. The pH was then adjusted to 2 and was maintained at that pH under constant stirring for 30 min at room temperature. After centrifugation, the supernatant was ultracentrifuged at 100 000 = g for 1 h at 48C. The pH of the resulting supernatant was adjusted to 7.2 and solid ammonium sulfate was added to two-thirds saturation Ž2.67 M. to precipitate the flagella. After centrifugation, the resulting pellet was resuspended and dialyzed against distilled water using 50 000 molecular weight cutoff dialysis tubing ŽSpectrum Medical Industries, Houston, TX.. Final purification was achieved using CL-6B gel-filtration chromatography ŽPharmacia Biotech, Piscataway, NJ.. Purity of the flagella preparation was ascertained by SDS-PAGE while the total protein was determined using the BCA protein assay ŽPierce Chemical Co., Rockford, IL.. The flagella were stored at 48C in 20 mM PBS pH 7.5. 2.4. Egg yolk IgG extraction Egg yolk IgG was extracted by a previously described procedure ŽAkita and Nakai, 1992.. Briefly, 20 ml of yolk was added to 180 ml of prechilled, pre-acidified distilled water Žacidified with 1 N HCl to obtain a final pH of 5–5.2 after dilution.. This solution was then incubated overnight at 48C to allow settling of insoluble material. After settling, a portion of the supernatant was then centrifuged, at 15 000 rpm, in a microcentrifuge for 15 min at 48C. Following centrifugation, the supernatant was filtered through a 0.22 m m syringe filter ŽMillipore, Bedford, MA.. 2.5. Iron chelate chromatography Iminodiacetic acid ŽIDA . epoxy-activated Sepharose 6B ŽPharmacia Biotech. was poured into a
C.R. Greene, P.S. Holt r Journal of Immunological Methods 209 (1997) 155–164
G10 = 150 Moduline medium pressure laboratory column ŽAmicon, Beverly, MA. fitted with an adjustable end piece to obtain a final packed bed volume of 7.5 ml. Following a deionized water wash, the column was converted to the IDA-Fe 3q form by charging with aqueous FeCl 3 P 6H 2 O Ž500 mg in 100 ml deionized water. ŽSulkowski, 1988.. After charging, the column was washed with deionized water and then equilibrated with 50 mM 2-w Nmorpholinox ethane-sulfonic acid pH 6 ŽMES. ŽSigma. ŽSulkowski, 1988.. At the completion of each run, the IDA gel was stripped Žchelate annihilation. with 50 mM EDTAq 500 mM NaCl pH 7 and regenerated as previously described. 2.6. Separation of yolk IgG into subpopulations A 5 ml sample of supernatant from the yolk IgG extraction procedure was transferred to SpectrarPor 6 dialysis tubing ŽSpectrum Medical Industries, Los Angeles, CA. and dialyzed overnight at 48C, with constant stirring, against 500 ml of 50 mM MES pH 6 buffer ŽIDA-Fe 3q equilibration buffer.. Following dialysis, the material was applied to a previously equilibrated IDA-Fe 3q column. After sample application, the column was washed with the equilibration buffer until the unabsorbed peak returned to the baseline. Bound immunoglobulin was then eluted using an ascending linear pH gradient consisting of 100 ml IDA-Fe 3q equilibration buffer and 100 ml of 50 mM N-w2-hydroxethylxpiperazine-N X-w2 -ethanesulfonic acidx pH 8 ŽHEPES. ŽSigma. ŽSulkowski, 1988.. Following gradient completion, the column was washed with 50 mM HEPES pH 8 and then subjected to chelate annihilation using 50 mM EDTAq 500 mM NaCl pH 7. 2.7. ELISA Purified SE flagella were bound to the solid phase using methyl glyoxal ŽCzerkinsky et al., 1983.. The flagella were diluted to 1 m grml using 0.3% Žvrv. methyl glyoxal pH 7 and 150 m l were added to the appropriate wells of Immulon 4 microtiter plates ŽDynatech Laboratories, Chantilly, VA.. The plates were then incubated for 2 h at 378C. Following incubation, the plates were twice washed with 20
157
mM PBS pH 7.4 and then blocked for 1 h at 378C with 250 m lrwell of 100 mM PB q 1% Žwrv. polyvinylpyrrolidone ŽSigma. pH 8. After blocking, the plates were washed twice with 20 mM PB q 0.05% Žvrv. Tween-20 pH 7.4 ŽPBST. and then two-fold serial dilutions, diluted in PBST, of each eluted subpopulation Ž150 m lrwell. were added to the appropriate wells of the microtiter plate and then incubated for 90 min at 378C. The plate was washed twice with PBST and then 150 m lrwell of alkaline phosphatase labeled affinity purified rabbit anti chicken IgG-Fc specific antiserum ŽJackson ImmunoResearch Laboratories, West Grove, PA., diluted 1:5000 in PBST, was added to the plate and incubated for 90 min at 378C. The plate was washed twice with PBST and then 150 m lrwell of 100 mM diethanolamine buffer pH 9.8 containing 1 mgrml p-nitrophenyl phosphate substrate ŽSigma. was added to each well. Color development was allowed to proceed for 30 min at room temperature and was then stopped by the addition of 50 m lrwell of 3 N NaOH. Absorbance was measured at 405 nm using a Multiscan MS microtiter plate reader ŽLabsystems, Needham Heights, MA.. 2.8. AÕidity index The avidity index ŽAI. was determined for each subpopulation using 8 M urea as previously described by Chargelegue et al. Ž1993.. Flagella, diluted to 1 m grml in 100 mM bicarbonate–carbonate coating buffer pH 9.6, was bound to the solid phase by overnight incubation at 48C. Following incubation, the microtiter plate was then blocked for 1 h at 378C as previously described. By prior titering, each eluted IgG subpopulation Žpeak. was diluted in PBST to give an OD405 nm reading of 1.0 " 0.1. Four replicates of each diluted subpopulation Žpeak. were allowed to react with the immobilized flagella by overnight incubation at 48C. The plates were then washed with PBST and one duplicate received 100 m l of 8 M urea in PBS while the other duplicate received 100 m l of PBST. Following incubation for 15 min at room temperature, the plate was then washed six times with PBST and 100 m l of alkaline phosphatase labelled affinity purified rabbit anti chicken IgG-Fc specific antiserum ŽJackson., diluted
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1:5000 in PBST, was added to each replicate and allowed to react for one hour at 378C. After twice washing the plate, each duplicate received 100 m l of 100 mM diethanolamine buffer pH 9.8 containing 1 mgrml p-nitrophenyl phosphate substrate ŽSigma.. Color development, after proceeding for 30 min at room temperature, was stopped by the addition of 50 m lrwell of 3 N NaOH. Absorbance was measured at 405 nm using a Multiscan MS microtiter plate reader ŽLabsystems.. The Avid-
ity Index ŽAI. was then calculated using the following formula: AI s ŽOD405 nm 8 M urearOD405 nm PBST control. = 100. 2.9. Immunoelectrophoresis Immunoelectrophoresis ŽIEP. was performed in 1% Žwrv. agarose ŽType II, Medium EEO, Sigma. in a Tris-Tricine buffer pH 8.6. Samples Ž5 m l. were electrophoresed in an EC 360 flatbed apparatus ŽE-C Apparatus Corp., St. Petersburg, Fl. at 200 constant
Fig. 1. ŽA. IDA-Fe 3q chromatography of pooled nonimmune yolks from 5 birds. ŽB–C. IDA-Fe 3q chromatography of nonimmune yolks from individual birds. In all chromatography runs, a 5 ml sample was applied to a previously equilibrated IDA-Fe 3q column at a flow rate of 1 mlrmin while the fraction collector was operated in the time collection mode Ž5 minrtube. and a sensitivity setting of 0.1 AUFS. Peak 1 is the unabsorbed fraction. Peaks 2 and 3 elute during gradient formation while peak 4 is the chelate annihilation phase.
C.R. Greene, P.S. Holt r Journal of Immunological Methods 209 (1997) 155–164
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2.10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Fig. 2. SDS-PAGE of pooled nonimmune yolks from IDA-Fe 3q chromatography. Samples Ž5 m grlane. were electrophoresised at 200 constant volts. Lane 1: molecular weight markers; lane 2: purified chicken IgG ŽJackson.; lane 3: peak 1; lane 4: peak 2; lane 5: peak 3; lane 6: peak 4r
volts for 45 min. After electrophoresis, the IEP films were incubated overnight in a humidity box and then processed and stained with 0.1% Žwrv. coomassie brilliant blue R-250 ŽSigma..
SDS-PAGE ŽLaemmli, 1970. was conducted using a vertical mini-gel electrophoresis unit ŽMighty Small II, Hoefer Scientific Instruments, San Francisco, CA.. Samples Ž5 m g. were dissolved in solubilization buffer and boiled for 90 s. They were then applied to a 0.75 mm thick gel of 12% poly- acrylamide with a 4% stacking gel and electrophoresed at 200 constant volts. The gel was then stained with coomassie brilliant blue R-250 ŽSigma., and apparent molecular weights were interpolated from the migration of phosphorylase Ž97.4 kDa., bovine serum albumin Ž66.2 kDa., ovalbumin Ž42.6 kDa., bovine carbonic anhydrase Ž31 kDa., soybean trypsin inhibitor Ž21.5 kDa. and lysozyme Ž14.4 kDa.. 2.11. Radial immunodiffusion Radial immunodiffusion ŽRID. ŽFahey and McKelvey, 1965. were done on calibrated immunodiffusion plates ŽICN Immunobiological, Lisle, IL. of 1% Žwrv. agarose ŽType II, Medium EEO, Sigma. in 30 mM PBS pH 8. Zone diameters were measured to the nearest 0.1 mm using a QUIPmeter ŽHelena Laboratories, Beaumont, TX..
Fig. 3. Immunoelectrophoresis of pooled nonimmune yolks from IDA-Fe 3q chromatography. Samples Ž5 m l. were electrophoresised for 45 min. After electrophoresis, each antiserum trough was filled with a 1:3 dilution of affinity purified rabbit anti chicken IgG H and L chain antiserum. After overnight incubation, the IEP films were processed and then stained with coomassie brilliant blue R-250.
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3. Results 3.1. IDA-Fe 3 q chromatography (nonimmune yolk) Prior to immunization, control or nonimmune eggs were collected from each bird. These eggs Žyolks. were subsequently pooled and a crude IgG fraction was obtained using the prechilled, pre-acidified methodology. A 5 ml sample of this crude extract was then fractionated on a previously equilibrated IDA-Fe 3q column ŽFig. 1.. Using an ascending lin-
ear pH gradient, four protein containing peaks were eluted: Peak 1 is unabsorbed fraction, peaks 2 and 3 elute during gradient formation, while peak 4 elutes during the chelate annihilation phase. This basic chromatographic profile is also exhibited in yolks from individual birds ŽFig. 1B–C. with some variation occurring in the heights of peaks 2 and 3. Analysis of elution pH ŽFig. 1A. indicates that peak 2 elutes from pH 6.19–6.72 while peak 3 elutes from 6.72–7.44. This indicates that the column has separated the IgG fraction into four subpopulations based
Fig. 4. ŽA. IDA-Fe 3q chromatography of pooled immune yolks from 5 birds. ŽB–C. IDA-Fe 3q chromatography of immune yolk from individual birds. In all chromatography runs, peak 1 is the unabsorbed fraction. Peaks 2 and 3 elute during gradient formation. Peak 4 is the chelate annihilation phase.
C.R. Greene, P.S. Holt r Journal of Immunological Methods 209 (1997) 155–164
161
umn can also bind and separate these additional yolk proteins which could then be further purified using a variety of chromatographic techniques. Examination of each IgG subpopulation by immunoelectrophoresis ŽFig. 3. demonstrates an electrophoretic mobility difference. This difference is clearly demonstrable between peaks 1 and 4 while minor differences exist between peaks 2 and 3. The IgG content of each peak was determined by RID’s: Peak 1 ranged from 72.86–80.81 m grml Ž"3.34; standard deviation., peak 2 ranged from 117–133 m grml Ž"11.73., peak 3 ranged from 77–113 m grml Ž"13.18., while peak 4 ranged from 98–106 m grml Ž"2.31.. 3.2. IDA-Fe 3 q chromatography (immune yolk) 3q
Fig. 5. SDS-PAGE of pooled immune yolks from IDA-Fe chromatography. Samples Ž5 m g. were electrophoresised at 200 constant volts. Lane 1: molecular weight markers; lane 2: purified chicken IgG; lane 3: whole yolk extract; lane 4: peak 1; lane 5: peak 2; lane 6: peak 3; lane 7: peak 4.
upon the elution pH. After chromatography, each peak was concentrated and subjected to SDS-PAGE ŽFig. 2. and IEP ŽFig. 3. analysis. Since the SDSPAGE ŽFig. 2. confirms the presence of other proteins, besides IgG, this would indicate that the col-
The crude IgG extract from five birds Žpooled yolks. immunized with an SE bactrin was chromatographed on a previously equilibrated IDA-Fe 3q ŽFig. 4A. column. As before, using an ascending linear pH gradient, four protein containing peaks were resolved. This same peak resolution Žchromatographic profile. occurred in the immune yolks from individual birds ŽFig. 4B–C.. The elution pH’s ŽFig. 4A. for peaks 2 Ž6.09–6.66. and 3 Ž6.66–7.34. are basically the same as those from the nonimmune yolk.
Fig. 6. Immunoelectrophoresis of pooled immune yolks from IDA-Fe 3q chromatography. After electrophoresis for 45 min at 200 constant volts, each antiserum trough was filled with a 1:3 dilution of affinity purified rabbit anti chicken IgG H and L anti- serum. After overnight incubation, the IEP films were processed and then stained with coomassie brilliant blue R-250.
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ELISA activity, against the SE flagella, from the pooled yolks ŽFig. 7A. and the yolks from individual birds ŽFig. 7B–C., was detected in all four protein containing peaks with the highest activity being found in peaks 2–4. This would indicate that the column has also separated the IgG into subpopulations based upon activity. Although the SDS -PAGE ŽFig. 5. demonstrates the presence of other proteins in each peak, the IEP analysis ŽFig. 6. shows, as with the
nonimmune yolk, an electrophoretic mobility difference, which is clearly evident between peaks 1 and 4. Using 8 M urea, the relative avidity index ŽFig. 8. was determined for each peak using both pooled yolks and yolks from individual birds. From these results, it is evident that the column also separated the IgG into sub populations based upon the avidity index. IgG concentrations for both pooled yolks and yolks from individual birds ranged from 77.29 to
Fig. 7. ŽA. ELISA results from pooled immune yolks. ŽB–C. ELISA results from individual immune yolks. Peak 1 is the unabsorbed fraction. Peaks 2 and 3 elute during gradient formation while peak 4 is the chelate annihilation phase. For the respective chromatographs, see Fig. 4.
C.R. Greene, P.S. Holt r Journal of Immunological Methods 209 (1997) 155–164
163
Fig. 8. ŽA. Relative avidity index from immune yolk pools. ŽB–C. Relative avidity index from individual immune yolks. Subpopulation 1 is the unabsorbed fraction. Subpopulation 2 and 3 elute during gradient formation while subpopulation 4 is the chelate annihilation phase. For the respective chromatographs see Fig. 4.
113.65 m grml Ž"14.68., for peak 2 173.57–181.68 m grml Ž"3.28., for peak 3 82.71–100.18 m grml Ž"9.43. and for peak 4 116.31–124.48 m grml Ž"5.77.. 4. Discussion Previous investigators ŽMcCannel and Nakai, 1990. have shown that yolk IgG can be separated
into subpopulations using DEAE ion-exchange chromatography. This separation was brought about by using two gradual linear gradients of increasing phosphate ion concentrations while maintaining a constant pH. In their elution scheme, those antibodies that were found in the unabsorbed fraction had the highest specific activity against the immunizing antigen, while subsequent fractions, eluted during gradient formation, demonstrated less specific activ-
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ity. Using Immobilized Metal Ion ŽFe 3q . Affinity Chromatography, we have improved the separation of yolk IgG into subpopulations as indicated by: Ža. the yolk IgG is separated into four well resolved peaks or subpopulations Žb. specific ELISA activity increases with antibody elution from the column and Žc. the Fe 3q column separates the yolk IgG into subpopulations based upon the relative avidity index. Immobilized metal ion affinity chromatography is a protein purification technique based upon the affinity that proteins have for certain metals. This protein-metal interaction is brought about by the amino acids histidine, tryptophan and cysteine being located on the surface of the protein molecule ŽPorath and Olin, 1983., with histidine functioning as the predominant ligand in the IMAC of proteins ŽHemdan et al., 1989.. In contrast, Fe 3q, a hard Lewis acid, displays primarily ion-exchange properties Žcation and anion. ŽSulkowski, 1988.. It has been shown that Fe 3q has a strong affinity for phosphorylated amino acids and proteins ŽAndersson and Porath, 1986.. In the chromatography of model proteins, Sulkowski Ž1988. found that bound proteins eluted in order of their isoelectric points that is, at pH 6 Žapplication pH., proteins that carry a net negative charge will pass through the column, while those that possess a net positive charge at the application pH will be retained by the column. In the Fe 3q chromatography of yolk IgG, peak 1 Žunabsorbed fraction. would have the highest net negative charge while peaks 2 and 3 are retained by the column indicating that they have a more net positive charge. A unique feature of yolk IgG is the extreme binding of peak 4 to the Fe 3q column. This binding is similar to that observed for a murine ascitic IgG2b monoclonal antibody Žunpublished data.. An additional unique feature, is that peak 1 Žhighest net negative charge. and peak 4 Žhighest net positive charge. demonstrate the most immunoelectrophoretic mobility differences which has also been shown by Schlamowitz et al. Ž1975. in his separation of rabbit IgG into subpopulations. In the chromatography of yolk IgG, the Fe 3q column has also separated the IgG into four subpopulations according to the relative avidity index. In
birds immunized with highly purified protein preparations, this technique would have a direct industrial application since the highest avidity antibodies could be resolved and used in polyclonal diagnostics. This technique could also be used to monitor the effacy of oil-emulsion and recombinant vaccines and to identify the subpopulation to which the avian protective antibody belongs. References Akita, E.M., Nakai, S., 1992. Immunoglobulins from egg yolk: Isolation and purification. J. Food Sci. 57, 629. Andersson, L., Porath, J., 1986. Isolation of phosphoproteins by immobilized metal ŽFe 3q . affinity chromatography. Anal. Biochem. 154, 250. Chargelegue, D., O’Toole, C.M., Colvin, B.T., 1993. A longitudinal study of the IgG antibody response to HIV-1 p17 gag protein in HIV-1q patients with hemophilia: Titre and avidity. Clin. Exp. Immunol. 93, 331. Czerkinsky, C., Rees, A.S., Bergmeier, L.A., Challacombe, S.J., 1983. The detection and specificity of class specific antibodies to whole bacterial cells using a solid phase radioimmunoassay. Clin. Exp. Immunol. 53, 192. Fahey, J.L., McKelvey, E.M., 1965. Quantitative determination of serum immunoglobulins in antibody-agar plates. J. Immunol. 91, 84. Hemdan, E.S., Zhao, Y.J., Sulkowski, E., Porath, J., 1989. Surface topography of histidine residues: A facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. USA 86, 1811. Ibrahim, G.F., Fleet, G.H., Lyons, M.J., Walker, R.A., 1985. Method for the isolation of highly purified Salmonella flagellins. J. Clin. Micro. 22, 1040. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. McCannel, A.A., Nakai, S., 1990. Separation of egg yolk immunoglobulins into subpopulations using DEAE ion exchange chromatography. Can. Inst. Food Sci. Technol. J. 23, 42. Porath, J., Olin, B., 1983. Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities of gel-immobilized iron and nickel ions. Biochem. 22, 1621. Schlamowitz, M., Kaplan, M., Shaw, A.R., Tsay, D.D., 1975. Preparation and characterization of rabbit IgG fractions. J. Immunol. 114, 1590. Sulkowski, E., 1988. Immobilized metal ion affinity chromatograph of proteins on IDA-Fe 3q. Macromol. Chem. Macromol. Symp. 17, 335.
An improved chromatographic method for the separation of egg yolk IgG into subpopulations utilizing immobilized metal ion žFe 3q / affinity chromatography Cam R. Greene ) , Peter S. Holt U.S. Department of Agriculture, Agricultural Research SerÕice, Southeast Poultry Research Laboratory, 934 College Station Road, Athens, GA 30605, USA Received 19 November 1996; revised 2 September 1997; accepted 5 September 1997
Abstract An improved methodology is described for the separation of yolk IgG into subpopulations using immobilized metal ion ŽFe 3q . affinity chromatography. The yolk IgG was first extracted using a prechilled, pre-acidified method. After extraction, the yolk IgG was then fractionated using an Fe 3q column. Using an ascending pH gradient, four IgG containing peaks were well resolved based upon the elution pH, specific activity and the relative avidity index. q 1997 Elsevier Science B.V. Keywords: Immobilized metal ion affinity chromatography; Yolk IgG; Subpopulations
1. Introduction Various animal species possess IgG subclasses with the exception of the chicken and the rabbit. Many IgG subclasses can be separated using protein
Abbreviations: AI, avidity index; ELISA, enzyme-linked immunosorbent assay; HEPES, N-w2-hydroxethylxpiperazine-N-w2ethanesulfonic acidx; IDA, imino-diacetic acid epoxy-activated Sepharose 6B; IMAC, immobilized metal ion affinity chromatography; IEP, immunoelectrophoresis; MES, 2-w N-morpholinex ethane sulfonic acid; PB, phosphate buffer; PBST, phosphate buffered salineqTween-20; RID, radial immunodiffusion; SE, Salmonella enteritidis; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis ) Corresponding author. Tel.: q1-706-5463441; fax: q1-7065463161.
A or DEAE ion-exchange chromatography. Because of its failure to bind to protein A, egg yolk IgG has been separated into subpopulations using DEAE ion-exchange ŽMcCannel and Nakai, 1990.. This was accomplished by maintaining a constant pH while increasing the phosphate ion concentration. Immobilized metal ion affinity chromatography ŽIMAC. is a protein purification technique based upon the affinity that proteins have for certain metals. This metal-protein binding is mediated through the presence of the amino acids histidine, tryptophan and cysteine being located on the surface of the protein molecule ŽPorath and Olin, 1983.. Elution of bound proteins from an IMAC column is usually brought about by lowering the pH, or increasing the salt concentration, or by both. The most commonly used metals in IMAC chromatography include Cu2q,
0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 1 5 5 - 5
156
C.R. Greene, P.S. Holt r Journal of Immunological Methods 209 (1997) 155–164
Ni 2q, Co 2q and Zn2q, while the Fe 3q cation occupies a special niche in IMAC chromatography. The Fe 3q ion has been shown to have a very strong affinity for phosphorylated amino acids and proteins ŽAndersson and Porath, 1986.. Using an Fe 3q column, Sulkowski Ž1988. found that Fe 3q can act as a carboxymethyl ŽCM. exchanger and that model proteins elute in order of their isoelectric points. Using this special property of Fe 3q, we were able to separate yolk IgG into four well resolved subpopulations based upon the elution pH and the relative avidity index.
2. Materials and methods 2.1. Immunizations Ten specific-pathogen-free White Leghorn laying hens Ž5 birds per experiment. were each immunized, subcutaneously, with an acetone-killed preparation of Salmonella enteritidis ŽSE. which was mixed with an equal volume of an incomplete adjuvant composed of mineral oil, Arlacel A and Tween-80. Each hen received a booster injection six weeks after the initial immunization. Eggs were collected three weeks after the booster injection and were stored at 48C until used. All animals used in this study were treated in a manner consistent with USDA animal welfare guidelines. 2.2. Bacterial growth conditions Stock cultures of SE, phage type 13A, were maintained at y208C in 15% Žvrv. glycerol-tryptic soy broth ŽDifco Laboratories, Detroit, MI.. For the flagella extraction, a stock culture was thawed and streaked on a nutrient agar plate ŽDifco. to obtain well isolated colonies. After growth at 378C for 18–24 h, individual colonies were transferred to U-shaped glass tubes ŽFisher Scientific, Atlanta, GA. containing GI motility medium ŽDifco. and were passaged five times to obtain highly motile SE which were then used to inoculate 6 = 500 ml of the chemically defined minimal medium of Ibrahim et al. Ž1985.. These cultures were then incubated at 358C
in an orbital shaker incubator ŽPrecision Scientific, Chicago, IL. at 80 rpm for 16 h. 2.3. Flagella extraction and purification Flagella were extracted by the method of Ibrahim et al. Ž1985.. Briefly, Salmonella cells were harvested by centrifugation and resuspended in a saline solution. The pH was then adjusted to 2 and was maintained at that pH under constant stirring for 30 min at room temperature. After centrifugation, the supernatant was ultracentrifuged at 100 000 = g for 1 h at 48C. The pH of the resulting supernatant was adjusted to 7.2 and solid ammonium sulfate was added to two-thirds saturation Ž2.67 M. to precipitate the flagella. After centrifugation, the resulting pellet was resuspended and dialyzed against distilled water using 50 000 molecular weight cutoff dialysis tubing ŽSpectrum Medical Industries, Houston, TX.. Final purification was achieved using CL-6B gel-filtration chromatography ŽPharmacia Biotech, Piscataway, NJ.. Purity of the flagella preparation was ascertained by SDS-PAGE while the total protein was determined using the BCA protein assay ŽPierce Chemical Co., Rockford, IL.. The flagella were stored at 48C in 20 mM PBS pH 7.5. 2.4. Egg yolk IgG extraction Egg yolk IgG was extracted by a previously described procedure ŽAkita and Nakai, 1992.. Briefly, 20 ml of yolk was added to 180 ml of prechilled, pre-acidified distilled water Žacidified with 1 N HCl to obtain a final pH of 5–5.2 after dilution.. This solution was then incubated overnight at 48C to allow settling of insoluble material. After settling, a portion of the supernatant was then centrifuged, at 15 000 rpm, in a microcentrifuge for 15 min at 48C. Following centrifugation, the supernatant was filtered through a 0.22 m m syringe filter ŽMillipore, Bedford, MA.. 2.5. Iron chelate chromatography Iminodiacetic acid ŽIDA . epoxy-activated Sepharose 6B ŽPharmacia Biotech. was poured into a
C.R. Greene, P.S. Holt r Journal of Immunological Methods 209 (1997) 155–164
G10 = 150 Moduline medium pressure laboratory column ŽAmicon, Beverly, MA. fitted with an adjustable end piece to obtain a final packed bed volume of 7.5 ml. Following a deionized water wash, the column was converted to the IDA-Fe 3q form by charging with aqueous FeCl 3 P 6H 2 O Ž500 mg in 100 ml deionized water. ŽSulkowski, 1988.. After charging, the column was washed with deionized water and then equilibrated with 50 mM 2-w Nmorpholinox ethane-sulfonic acid pH 6 ŽMES. ŽSigma. ŽSulkowski, 1988.. At the completion of each run, the IDA gel was stripped Žchelate annihilation. with 50 mM EDTAq 500 mM NaCl pH 7 and regenerated as previously described. 2.6. Separation of yolk IgG into subpopulations A 5 ml sample of supernatant from the yolk IgG extraction procedure was transferred to SpectrarPor 6 dialysis tubing ŽSpectrum Medical Industries, Los Angeles, CA. and dialyzed overnight at 48C, with constant stirring, against 500 ml of 50 mM MES pH 6 buffer ŽIDA-Fe 3q equilibration buffer.. Following dialysis, the material was applied to a previously equilibrated IDA-Fe 3q column. After sample application, the column was washed with the equilibration buffer until the unabsorbed peak returned to the baseline. Bound immunoglobulin was then eluted using an ascending linear pH gradient consisting of 100 ml IDA-Fe 3q equilibration buffer and 100 ml of 50 mM N-w2-hydroxethylxpiperazine-N X-w2 -ethanesulfonic acidx pH 8 ŽHEPES. ŽSigma. ŽSulkowski, 1988.. Following gradient completion, the column was washed with 50 mM HEPES pH 8 and then subjected to chelate annihilation using 50 mM EDTAq 500 mM NaCl pH 7. 2.7. ELISA Purified SE flagella were bound to the solid phase using methyl glyoxal ŽCzerkinsky et al., 1983.. The flagella were diluted to 1 m grml using 0.3% Žvrv. methyl glyoxal pH 7 and 150 m l were added to the appropriate wells of Immulon 4 microtiter plates ŽDynatech Laboratories, Chantilly, VA.. The plates were then incubated for 2 h at 378C. Following incubation, the plates were twice washed with 20
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mM PBS pH 7.4 and then blocked for 1 h at 378C with 250 m lrwell of 100 mM PB q 1% Žwrv. polyvinylpyrrolidone ŽSigma. pH 8. After blocking, the plates were washed twice with 20 mM PB q 0.05% Žvrv. Tween-20 pH 7.4 ŽPBST. and then two-fold serial dilutions, diluted in PBST, of each eluted subpopulation Ž150 m lrwell. were added to the appropriate wells of the microtiter plate and then incubated for 90 min at 378C. The plate was washed twice with PBST and then 150 m lrwell of alkaline phosphatase labeled affinity purified rabbit anti chicken IgG-Fc specific antiserum ŽJackson ImmunoResearch Laboratories, West Grove, PA., diluted 1:5000 in PBST, was added to the plate and incubated for 90 min at 378C. The plate was washed twice with PBST and then 150 m lrwell of 100 mM diethanolamine buffer pH 9.8 containing 1 mgrml p-nitrophenyl phosphate substrate ŽSigma. was added to each well. Color development was allowed to proceed for 30 min at room temperature and was then stopped by the addition of 50 m lrwell of 3 N NaOH. Absorbance was measured at 405 nm using a Multiscan MS microtiter plate reader ŽLabsystems, Needham Heights, MA.. 2.8. AÕidity index The avidity index ŽAI. was determined for each subpopulation using 8 M urea as previously described by Chargelegue et al. Ž1993.. Flagella, diluted to 1 m grml in 100 mM bicarbonate–carbonate coating buffer pH 9.6, was bound to the solid phase by overnight incubation at 48C. Following incubation, the microtiter plate was then blocked for 1 h at 378C as previously described. By prior titering, each eluted IgG subpopulation Žpeak. was diluted in PBST to give an OD405 nm reading of 1.0 " 0.1. Four replicates of each diluted subpopulation Žpeak. were allowed to react with the immobilized flagella by overnight incubation at 48C. The plates were then washed with PBST and one duplicate received 100 m l of 8 M urea in PBS while the other duplicate received 100 m l of PBST. Following incubation for 15 min at room temperature, the plate was then washed six times with PBST and 100 m l of alkaline phosphatase labelled affinity purified rabbit anti chicken IgG-Fc specific antiserum ŽJackson., diluted
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1:5000 in PBST, was added to each replicate and allowed to react for one hour at 378C. After twice washing the plate, each duplicate received 100 m l of 100 mM diethanolamine buffer pH 9.8 containing 1 mgrml p-nitrophenyl phosphate substrate ŽSigma.. Color development, after proceeding for 30 min at room temperature, was stopped by the addition of 50 m lrwell of 3 N NaOH. Absorbance was measured at 405 nm using a Multiscan MS microtiter plate reader ŽLabsystems.. The Avid-
ity Index ŽAI. was then calculated using the following formula: AI s ŽOD405 nm 8 M urearOD405 nm PBST control. = 100. 2.9. Immunoelectrophoresis Immunoelectrophoresis ŽIEP. was performed in 1% Žwrv. agarose ŽType II, Medium EEO, Sigma. in a Tris-Tricine buffer pH 8.6. Samples Ž5 m l. were electrophoresed in an EC 360 flatbed apparatus ŽE-C Apparatus Corp., St. Petersburg, Fl. at 200 constant
Fig. 1. ŽA. IDA-Fe 3q chromatography of pooled nonimmune yolks from 5 birds. ŽB–C. IDA-Fe 3q chromatography of nonimmune yolks from individual birds. In all chromatography runs, a 5 ml sample was applied to a previously equilibrated IDA-Fe 3q column at a flow rate of 1 mlrmin while the fraction collector was operated in the time collection mode Ž5 minrtube. and a sensitivity setting of 0.1 AUFS. Peak 1 is the unabsorbed fraction. Peaks 2 and 3 elute during gradient formation while peak 4 is the chelate annihilation phase.
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2.10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Fig. 2. SDS-PAGE of pooled nonimmune yolks from IDA-Fe 3q chromatography. Samples Ž5 m grlane. were electrophoresised at 200 constant volts. Lane 1: molecular weight markers; lane 2: purified chicken IgG ŽJackson.; lane 3: peak 1; lane 4: peak 2; lane 5: peak 3; lane 6: peak 4r
volts for 45 min. After electrophoresis, the IEP films were incubated overnight in a humidity box and then processed and stained with 0.1% Žwrv. coomassie brilliant blue R-250 ŽSigma..
SDS-PAGE ŽLaemmli, 1970. was conducted using a vertical mini-gel electrophoresis unit ŽMighty Small II, Hoefer Scientific Instruments, San Francisco, CA.. Samples Ž5 m g. were dissolved in solubilization buffer and boiled for 90 s. They were then applied to a 0.75 mm thick gel of 12% poly- acrylamide with a 4% stacking gel and electrophoresed at 200 constant volts. The gel was then stained with coomassie brilliant blue R-250 ŽSigma., and apparent molecular weights were interpolated from the migration of phosphorylase Ž97.4 kDa., bovine serum albumin Ž66.2 kDa., ovalbumin Ž42.6 kDa., bovine carbonic anhydrase Ž31 kDa., soybean trypsin inhibitor Ž21.5 kDa. and lysozyme Ž14.4 kDa.. 2.11. Radial immunodiffusion Radial immunodiffusion ŽRID. ŽFahey and McKelvey, 1965. were done on calibrated immunodiffusion plates ŽICN Immunobiological, Lisle, IL. of 1% Žwrv. agarose ŽType II, Medium EEO, Sigma. in 30 mM PBS pH 8. Zone diameters were measured to the nearest 0.1 mm using a QUIPmeter ŽHelena Laboratories, Beaumont, TX..
Fig. 3. Immunoelectrophoresis of pooled nonimmune yolks from IDA-Fe 3q chromatography. Samples Ž5 m l. were electrophoresised for 45 min. After electrophoresis, each antiserum trough was filled with a 1:3 dilution of affinity purified rabbit anti chicken IgG H and L chain antiserum. After overnight incubation, the IEP films were processed and then stained with coomassie brilliant blue R-250.
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3. Results 3.1. IDA-Fe 3 q chromatography (nonimmune yolk) Prior to immunization, control or nonimmune eggs were collected from each bird. These eggs Žyolks. were subsequently pooled and a crude IgG fraction was obtained using the prechilled, pre-acidified methodology. A 5 ml sample of this crude extract was then fractionated on a previously equilibrated IDA-Fe 3q column ŽFig. 1.. Using an ascending lin-
ear pH gradient, four protein containing peaks were eluted: Peak 1 is unabsorbed fraction, peaks 2 and 3 elute during gradient formation, while peak 4 elutes during the chelate annihilation phase. This basic chromatographic profile is also exhibited in yolks from individual birds ŽFig. 1B–C. with some variation occurring in the heights of peaks 2 and 3. Analysis of elution pH ŽFig. 1A. indicates that peak 2 elutes from pH 6.19–6.72 while peak 3 elutes from 6.72–7.44. This indicates that the column has separated the IgG fraction into four subpopulations based
Fig. 4. ŽA. IDA-Fe 3q chromatography of pooled immune yolks from 5 birds. ŽB–C. IDA-Fe 3q chromatography of immune yolk from individual birds. In all chromatography runs, peak 1 is the unabsorbed fraction. Peaks 2 and 3 elute during gradient formation. Peak 4 is the chelate annihilation phase.
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umn can also bind and separate these additional yolk proteins which could then be further purified using a variety of chromatographic techniques. Examination of each IgG subpopulation by immunoelectrophoresis ŽFig. 3. demonstrates an electrophoretic mobility difference. This difference is clearly demonstrable between peaks 1 and 4 while minor differences exist between peaks 2 and 3. The IgG content of each peak was determined by RID’s: Peak 1 ranged from 72.86–80.81 m grml Ž"3.34; standard deviation., peak 2 ranged from 117–133 m grml Ž"11.73., peak 3 ranged from 77–113 m grml Ž"13.18., while peak 4 ranged from 98–106 m grml Ž"2.31.. 3.2. IDA-Fe 3 q chromatography (immune yolk) 3q
Fig. 5. SDS-PAGE of pooled immune yolks from IDA-Fe chromatography. Samples Ž5 m g. were electrophoresised at 200 constant volts. Lane 1: molecular weight markers; lane 2: purified chicken IgG; lane 3: whole yolk extract; lane 4: peak 1; lane 5: peak 2; lane 6: peak 3; lane 7: peak 4.
upon the elution pH. After chromatography, each peak was concentrated and subjected to SDS-PAGE ŽFig. 2. and IEP ŽFig. 3. analysis. Since the SDSPAGE ŽFig. 2. confirms the presence of other proteins, besides IgG, this would indicate that the col-
The crude IgG extract from five birds Žpooled yolks. immunized with an SE bactrin was chromatographed on a previously equilibrated IDA-Fe 3q ŽFig. 4A. column. As before, using an ascending linear pH gradient, four protein containing peaks were resolved. This same peak resolution Žchromatographic profile. occurred in the immune yolks from individual birds ŽFig. 4B–C.. The elution pH’s ŽFig. 4A. for peaks 2 Ž6.09–6.66. and 3 Ž6.66–7.34. are basically the same as those from the nonimmune yolk.
Fig. 6. Immunoelectrophoresis of pooled immune yolks from IDA-Fe 3q chromatography. After electrophoresis for 45 min at 200 constant volts, each antiserum trough was filled with a 1:3 dilution of affinity purified rabbit anti chicken IgG H and L anti- serum. After overnight incubation, the IEP films were processed and then stained with coomassie brilliant blue R-250.
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ELISA activity, against the SE flagella, from the pooled yolks ŽFig. 7A. and the yolks from individual birds ŽFig. 7B–C., was detected in all four protein containing peaks with the highest activity being found in peaks 2–4. This would indicate that the column has also separated the IgG into subpopulations based upon activity. Although the SDS -PAGE ŽFig. 5. demonstrates the presence of other proteins in each peak, the IEP analysis ŽFig. 6. shows, as with the
nonimmune yolk, an electrophoretic mobility difference, which is clearly evident between peaks 1 and 4. Using 8 M urea, the relative avidity index ŽFig. 8. was determined for each peak using both pooled yolks and yolks from individual birds. From these results, it is evident that the column also separated the IgG into sub populations based upon the avidity index. IgG concentrations for both pooled yolks and yolks from individual birds ranged from 77.29 to
Fig. 7. ŽA. ELISA results from pooled immune yolks. ŽB–C. ELISA results from individual immune yolks. Peak 1 is the unabsorbed fraction. Peaks 2 and 3 elute during gradient formation while peak 4 is the chelate annihilation phase. For the respective chromatographs, see Fig. 4.
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Fig. 8. ŽA. Relative avidity index from immune yolk pools. ŽB–C. Relative avidity index from individual immune yolks. Subpopulation 1 is the unabsorbed fraction. Subpopulation 2 and 3 elute during gradient formation while subpopulation 4 is the chelate annihilation phase. For the respective chromatographs see Fig. 4.
113.65 m grml Ž"14.68., for peak 2 173.57–181.68 m grml Ž"3.28., for peak 3 82.71–100.18 m grml Ž"9.43. and for peak 4 116.31–124.48 m grml Ž"5.77.. 4. Discussion Previous investigators ŽMcCannel and Nakai, 1990. have shown that yolk IgG can be separated
into subpopulations using DEAE ion-exchange chromatography. This separation was brought about by using two gradual linear gradients of increasing phosphate ion concentrations while maintaining a constant pH. In their elution scheme, those antibodies that were found in the unabsorbed fraction had the highest specific activity against the immunizing antigen, while subsequent fractions, eluted during gradient formation, demonstrated less specific activ-
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ity. Using Immobilized Metal Ion ŽFe 3q . Affinity Chromatography, we have improved the separation of yolk IgG into subpopulations as indicated by: Ža. the yolk IgG is separated into four well resolved peaks or subpopulations Žb. specific ELISA activity increases with antibody elution from the column and Žc. the Fe 3q column separates the yolk IgG into subpopulations based upon the relative avidity index. Immobilized metal ion affinity chromatography is a protein purification technique based upon the affinity that proteins have for certain metals. This protein-metal interaction is brought about by the amino acids histidine, tryptophan and cysteine being located on the surface of the protein molecule ŽPorath and Olin, 1983., with histidine functioning as the predominant ligand in the IMAC of proteins ŽHemdan et al., 1989.. In contrast, Fe 3q, a hard Lewis acid, displays primarily ion-exchange properties Žcation and anion. ŽSulkowski, 1988.. It has been shown that Fe 3q has a strong affinity for phosphorylated amino acids and proteins ŽAndersson and Porath, 1986.. In the chromatography of model proteins, Sulkowski Ž1988. found that bound proteins eluted in order of their isoelectric points that is, at pH 6 Žapplication pH., proteins that carry a net negative charge will pass through the column, while those that possess a net positive charge at the application pH will be retained by the column. In the Fe 3q chromatography of yolk IgG, peak 1 Žunabsorbed fraction. would have the highest net negative charge while peaks 2 and 3 are retained by the column indicating that they have a more net positive charge. A unique feature of yolk IgG is the extreme binding of peak 4 to the Fe 3q column. This binding is similar to that observed for a murine ascitic IgG2b monoclonal antibody Žunpublished data.. An additional unique feature, is that peak 1 Žhighest net negative charge. and peak 4 Žhighest net positive charge. demonstrate the most immunoelectrophoretic mobility differences which has also been shown by Schlamowitz et al. Ž1975. in his separation of rabbit IgG into subpopulations. In the chromatography of yolk IgG, the Fe 3q column has also separated the IgG into four subpopulations according to the relative avidity index. In
birds immunized with highly purified protein preparations, this technique would have a direct industrial application since the highest avidity antibodies could be resolved and used in polyclonal diagnostics. This technique could also be used to monitor the effacy of oil-emulsion and recombinant vaccines and to identify the subpopulation to which the avian protective antibody belongs. References Akita, E.M., Nakai, S., 1992. Immunoglobulins from egg yolk: Isolation and purification. J. Food Sci. 57, 629. Andersson, L., Porath, J., 1986. Isolation of phosphoproteins by immobilized metal ŽFe 3q . affinity chromatography. Anal. Biochem. 154, 250. Chargelegue, D., O’Toole, C.M., Colvin, B.T., 1993. A longitudinal study of the IgG antibody response to HIV-1 p17 gag protein in HIV-1q patients with hemophilia: Titre and avidity. Clin. Exp. Immunol. 93, 331. Czerkinsky, C., Rees, A.S., Bergmeier, L.A., Challacombe, S.J., 1983. The detection and specificity of class specific antibodies to whole bacterial cells using a solid phase radioimmunoassay. Clin. Exp. Immunol. 53, 192. Fahey, J.L., McKelvey, E.M., 1965. Quantitative determination of serum immunoglobulins in antibody-agar plates. J. Immunol. 91, 84. Hemdan, E.S., Zhao, Y.J., Sulkowski, E., Porath, J., 1989. Surface topography of histidine residues: A facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. USA 86, 1811. Ibrahim, G.F., Fleet, G.H., Lyons, M.J., Walker, R.A., 1985. Method for the isolation of highly purified Salmonella flagellins. J. Clin. Micro. 22, 1040. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. McCannel, A.A., Nakai, S., 1990. Separation of egg yolk immunoglobulins into subpopulations using DEAE ion exchange chromatography. Can. Inst. Food Sci. Technol. J. 23, 42. Porath, J., Olin, B., 1983. Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities of gel-immobilized iron and nickel ions. Biochem. 22, 1621. Schlamowitz, M., Kaplan, M., Shaw, A.R., Tsay, D.D., 1975. Preparation and characterization of rabbit IgG fractions. J. Immunol. 114, 1590. Sulkowski, E., 1988. Immobilized metal ion affinity chromatograph of proteins on IDA-Fe 3q. Macromol. Chem. Macromol. Symp. 17, 335.