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Dual Microcolumn Immunoaffinity Liquid Chromatography: An Analytical Application to Human Plasma Proteins Cheryl L. Flurert and Milos Novotny' Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Duaccolumnmlcrocdumn hmuroallkrlty chromatographywas accompbhod by coup#ngImmunoaMnlty (IAC) and reversedphase high-performance llquld chromatography (RP-HPLC) analyrk columns. The IAC support was prepared by hydrophobkally adeorblng the approprlate antibody onto an odyl rlllca-basedstatlonary phase containedIn a 2 5 0 - ~ 1 . d . fused-sl11ca microcolumn. Once the antlgen was captured, the antigen-subtracted human plasma could be sent to the RP-HPLC microcolumnfor plasma proflllng. The antigen was quantitated by desorbing it from the IAC column and transferring it to the RP-HPLC system. Humanplasma proflies were generated aner removal of albumin, and transferrin and af-antitryprln were quantitated with nanogram sensitlvlty. All analyses were accomplished with less than 1 pL of human plasma.
INTRODUCTION Human plasma contains well over 100proteins that perform specific functions. Examples of these proteins include enzymes, components of the immune system, factors in blood coagulation, or transport proteins for vital metabolites, metal ions, hormones, and lipids. Due to the myriad of functions such proteins perform, changes in their concentrations, both total and relative, are often checked when a disease is suspected. Clinical chemists routinely use either agarose' or cellulose acetate2 gel electrophoresis to produce plasma profiles, although researchers have generated protein profiles from capillary electrophoresis (CE),3high-performance liquid chromatography (HPLC),4-6and a combination of CE and HPLC.' If analysis of whole serum/plasma or individual proteins is required, immunoelectrophoretic techniques198are often used. However, proteins present a t lower concentrations, such as complement components and coagulation factors, are currently quantitated with the more sensitive immunoassaya.g Recently, a highly selective affinity method that combines the principles of immunological interactions and highperformance chromatography has been introduced and is called,appropriately, immunoaffinity chromatography (IAC).
* To whom all correspondence should be addressed.
+ Present address: National Forensic Chemistry Center, U.S.Food and Drug Administration, 1141 Central Parkway, Cincinnati, OH 45202. (1)Grant, G. H.;Kachmar, J. F. In Fundamentals of Clinical Chemistry,2nd ed.; Tietz, N. W., Ed.; W. B. Saunders Co.: Philadelphia, 1982; pp 298-376. (2)Killingsworth, L. M. High Resolution Protein Electrophoresis: A Clinical Overview with Case Studies; Helena Laboratories: Beaumont, TX. - - -, 1QAFi. -(3) Manabe, T.; Yamamoto, H.; Okuyama, T. Electrophoresis 1989, 10,172. (4) Schlabach, T. D.; Abbott, S. R. Clin. Chem. 1980,26,1504. (5) Tomono, T.; Ikeda, H.; Tokunaga, E. J.Chromatogr. 1983,266,39. (6)Chang, S.H.; Gooding, K. M.; Regnier, F. E. J. Chromatogr. 1976, 125,103. (7)Yamamoto, H.; Manabe, T.; Okuyama, T. J. Chromatogr. 1990. 515,659. (8) Clarke, H. B. M.; Freeman, T. Clin. Sci. 1968,35, 403. (9)Monroe, D. Anal. Chem. 1984,56,920A.
0003-2700/93/0365-0817$04.00/0
The use of IAC has been extensive in the isolation of proteins for further study, such as a-fetoprotein,l0 IgG," IgE,12 transferrin,13J4 human growth hormone,15 anti-iodotypic antibodies,16 j3z-microglobulin,17and a variety of membrane receptors.18Jg The goal of this article is to describe the development and use of IAC systems a t a much reduced scale. When required, individual proteins can be targeted for quantitation and/or isolation for structural characterization. Because desorption kinetics between the antibody and antigen are slow and lead to broad peaks,18 a subsequent reversed-phase HPLC (RPHPLC) s y ~ t e mis~ used ~ , ~to~ concentrate the antigen and provide sharper peaks for easier detection and quantitation. In addition, subsequent separations provide profiles of human plasma that could potentially be used in diagnosticprocedures, while utilizing the highly sensitive detection capabilities and low sample volume requirements inherent to microcolumn HPLC.
EXPERIMENTAL SECTION Reagents. HPLC grade acetonitrile and trifluoroacetic acid (TFA)were obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). HPLC grade HPOland biotech grade glycine were obtained from Fisher Scientific (Fair Lawn, NJ). All other materials used were reagent grade. Distilled and deionized water was obtained locally. Mobile phases were filtered through 0.2-pm Nylon 66 membrane filters (Millipore Corp., Bedford, MA). Human transferrin (hTf, No. T-24031, human al-antitrypsin (halAT, No. A-9024), human albumin (hAlb, No. A-3782), conalbumin (CON), and lysozyme (LYZ)were purchased from Sigma Chemical Co. (St. Louis, MO). Normal human plasma, from a pool of adult donors, was obtained as the lyophilized powder of a 10-mLaliquot from Calbiochem (La Jolla, CA). Goat anti-human transferrin (anti-hTf,No. T-6265),goat anti-human albumin (anti-hAlb, No. A-11511, and rabbit anti-human alantitrypsin (anti-halAT, No. A-0409), also from Sigma, were obtained as lyophilized powders of the immunoglobulinG (IgG) fraction of antiserum. They were reconstituted in 2.0 mL of distilledHzO and filtered through 0.22-pm Millex-GV low proteinbinding syringe filters (Millipore). Preparation of Hydrophobic-Adsorption IAC Columns. Chromegabond MC-8 (30 cm X 250-pm i.d.1 (ES Industries, Marlton, NJ) octyl columns were equilibrated with 0.10 M H3PO4-0.10 M KHzP04 buffer, pH 2. An aliquot of the appropriate (10)StefanovB,I.; HorejZiI,V.; KrigtofovB,H.; Angelisov&P.; pikovsky, V.; Hilgert, I. J. Immunol. Methods 1988, 111, 67. (11)Hage, D. S.;Walters, R. R. J. Chromatogr. 1987,386,37. (12)Phillips, T. M.; More, N. S.; Queen, W. D.; Thompson, A. M. J. Chromatogr. 1985,327,205. (13)Ohlson, S.;Gudmundsson, B. M.; Wikstroem, P.; Larsson, P. 0. Clin. Chem. 1988,34,2039. (14)Janis, L. J.; Regnier, F. E. A d . Chem. 1989,61,1901. (15)Lejeune, R.; Thunus, L.; Gomez, F.; Frankenne, F.; Cloux, J.-L.; Hennen, G. Anal. Biochem. 1990,189,217. (16)Phillips, T.M. Clin. Chem. 1988,34,1689. (17)Mogi, M.; Harada, M.; Adachi, T.; Kojima, K.; Nagatsu, T. J. Chrornatogr. 1989,496,194. (18)Phillips, T. M. In The Use of HPLC in Receptor Biochemistry; Kerlavage, A. R., Ed.; Alan R. Lias, Inc.: New York, 1989;pp129-154 (see also references therein). (19)Phillips, T. M.J. Chromatogr. 1991,550,741. (20)Janis, L. J.; Regnier, F. E. J. Chromatogr. 1988,444, 1. 0 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993
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Flgurr 1. Schematic of the apparatus used In duakolumn Immunoafflnlty chromatographlcstudies. An expianatlon of the symbols Is found In the text.
IgG solution was added to a microreservoir tube and was flushed onto the column at a flow rate between 2 and 3 pL/min. Column saturation was indicated by the observation of a concentration plateau on the chart recorder paper. The microreservoir tube was removed, and saturation was verified by the injection of 200-nL aliquots of the IgG solution, which repeatedly yielded peaks of the same height. Using frontal analysis?' it was determined that, typically, 600-700 pg of IgG were adsorbed. The column then was equilibrated with the sample adsorption buffer,O.lOM KH2PO&lO MK2HP04,pH 7. Amicroreservoir tube was filled with a 10 mg/mL solution of either CON or LYZ and was flushed through the IgG column to coat any exposed nonspecific binding sites.2o Saturation was verified by 200-nL injections of the appropriate solution. IAC Apparatus. The experimental apparatus for the IAC studies is shown in Figure 1. Pump C (Model pLC-500, Isco, Inc., Lincoln, NE) contained the sample adsorption buffer, and reservoir R contained the sample desorption buffer. Valve VI (Model ECSD4UW, Valco Instruments, Houston, TX) could be switched to allow either the sample adsorption buffer (position 1)or the sample desorption buffer (position 2) to flow through the IAC column. The two-wayvalve VZ(ScientificSystems, Inc., State College,PA) was closed when V1was in position 1and open when in position 2. The buffers were routed through manifold M (Valco,Model Z6M1) into the "pump port" of the electricallyactuated injector V3 (Valco, Model ECIIW), fitted with a 0.2-clL rotor. V3 was used to load the plasma onto the IAC column. During sample analysis, the eluent from the IAC column was delivered to the RP-HPLCanalysiscolumnusing 50-clrn4.d.fused silica that connected the outlet of the IAC column to the inlet port of valve Vg (Valco,Model ECSTGUW). The gradients were generated by pumps A and B using a modifiedz3commercial mixing device (a Lee Micro-miser, Lee Co., Westbrook, CT). The splitter required a 50-cm segment of 25-pm4.d. fused silica to obtain a 1O:l ratio. The flow rate through the RP-HPLC column was between 2 and 3 pL/min. Detection occurred at 215 nm with an Isco pLC-10 detector. IAC Studies. When antigen-subtracted plasma analysis was desired, the plasma was loaded onto the IAC column with sample (21)Jacobson, J.; Frenz, J.; Horvhth, Cs.J.Chromatogr. 1984,316,53. (22) Flurer, C. L.; Borra, C.; Andreolini, F.; Novotny, M. V. J. Chromatogr. 1988,448, 73. (23)Banks, J. F., Jr.; Novotny, M. Unpublished experiments.
adsorption buffer. The eluent, containing all plasma proteins except the antigen, was directed to VS. After the RP-HPLC column was equilibrated with the eluent, ensuring protein adsorption,the columnwas reequilibrated with the initial mobile phase, and the gradient program was initiated. Quantitation of the antigen required its desorption from the IAC column. VI was switched to position 2, and VZwas opened so that the desorption buffer could flow through the IAC column and onto the RP-HPLC column via v& Again, once this was accomplished,the RP-HPLC analysis column was reequilibrated with the initial mobile phase required for gradient analysis, and the gradient program was initiated. RP-HPLC Analysis Columns. Reversed-phase studies utilized 30-cm X 250-pm-i.d. fused-silica (Polymicro Technologies, Phoenix, AZ) microcolumns packed with Chromegabond MC-8. The proceduresfor the preparation of these columns are described elsewhere.22RP-HPLC analyses were carried out using CH&N gradients in 0.1 % TFA. Sample adsorption for all experiments required 0.10 M KHzP044.10 M KzHPO,, pH 7. The sample desorption solutionsutilizedwere 0.032 M glycine4032 M NaCl0.068 M HC1 buffer, pH 2, for anti-hTf studies, and 0.1% TFA, pH 2, for anti-halAT analyses. Antigen calibration studies occurred off-line with the RPHPLC analysiscolumn. The mixing device was attached directly to the splitter tee, and the third port of the splitter was connected to an injectorvalve (ValcoModel ECI4W). The antigen standard solutions were injected onto the column using a 0.2-pL rotor and were detected at 215 nm.
RESULTS AND DISCUSSION The previous development of microcolumn RP-HPLC methods22 introduced the possibility of hydrophobically adsorbing immunoglobulins onto the lipophilic octyl stationary phase. The aqueous acidic buffer that is used to immobilize antibodies onto other IAC adsorbents14 mimics the typical conditionsused as the initial mobile phase in RPHPLC. The hydrophobic moieties of the IgG molecule should adsorb to the octyl stationary phase and remain there under the aqueous buffer conditions used to perform IAC analyses. In contrast, although the Protein G system described by Janis and RegnierI4yielded excellent results, the antibody desorbed with the antigen by experimental design, requiring the continual preparation of the IAC column before each run. The on-column hydrophobic adsorption procedure also allows the initial packing of the column under optimum conditions without concern for deactivation of the IgG. When covalent immobilization is used, the antibody must be attached to the solid support before the chromatographic column is packed. Under these circumstances, great care must be taken when choosing solvent and pressure conditions for column packing to avoid deactivation of the antibody.'* Although IAC interactions are highly selective, the technique suffersfrom the fact that adeorption/desorption kinetics between the antibody and the antigen can be very s ~ o w . ~ ~ Broad chromatographic peaks result, and quantitation is difficult. A dual-column system, however, offers improved ~ ~ ~antigen ~~ can resolution and ease of q ~ a n t i t a t i o n . 1 * -The be desorbed from the IAC column and concentrated onto an analytical column. This approach allows not only a more direct antigen quantitation through the use of calibration plots but also a choice of chromatographic schemes for the analytical system to optimize separation of antigen isoenzymes, subunits, or microheterogeneous forms. (24) Rybacek, L.; D'Andrea, M.; Tarnowski, S. J. J.Chromatogr. 1987, 397, 355. (25)Gianazza, E.;Aruaud, P. Biochem. J. 1982,201, 129.
(26)Gianazza, E.;Aruaud, P. Biochem. J. 1982,203,637. (27)Affi-Gel Blue Affinity Chromatography Gel forEnzymeand Blood Proteinhrifications. Bulletin 1107;Bio-Rad Laboratories: Richmond, CA, 1986.
ANALYTICAL CHEMISTRY, VOL. 85, NO. 8, MARCH 15, 1003 818
Static Properties of Microcolumn IAC Matrices. Maximum antigen capacity was determined with albumin. The albumin present in 1 pL of plasma, the equivalent of 35-60 e(g,28was enoughto saturate the anti-hAlbIAC column. This 109% antigen loading capacityhas been observed in other immunoaffinity systems11.20and is probably due to a combination of improper orientation of the IgG molecule on the octyl surface, and antibody deactivationby the acidic buffers used for IgG adsorption. This limits the “usable” IgG to 60 or 70 pg. Although the column lifetime was not determined, the microcolumns were used 15-20 times during method development before being discarded. On the basis of the size of the IgG peak in the blank, for example in Figure 5C, it is estimated that 500-700 ng of IgG are lost per analysis, which representa a 1% loss through column “bleeding”. Limiting column use to 20 runs would result in a 20% loss in antigen binding capacity, and is typical of IAC column lifetimes under acidic elution conditions.29 Generation of Plasma Profiles Following Albumin Removal. Albumin is an extraordinary protein in that it performs a multiplicity of functions. It transports large organic anions such as long-chain fatty acids and bilirubin, binds toxic heavy metal ions, transports poorly soluble hormones when the capacities of more specific binding proteins are exceeded, and provides a reserve store of protein.‘ A decrease in the albumin concentration (hypoalbuminemia) is very common in disease and can be indicative of malnutrition, liver disease, or an acute phase reaction, which occurs after tissue damage. Because of ita prevalence in plasma, albumin can often obscure the determination of certain minor proteins. Albumin can be removed from serum by using ita affinity for the dye cibacron blue F3GA. Cibacron blue has been used to purify a number of enzymes and plasma proteins.25,27 The adsorption of other plasma proteins to the dye could result in the loss of valuable information when it is used for bulk albumin removal, for example, as a precursor to further plasma analysis or protein isolation. As shown here and elsewhere,” a more precise removal of serum albumin was accomplished using immunoaffmity interactions. An anti-hAlb IAC column was prepared in order to demonstrate the ease of albumin removal, in contrast to the cibacron blue method. Figure 2 is an example of the RPHPLC analysis of 600 nL of plasma with hAlb present (2A) and 400 nL of plasma after passing through the anti-hAlb IAC column (2B).Removal of hAlb allows an increase in detection sensitivity if necessary, which permita the visualization of plasma proteins that are otherwise obscured by hAlb or that are not sufficiently concentrated to be seen at the same sensitivity as hAlb. The major drawback of the use of microcolumn IAC is the limitation of the amount of IgG that can be adsorbed which, in turn, will limit the amount of antigen that can be removed from the sample. Quantitation of the more abundant proteins such as albumin and IgG can be accomplished more effectively and rapidly through the use of short, 4.1-mm4.d. columns.11 Quantitation of Clinically-Relevant Proteins. The first clinically important protein chosen for quantitation was human transferrin, the major iron-transport protein in plasma. It carries two femc ions per molecule to the reticuloendothelial system and serves as the mediator of the plasma concentration of free iron. As such, hTf prevents iron intoxication and minimizes iron loss through urinary excretion.28 The con(28) Putnam, F. W. In The Plasma Proteins: Structure, Function, and Genetic Control, 2nd ed.;Putnam, F. W., Ed.;Academic Press, Inc.: New York, 1984, Vol. IV, pp 1-166. (29) Phillips, T. M. LC New York, Mag. 1986, 3, 962.
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centration of hTf increases in iron deficiency anemia and in the last months of pregnancy. A decrease in concentration, usually accompanied by a decrease in hAlb, is seen in many diseases due to either impaired synthesis, as in cirrhosis, starvation, or chronic infection, or increased excretion, as in nephrotic syndrome.’ This protein, hTf, is also an acute phase reactant. The calibration studies for hTf were accomplished by injecting hTf standards directly onto the RP-HPLC column, and Figure 3 representa the detection limit of 10 ng of hTf. Although this value compares favorably with the 60-ng limit obtained by Janis and Regnier,I4 it is far from the picogram sensitivity achieved in the microcolumn studies described elsewhere.22 The primary difference is due to the fact that this system requires gradient elution, whereas the detection limit studies described by Flurer et al.22 were run under isocratic conditions. The increase in the baseline, seen in Figure 3, is not a factor in isocratic systems. In order to determine hTf in normal plasma, 400 nL of 1:lO diluted plasma was injected onto the anti-hTf column. The hTf was desorbed onto the RP-HPLC column and eluted under CH&N gradient conditions. The hTf peak area was compared to the calibration plot (peak area hTf = (6.09 X 10-*)hTfin ng) + 1.04 X 1 t 2 ;correlation coefficient = 0.985), and the value indicated the presence of 99 ng in 40 nL of plasma. On the basis of normal concentration ranges of 2.03.2 mg/mL,28or 80-128 ng in 40 nL of plasma, the calculated value of hTf is certainly within expected limits for normal plasma. The second protein studied here was human ul-antitrypsin (halAT). halAT is a marker protein of inflammation, and it may increase in concentration up to 4-fold during acute phase reaction28 and in pregnancy.’ The concentration decreases in infanta with idiopathichyaline membranedisease. Congenital deficiencies are associated clinically with the development of emphysema at an unusually early age, and
820
ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993 70
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Figure 5. RP-HPLC analysis of eluent from antChcrlAT after rinsing the IAC column wlth 0.1% FA:(A) 200 nL of plasma; (B) 200 nL of 5 mg/mL halAT standard solution; (C) blank. Chromatographlc conditions are as in Figure 2, with CH3CN gradlent as indicated.
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analyses of samples desorbed from the anti-halAT column. Parts A and B of Figure 5 are the resulta of passing 200 nL of halAT-containing colutions (plasma and standard, respectively) through the anti-halAT column, and Figure 5C represents the blank obtained by washing the IAC column with desorption buffer. The first peak, presumably halAT, elutes at 65% CH3CN and appears in both samples but not in the blank. The peak area corresponds to a range of 664704 ng on Figure 4. With normal concentrations of halAT between 2 and 4 mg/mL in plasma,28a 200-nL aliquot should contain 400-800 ng. Therefore, the result obtained in Figure 5A appears to represent a normal level.
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the increased incidence of neonatal hepatitis, which usually progresses to cirrhosis.’ The calibration studies for halAT were done directly on the RP-HPLC analysis column, using a standard that was not 100% halAT. The supplier, Sigma Chemical Co., states that the product is 8 5 9 0 % halAT, with serum albumin as the major contaminant. A separate injection of hAlb indicated that the peak eluting at 55% CH3CN (data not shown) was due to hAlb. The calibration plot seen in Figure 4 was generated for 85% concentration levels, and yielded a leastsquares regression analysis of peak area halAT = (1.76 X lO-’)(halAT in ng) - 5.09 X 10-3, with a correlation coefficient of 0.9956. The concentrations calculated for 90% purity did not change the calibration plot to any great extent and were not included in Figure 4. The three chromatograms shown in Figure 5 represent
In most cases, immunoaffinity chromatography is not designed to replace existing rapid diagnosticmethods but to give sensitive alternatives for more in-depth analyses and to provide isolation techniques for probing structural characteristics such as microheterogeneitiesor amino acid substitutions. The disadvantages of the hydrophobic adsorption procedure arise from the bleeding of the IgG from the IAC column, which could interefere with the examination of monoclonal gammopathies. The acidic conditions under which IgG immobilization takes place undoubtedly lead to deactivation of the antibody and ita improper orientation on the surface of the silica. The miniaturization of IAC to the microcolumn scale also limits the amount of antigen that can be adsorbed, because not as much antibody is present. However, this fact should rarely be a problem when the microcolumns are used for the isolation of trace cornponenta. The use of microcolumns for the hydrophobic adsorption of antibodies offers several advantagesover other procedures. First, the covalent immobilization of antibodies is not necessary, which offers a more rapid and straightforward preparation of the IAC column. An even more significant contribution is the decrease in the amount of antibody
ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993
required, due to the reduced column volume with the use of microcolumns. Less IgG is needed for the initial adsorption procedure, typically 700 pg per column, as opposed to 35-40 mg per conventional4.6-mm4.d. column,2O 16 mg per 6.4 mm X 4.1-mm4.d. column,11 or 1mg antibody per analysis on a Protein G The smaller amounts of IgG required could lead to the effective use of more expensive monoclonal antibodies. The miniaturization of the IAC system to the microcolumn scale also reduces the sample volumes required for both profiling and quantitation. The analysisof human transferrin occurred with 40 nL of plasma, and that of human alantitrypsin with 200 nL of plasma. Additionally, the utilization of an anti-human albumin affinity column led to the
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selective removal of albumin from 400 nL of plasma, for the generation of albumin-subtracted plasma profiles. This ability to accomplish analyses on less than 1pL of biological fluid could be a great benefit when dealing with samples such as cerebrospinal fluid.
ACKNOWLEDGMENT This work was supported by Grant No. GM24349 from the National Institute of General Medical Sciences and a Grantin-aid from Genentech, Inc.
RECEIVED for review June 22, 1992. Accepted December 15, 1992.
