Conjugation of a modified form of human C-reactive protein to affinity membranes for extracorporeal adsorption

ClinicalA4ateuials11 (1992) 105-117

Conjugation of a Modified Form of Human C-Reactive Protein to Affinity Membranes Extracorporeal Adsorption L. A. Potempa,a u Immtech International, Illinois, USA

M. Motie,b B. Andersoqa Inc., Evanston,

Illinois,

USA,

E. Klein” & U. Baurmeisterd

and Northwestern

University

Medical

b Immtech International, Inc., Evanston, Illinois, USA ‘University of Louisville, Louisville, Kentucky, USA ’ AKZO Fibers and Polymers Division, Wuppertal, Germany

Abstract: C-reactive protein (CRP) is the prototypic acute phase reactant in man and a number of other species. Zn vitro, it has been reported to activate the complement system and promote reactions of phagocytosis. In viva, CRP has been shown to protect mice from lethal bacteremia and to possibly have a role in protection from plasmodial infection, endotoxic shock and exposure of nuclear antigens to the immune system in autoimmune disease. We have shown that CRP can assume a second molecular form, distinguishable from the native, pentameric molecule in terms of antigenicity, solubility and binding reactivity. This form, termed ‘modified-CRP’ (m-CRP), binds aggregated and immune complexed IgG and not monomeric IgG and we postulate that a natural biological function of CRP may involve binding of immune complexes for facilitated removal from plasma. In the present study, we have covalently immobilized m-CRP on to various solid-phase supports for selective extracorporeal binding and removal of immune complexes during apheresis. Modified-CRP was immobilized in the absence and presence of &amine spacers to agarose beads, and to cellulose, polyamide and modified polyvinylidene difluoride (PVDF) fibers using carbonyldiimidazole (CDI), cyanuric chloride (CyCl,), cyanogen bromide and reductive amination chemistries. Staphylococcal protein A was derivatized in certain experiments as a control protein known to bind IgG. Various elution conditions were established to identiffy those proteins selectively removed from patient plasmapheresis fluids by experimental surfaces. We report that m-CRP can be covalently linked to affinity surfaces and that such surfaces have the capacity to bind two populations of IgG from plasma. Using a goat antiserum known to contain specific antibodies to m-CRP, one population of IgG was bound and eluted with a standard acid wash, suggesting a relatively weak binding avidity between IgG and immobilized protein. Using isolated IgG or patient plasmapheresis fluid, we have identified a second population of IgG that is not elutable in acid, instead requiring chaotrope, NaOH or SDS elution methods. Other adhesive glycoproteins were also recognized in the chaotrope eluate of both experimental and control surfaces, suggesting substantial nonspecific adsorption of such proteins on to some surfaces. Thus, we have identified conditions for the immobilization of m-CRP onto solid-phase support surfaces for the extracorporeal removal of immune complexes during apheresis. We continue to define those proteins specifically removed from various test surfaces to define whether such selective removal will have a therapeutic benefit in the treatment of diseases with known immune complex pathologies.

105 ClinicalMaterials O267-6605/92/$05.00 0

1992

Elsevier Science Publishers Ltd, England

School,

Chicago,

106

Lawrence

et aI,

A. Potempa

INTRODUCTION C-reactive protein (CRP) is a prototypic acute phase protein in man an a number of lower vertebrates. Its serum level known to increase u to a thousandfold in the first 24-48 h of most nonspecific inflammatory responses.lm3 While serum CRP levels correlate with the most intense inflammatory processes, the role that CRP plays in the stimulation, amplification or regulation of such processes is largely unknown. 1~ vitro, C been shown to act as an opsonin and agglutimn a to activate the primary complement inhibiting the alternative compleme In rive, CRP has the capacity to protect mice from lethal infection,g,10 modify antibody responses to the phosphorylcholinell and chromatin’2 antigens, and, when included in liposomes, inhibit metastasis of certain tumors in the rn~use.~~~‘~ These activities suggest that CRP is an important mediator of the immune response. CRP is a molecule composed of five, identical, noncovalently associated globular subunits, each of Mr 23000, arranged in cyclic symmetry.l” Eat subunit has one binding site for the primary bindin ligand, phosphorylcholine (PC) I7 CRP binding to PC is modulated by calcium which is known to influence the conformation of CRP.“, I9 Indeed, idiotype are certain murine antibodies to the known to cross-react only when P is kept in calcium-containing buffers. A calcium-binding site sequence within CRP has been defined as being similar to the calcium-binding sites in calmodulin and other related calcium-binding proteins2” Inasmuch as each subunit has been reported to bin two calcium ions, it is currently unknown whether there is a second calcium-binding site or whether d to the defined sequence. Of both calcium io structure/function activities, importance to these calcium ions are bound to CRP with significantly different affinities. While one calcium is easily chelated and influences CRP’s capacity to reversibly bind to PC ligands, the second calcium cannot be removed by simple chelation. This highaffinity calcium has been reported to influence CRP quaternary structure and the capacity of GRP to undergo an irreversible conversion into a distinct molecular form which is referred to as ‘modifiedCRF’ (m-CRP) and which expresses epitopes that are distinct from the native, pentameric form of the molecule (irrespective of the presence or absence of calcium).“1.“2 Various studies have suggested that molecules identical to or cross-reactive with m-CRP

are

~a~~ra~~y 56c~~~i~g and

may

be importam

inverse reaction, i.e. i~~~~iti5~~ of b~~di~~ to its ~e~e~t~~~~~with C

remains undefined e report here that m-C and nst native-CR the capacity to bind regated or immune complexed e report on &forts to im ize ty matrices f5r extrac eal CRP on t removal of immune corn lexes fraom plasma.

uman

column

was extensively

s eluted

in

w

tris-

Conjugation of human C-reactive protein to afinity membranes

Acrodiscs and concentration was assigned based on a milligram per milliliter extinction coefficient for isolated CRP of 1.98. Final preparations produced a single band of Mr 23000 on SDS-PAGE stained with. Coomassie Brilliant Blue and were negative by immunodiffusion analyses for IgG and SAP. By these criteria, final CRP preparations were > 99 % pure. Modification

of CRP (m-CRP)

Solutions of isolated CRP at 1 mg/ml were adjusted to have sufficient chelator to remove all calcium. Generally, ethylene diamine tetra acetic acid (EDTA) was added to a final concentration of 10 mM. Ultrapure urea was then dissolved in the CRP solution to a final concentration of 8 M. After l-2 h at 37 “C, urea was removed by exhaustive -05 M Na bicarbonate (pH 8.3). Dialyzed solutions were sterile filtered through 0.2 pm Acrodiscs and protein concentrations were assigned based on the CRP extinction coefficient. Enzyme-linked

immunosorbent

107

The protein solution was heat aggregated at 63 “C for 30 min and the resulting opalescent solution was passed through a Bio-Gel A 1.5 m (Bio-Rad, Richmond, Calif.) (1.5 cm x 90 cm) at 4 “C in 20 mM tris-HCl, O-3M NaCl (pH 7.4) containing 0.02% NaN,. Soluble immunoglobulin aggregates were separated from monomeric IgG and IgG fragments and pooled fractions were assigned concentrations based on a milligram per milliliter extinction coefficient for IgG of 1.4. Preparation of goat antiserum

Goat antiserum was raised to m-CRIP essentially as described.22 This antiserum is generally referred to as ‘anti-neo-CRP’ in order to differentiate it from antiserum that is more specific for the native, pentameric form of the molecule. For some experiments, antiserum was fractionated with 45 % ammonium sulfate and the precipitate exhaustively dialyzed into 10 mM Na phosphate buffer containing 0.15 M NaCl (pH 7.3) with NaN, added to a final concentration of 0.02 % as a preservative.

assays (ELISA) SDS-PAGE

One hundred microliters of m-CRP at 2 pug/ml in 0.05 M bicarbonate (pH 9.0) was immobilized on Nunc, polystyrene microtiter plates (Scientific Supply Co., Schiller Park, Ill) overnight ak 4 “C. Wells were backcoated with 1 % BSA in trisbuffered saline and 100 ~1 test samples were incubated for l-2 h at 37 “C. Excess sample was aspirated and wells were washed at 25 mM tris-HCl, 0.15 M NaCl (pH 74) containing O-05% Tween 20. Peroxidase-labeled conjugates (100 ~1) were added at appropriate dilutions and, after incubation and removal of nonbound conjugate, 100 ,ul of peroxidase substrate solution [prepared by dissolving 150 ~1 ABTS (Sigma Chemical Co., St Louis, Ma) at 44 mg/ml, into 10 ml 0.05 M citrate (pH 40), and adding 40 ,~l 3 % H,O, immediately prior to use] were added per well. After a suitable incubation at room temperature, the absorbance of each well was at A 414 nm on a Titertek Multiskan Plus (Flow Laboratories, McLean, Va). Preparation of human aggregated and monomeric IgG

Human gamma-globulins (Sigma Chemical Co.) were adjusted to 20 mg/ml in O-15M NaCl which was made slightly basic by the addition of NaOH.

and Western blot analysis

Discontinuous SDS-PAGE was performed according to the method of Laemmli30 on a Mini-Protean II Slab Gel electrophoresis apparatus (Bio-Rad, Richmond, Calif.). The separating gel most generally used was 12% acrylamide, 0.3 % bisacrylamide while the stacking gel was 4% acrylamide, 0.1% bis-acrylamide. Samples were solubilized for 5 min in a boiling water bath in SDSsolubilizing buffer either containing or lacking 2mercaptoethanol. Proteins were separated using constant 100 V electrophoresis for l-2 h. After electrophoresis, gels were either stained in 25 % methanol, 10 % acetic acid containing 0.05 % 250, or were transCoomassie Brilliant Blue blotted onto nitrocellulose ts using a semi-dry enmark) according electroblotter (JKA-Biotech, to the specifications of the manufacturer. Transferred nitrocellulose sheets were blocked with 1% BSA in buffer and were developed with appropriate reagents and peroxidase-labeled conjugates. After washing away excess conjugate, Western blots were developed using HRP color developing reagent (Bio-Rad) (prepared by dissolving 30 mg reagent in 10 ml cold methanol, then adding 30 ~1 of 30 % H,O, in 50 ml deionized water immediately prior to use).

108

Lawrence A. Potempa et al.

Solid-phase surfaces Bio-Gel A 0.5 m agarose-based beads were purchased from Bio-Rad Laboratories. High-flux RCHP400 cellulose capillary membranes of 200 pm inner diameter, type V386c polyamide capillary membranes of 300 ,um inner diameter and polyvinylidene difluoride capillary membranes modified with aminohexyl groups (PVDF-NH,) of 1000 iurn inner diameter were supplied with the generous cooperation of Dr Ernst Spindler of ENKA AG, Business Unit Membrana, Wuppertal, Germany. Coupling chemistries Cyanogen bromide Bio-Gel A 0.5 m was exhaustively washed in deionized water before being equilibrated in 2 M Na carbonate (pH 11.5). Cyanogen bromide (20 gm) dissolved in 10 ml acetonitrile was slowly added to 100 ml beads and the slurry was stirred for 30-60 min. During this time, the pH of the solution was maintained at 11-O y the addition of 10 N NaOH and the temperature was maintained at 20 “C by the addition of ice chips. Using a Buchner funnel, beads were washed twice with 8.1 N Na bicarbonate (pH 9.5) and water before being equilibrated in 0.05 M Na bicarbonate (pH 8.3) for addition of m-CRP (equilibrated in the same buffer). Beads were rocked gently overnight at 4 “C. Nonbound protein was removed by aspiration and was quantified by BCA protein assay Chemical Co., Rockford, Ill). The amount was determined by the difference compared to the amount of protein offered to cyanogen bromideactivated beads. Excess reactive sites on the activated Bio-Gel were blocked with O-1 M glycine in O-05 M Na bicarbonate (pH 9.0) for 4 h at room temperature. Derivatized beads were equilibrated in 25 mM tris-HCl-O.15 M NaCl (pH 7.4) containing O-02 % NaN,. Carbonyldiimidazole Capillary membrane surfaces were dehydrated continuous flow in glass tubes with sequential passage of 33, 66 and 100 % acetone. Carbonyldiimidazole (CDI) (Sigma Chemical Co.) dissolved in acetone was passed through capillary membranes at 0.005-0.1 M, generally at a two- to a hundredfold excess to the assayed number of reactive amine groups on the membranes.“l After 15-30 min at room temperature, excess reagent was removed and membranes were washed with acetone. In some

acetone were passe were ~~a~tivate~ in

bound protein residual protein

was usi

residual acetone which has a s~~~~~~~~~~ a~s~~~~.~~~ in the ultraviolet region. Excess reactive groups on ocked by passage 01 5 M Na bicarbo anes were extensively washe -but%ered saline (pH 7~4) containControl ~e~b~~~~s were treated identically using bu,SFers lacking protein.

for

CDI

molar

activation.

to CyCQ

Cya,nuric chloride at Q-1 % in

was

washing away excess tinually perfused at 2

reactive grsups were amine in 0~05 M Na ~~rb0~ate membranes were ~q~~~ib~~te~ in bufYered saline (pH 44) containin Control ~~e~~b~~l~~s were treated I buffers lacking protein,

(CyCl,) acetone

Conjugation of human C-reactive protein to afinity membranes

109

perfusion of either 0.1 % Na borohydride olr 0.1 M ethanolamine which was perfused in the presence of 0.1% Na cyanoborohydride. Affinity membranes were washed and equilibrated in phosphatebuffered saline (pH 7.4) containing 0*02% NaN,. Control columns were prepared identically in buffers lacking protein. Plasmapheresis fluids Plasmapheresis fluids were collected by certified physicians as part of therapeutic plasmapheresis procedures in full compliance with human subject guidelines. Some fluids were obtained with the kind cooperation of Dr Peter Dau of Evanston Hospital, Evanston, Ill. Other fluids were collected tlhrough the Kidney Disease Center at the University of Louisville Medical Center.

0.006

1.1E TO-

G o-9 t? 0.6m’ ooe $ 0,4-

Some of the more routinely used assays to quantify immune complexes (ICs) in serum use solid-phase immobilized Clq component of the complement system as the binding ligand.32 The level of ICs in a particular sample is generally quantified in terms of equivalent levels of aggregated IgG binding which is used as a positive control. In Fig. 1, panel A, we demonstrate typical binding curves which show aggregated but not monomeric IgG binding to immobilized Cl q. For 2 pg Clq immobilized per well, the mid-point of aggregated IgG binding was found to be 4 pug/ml IgG. We next compared the binding of identical Ig reagents to solid phase immobilized m-CRP. As can be seen, essentially identical binding curves were generated. For O-5 ,ug m-CRP immobilized per well, the mid-point of aggregated IgG binding was also found to be 4 pug/ml IgG, indicating on a weight basis that mis more efficient than Clq in binding aggreIgG. On a molar basis, however, based on an Mr of m-CRIB of23 000 (of the free CRP subunit),” approximately four moles of m-CRP for every mole of Clq is required for equivalent levels of aggregated IgG binding. Since Clq is known to have six ular IgG binding sites per molecule which contribute to the functional affinity of Clq for aggregated IgG,33 the requirement for at least four moles of m-CRP to obtain equivalent binding suggests aggregated forms of m-CRP may con-

O-8 4 IGG in TE-albumin

20

100

._

s o-7-

Demonstration of aggregated IgG and immune complex binding to m-CRP

016 UGlML

1.2 iB

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RESULTS

0.031

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Fig. 1. Binding of heat-aggregated IgG to Clq and an-CRP. Panel A: 100 ~1 of either isolated Clq at 20 ,~g/ml or m-CRP at 5 pg/ml was immobilized onto polystyrene EIA wells. Human heat-aggregated IgG was chromatographically separated into monomeric IgG (Mr 160000) and soluble, aggregated IgG (Mr > 350000). IgG aliquots diluted intfo buffer containing 1 % BSA were incubated on immobilized protein, and, after washing, bound Ig was detected with an F(ab’), fragment of goat antihuman IgG-peroxidase conjugate and ABTS. Aggregated IgG binding on Clq (a---A); monomeric IgG binding on Clq (O--O); aggregated IgG binding on m-CRP +) ; monomeric IgG binding on m-CRP (o---0). (+-Panel B: 100 ~1 of 20 pg/ml native CRP was captured on to 0.5 pg plate-immobilized PC-KLH in buffer containing 2 mM CaCl,. Aggregated IgG in buffer containing 1% BSA was added and, after washing, bound immunoglobulin was detected with an F(ab’)2 fragment of goat antibuman IgG-peroxidase and ABTS (A ---A). Binding of aggregated IgG to 0.5 pg plate-immobilized m-CRP is shown for comparison (+ ---+).

tribute to enhanced binding reactions. For both Clq and m-CRP, monomer IgG did not show significant binding to m-CRP at the concentration tested. Binding of aggregated IgG was found to be specific to modified-CRP and not native, pentamerit CRP. To demonstrate this, native-CRP was immobilized onto EIA wells by a sandwich method to prevent spontaneous conversion of CRP to mCRP upon contact with the polystyrene plate

110

Lawrence

surface.22 In this method, native-CRP was bound in the presence of calcium to its classically defined ligand-phosphorylcholine (PC)-which was conjugated onto keyhole limpet hemocyanin (PCKLH). Using monoclonal antibodies specific for either native-CRP or m,34 we verified that native protein was bound a not express m-CRP antig demonstrates that while a CRP, no binding to nati We next tested the capacity of m-CRP to defined ICs of various a utilized the Ig fraction of rabbit antiperoxidase antiserum which was tions with isolated peroxidase eplzyme to form soluble ICs. Concentrations of reagents were adjusted to have antibody-antigen molar ratios of approximately 1: 1, 2 : B and 4: 1I As a control to assess the capacity of uncomplexed antibody (i.e. monomeric immunoglobulin) to bind incubated antiperoxidase alone on im CRP at identical concentrations as prepare soluble ICs. Figure 2 remonstrates that antibody in the form of sol le PCs (4: 1 antibodyantigen ratio) binds m-C while ~o~c~rnp~~~e~ antibody does not. Controls in which peroxidase

A

cs with nst

higher raeam shown). These

not mQ~Qm~ric ieg, alecules

in the

s in additicnl i”Q human

%gs.

m-C arose was activated

fraction of a goat antise passed across the resin washe away. Figure rorn~t~g~a~ which

with cyano

3 illustrates :iilc elation k was recovered

With 5 % ac=etiar:acid.

analyses.

Two

minor

identities

bands

of

of which are ~~~~~~w~,

Agarse-mCRP goat Antisewn 0075

15

---~_~~~I 3 6 12 UGiML Antbperoxidase

~

i i4

48

Fig. 2. Binding of rabbit antiperoxidase-peroxidase soluble immune complexes to solid-phase m-CRP. Indicated concentrations of rabbit antiperoxidase diluted in buffer containing 1% BSA was preincubated in the fluid phase with various concentrations of isolated peroxidase to form soluble ICs of approximately 4 : 1 antibody-antigen molar ratios. Formed soluble ICs were incubated on plate-immobilized m-CRP and, after washing, bound protein was detected by adding ABTS (O---0). Alternatively, identical concentrations of nonimmune complexed antiperoxidase in buffer containing 1% BSA were incubated on plate-immobilized m-CRP. After washing, peroxidase antigen was added at the same antibodyantigen ratios as used with pre-formed soluble ICs to wells that had been offered antiperoxidase. Bound antibody was detected using the peroxidase substrate ABTS (O---n). Control experiments indicated peroxidase alone did not bind m-CRP.

Fractlov

number

Fig. 3. Elut~on chromatogram of goat anri-m-CRP di-sm mCRP-agarose. A 4 ml lg fraction of goat i-mP was passed through IO ml of CNBr-activated -Ge 03 m containing 0.5 mg/ml m-CRP. After washing, bound proteins were eluted with 5 % acetic acid followed by 2 M KSCN.

111

Conjugation of human C-reactive protein to afinity membranes Agarose-mCRP 040 E 6 0.35 g 0.30, xi 025 8 6 0.20_o 6 O-15 2 O.lO-

-

_ kd _.97.4 66.2

-

45

-

31

-

21.5

-

14.4

Fig. 4. SDS-PAGE analysis of eluted fractions recovered from m-CRP-immobilized experimental surfaces. Panel A : eluates from m-CRP-agarose beaded affinity surfaces (10 ml beaded resin containing O-5 mg m-CRP/ml beads). Column 1: acetic acid eluate from goat anti-m-CRP antiserum. Column 2: thiocynate eluate from human plasmapheresis fluid. 12% Laemmli gels were stained with Coomassie Brilliant Blue. Panel B: eluates from PVDF-NH, fibrous surfaces. Column 3: thiocyanate eluate from human plasmapheresis fluid proteins adsorbed to control unsubstituted PVDF-NH, surfaces. Column 4: thiocyanate eluate from goat anti-m-CRP Ig fraction bound to m-CRP-PVDF-NH, fibrous surface. Test surfaces contained 0.028 m2 of surface area based on the inner diameter of the fibers. Test surface contained 3 mg immobilized m-CRP. SDS-PAGE standards are shown for reference.

results suggest CNBr-activated m-CRP-agarose is a reasonable affinity matrix for the isolalion of specific antibody. In an effort to see if other proteins might be bound to the affinity matrix which resisted acid elution, this same resin was washed with 2 M KSCN. A small peak of material was recovered which, when dialyzed to remove excess chaotrope, resulted in some protein aggregation. This material was resolubilized in bicarbonate buffer at pH 9-O and was analyzed by SDS-PAGE analyses. Predominant IgG bands were again observed and, as above, IgG was verified antigenically in this isolate. In addition to the IgG bands of Mr 50000 and 25000, bands of Mr 75 000, 65 000 and some high Mr material which did not enter the gel were also observed (data not shown). The m-CRP-agarose resin was reequilibrated in buffer and plasmapheresis fluid from a patient with

i

0.051

i.

I

o_&I5!%dc-_ ,.,,,,,,,,,_-r-,~L~+-,+-yity ‘2 2

12

22

32

42 52 62 72 82 Fraction number

92

102

112 12

Fig. 5. Elution of bound materials from human plasmapheresis fluid from m-CRP-agarose. A 100 ml plasmapheresis fluid from a patient with myastinia gravis was passed over m-CRPBio-Gel A 0.5 m (10 ml beaded resin containing 05 mg mCRP/ml beads). Bound protein was elmed with 2 M thiocyanate.

myastinia gravis (MG) was passed. After washing, we were unable to detect a signific,ant eluate with 5% acetic acid. However, as shown in Fig. 5, protein was recovered from this affinity resin using 2 M Na thiocyanate. When this eluate was dialyzed and concentrated, we again found precipitated material similar to what had been observed with the m-CRP-agarose chaotrope eluate from the goat antiserum. When analyzed by SDS-PAGE, predominant bands were observed at the top of the gel, and at apparent Mr of 75 000, 65000, 50000 and 28000 (Fig. 4, panel A, column 2). In addition, a broad band at Mr 40000 was observed. Western blot analyses using an antihuman IgG,A,M conjugate indicated that bands at Mr 50 000 and 75 000 were positive for immunoglobulins, suggesting that isotypes other than IgG were recovered from mCRP-agarose. Based on the molecular weights of the isolated bands, and based on the previous report than CRP has a specificity for certain adhesive proteins,35,36 we analyze:d the plasmapheresis fluid chaotrope eluate by Western blot analysis and by radial immunodiffusion and identified the band at Mr 65000 as vitronectin. No albumin was observed in these eluates; also, no mCRP was detected in recovered protein, suggesting no leaching of surface-immobilized protein during all manipulations. m-CRP-PVDF

PVDF-NH, high porosity fibrous surfaces were activated with carbonyldiimidazole (CDI). m-CRP was immobilized at a density of 110 mg protein/m2

112

Lawrence

A

surface area and surfaces containing 3 mg protein were prepared for similar testing, as was done with the agarose resin. Excess reactive groups on mCRP-PVDF were bloc with ethanolamine. Goat anti-m-CRP perfused through the fibers at a rate of l-3 ml/mm, an bound protein was eluted with ch not attempt an acid elution step in SDS-PAGE analysis of the eluted protein showe predominant bands at the t of the gel and at calculated Mr of 93000, 850 73000, 55000 an 25 000 (see Fig. 4, panel B, column 4). Goat IgG was verified in the eluate, and when no~~ednc~ng S PAGE gels were run, the Mr bands at 50000 25000 were not observed, instead mi top of the gel, suggesting that these heavy and light chains of the is01 globulin. We performed a this isolate using antihum We found sufficient species cro suggest that the band at apparent goat vitronectin. We next perfused plasmapheresis fluid from a patient with systemic lup erythematosis (SLE) through m-CRP-PVDF a , after washing, were unable to demonstrate pr in (by absorbance at 280 nm) in an acid eluate. Similar to our results with m-CRP-agarose, however, a chaotrope eluate was recovered. Similar bands as were seen in the chaotrope eluates described above were observed. 50000 and 25000 Bands at apparent Mr of 75 stern blot analysis stained positively for Igs by using anti-TgG,A,M conjugates, again suggesting that m-CRP immobilized surfaces om patient plasma. at Mr 65000 were also seen (data not To define nonspecific adsorption properties of the PVDF-NH, surface, a similar aliquot of uid was perfuse plasmapheresis derivatized fibers. Figure 6 compares th pheresis fluid Na thiocyanate elutio and underivatized grams from ope eluate from PVDF-NH,. control fibers was anal strongly stained band at band at Mr 65000 were observe column 3). Neither protean stained positively for Igs by either immunodiffu The main band at antivitronectin, sugge tein may be selectively a surfaces.

PoLempa

et al.

Fig. 6. Elmion of bound materials

from human piasmapheresis -NH, surfaces. A PO0 ml of plasmapheresis ent diagnosed with systemic lupus erythematosis was perfused through PVDF fibers at 1-3 ml/min using a peristaltic pump. m-CRP was immobiiized on experimental surfaces using CD1 activation chemistry and surfaces having @028 rn2 surface area and 3 mg immobilized mCRP were used. Bound proteins were elated with 2 M thiocyanate. Elution ofm-CRP-immobilized fibers (a-mPg); elution

of control,

underivatized

ers by fiber ~mrnobi~~zatio~ with ca chloride or reductive

fibers ( + - ~~f

:

aetivath

olyamide surfaces immobilized

g ~~mo~il~zed

protein

minor bands in e eluate, these b

s were now

Conjugation of human C-reactive protein to a&ity O-26 ~ 0.24 022-

Polyamide-Protein

A

f

: 0.20 $

0.1%

CJ 0.16 s

o-14

g 0.122

O-IO

5 O-08 2 0.06 0.04

Fraction

number

Fig. 7. Elution of bound materials from plasmapheresis fluid from staphylococcal protein A-immobilized polyamide fibrous surfaces. A 100 ml human plasmapheresis fluid from a patient with systemic lupus erythematosis was perfused through 0.05 mZ of polyamide fibers containing 8 mg protein A. Protein A was immobilized using glutaraldehyde and cyanoborohydride reductive amination. Bound protein was eluted with 5% acetic acid followed by 2 M thiocyanate.

membranes

113

identify a small amount of IgG in the chaotrope eluate. We repeated our analyses using control polyamide fibers which were treated with glutaraldehyde and cyanoborohydride but which lacked protein. We found vitronectin and fibronectin in chaotrope eluates. Prior to perfusing the chaotrope solution, control fibers were perfused with acetic acid but no protein was recovered. As was observed in previously generated chaotrope eluates, it was important to carefully observe dialyzed, concentrated eluates for precipitated protein. We believe such a phenomenon may be due to the adhesive, ‘sticky ’ nature of the proteins being recovered and the effects of the high concentration of the chaotrope salt solution needed to remove such proteins from the experimental surfaces tested. We attempted to immobilize m-GRP on to polyamide using the same reductive amination chemistry as was used successfully for protein A. Although we were able to immobilize protein on to activated surfaces, we were unable to verify that antigenic protein was accessible w

83 56

kd

42 ;*, ,s$

36.5

12 Fig. 8. SDS-PAGE analysis of plasmapheresis fluid eluates from staphylococcal protein A polyamide fibers. Column 1: 5 % acetic acid eluate. Column 2: thiocyanate eluate. 10 % Laemmli gels are stained with Coomassie Brilliant Blue. SDSPAGE standards are shown for reference.

12 predominant (Fig. 8, column 2). In addition, a strong band at Mr 210000 and lightly staining bands at Mr 50000,420OO and 36000 were seen (all using reducing conditions for SDS-PAGE analysis). By antigenic analyses, we have identified the Mr band at 210000 as fibronectin and the Mr band at 72000 as vitronectin. Using sensitive assays, we did

Fig. 9. SDS-PAGE analysis of eluates from m-CRP-polyamide fibers. 3.8 mg of m-CRP was immobilized onto 0.028 m2 of polyamide fibers using cyanuric chloride. Column 1: 5 % acetic acid elute from m-CRP-polyamide fibers after 4 ml goat antim-CRP was perfused. Column 2 : 5 % acetilc acid eluate from m-CRP-polyamide after 100 ml of human plasmapheresis fluid from a patient with systemic lupuserythematosis was perfused. 10 % Laemmli gels were stained with Coomassie Brilliant Blue. SDS-PAGE standards are shown folr reference. ECM

II

114

Lawrence A. Botempa et al,

anti-m-CRP antiserum through m-CRP-polyamide. We thus used cyanuric chloride as an alternative activation chemistry to immobilize m-CRP on to polyamide. Using cyanuric chloride, antigenically active mCRP was immobilized on polyamide at a density of 152 mg/m’ of fiber or a surface containing 3.8 mg total protein When goat anti-m-CRP antiserum was perfused, an acid eluate was generated. Goat immunoglobulin was the predominant protein recovered in this isolate (Fig. 9, column I). also observed a significant band at apparent r of 67000 which we have identified as that little or no vitronectin or ii recovered in this eluate. When plasmapheresis fluid from a patient with SLE was perfused through these fibers, we unexpectedly observed an acid peak and failed %ogenerate a significant chaotrope peak. Control fibers lacking m-CRP produced a very small acid eluate when perfused with the sa e plasmapheresis fluid (Fig. 10). The chaotrope “peak’ from % control fibers was simi m-CRP-containing fibers. m-CRP-polyamide was ana bands at apparent M 5 1000 and 44 000 were We verified the pres vitronectin an aPbumin in this sample. When he control, acid eliuate ‘peak’ were only a small concentrated and run on SDSen chaokrope amount of albumin was observ eluates were analyzed, no other significant bands were identified.

reac%ive protein can

on to a. variety of beaded and fibrous surfaces e bvere sucon to beaded romide activa%ion chemfrom goat antiserum. 22.88 In s, we did no% attempt to m”sa vvitkt acid so!utions, and eBu%especific antibody ins%eadused high i i&:s’trength solutions %Qrecover lin.. ~~~~~~~~ analyses OS such eluates did verify ihat the maj eluked protein was ~~~~~~~~~~~~~~~~~~. the hght of ahe findings presented here, m-CRP has the capacity lo bind two populations of imm.unoglobulin from s~erum or pkasma. On &ted with solu%ions of pH < 3.0 w resists acid ehttion and mom Pikcliy represents a ~o~~~~ti~~ of ag gated or i~~~~~~~~complexed hese previous s~tudics did not

o-45

Igs, other protems were recovered, especially in the 13 agarose surfaces. were dialyzed to remove excess sal%, egation was observed in the isolated regates was found to

O-40 E 0-35 C g 0.30 Cd ii;025 a, g O-20 2 $ o-15 {OlO

Fraction

number

Fig. 10. Elution

of bound materials from plasmapheresis fluids from polyamide surfaces. Elution chromatographic profiles from m-CRP-polyamide (+-+) or control underivatized polyamide ([7--n) after 100 ml human plasmapheresis fluid from a patient with systemic lupus erythematosis was perfused through 028 m2 fiber surface area. Elution solutions were 5 % acetic acid and 2 M thiocyanate.

get

a~%~~~~.~~ and func%iorraKprotein. to blrad on

Conjugation

of human C-reactive

both PVDF and polyamide. We have analyzed bound/eluted fractions from each surface and find that, in addition to immunoglobulin, the adhesive proteins vitronectin and fibronectin are also isolated from experimental surfaces. It was noted that in no instance throughout the experiments presented herein did we find leached m-CRP in our recovered protein, indicating that the linkage chemistries were sufficient to withstand the numerous treatment steps used. We were able to successfully immobilize m-CRP on PVDF-NH2 using carbonyldiimidazole. The acid eluate after goat antiserum specific for m-CRP was perfused through these fibers contained predominantly IgG which, when tested by ELISA, appeared to be specific antibody to m-CRP. When human plasmapheresis fluid was next passed across this surface, no significant acid eluate was observed. Based on the premise that specific antibody that binds m-CRP through Fab specificity is dissociable in acid while ICs that bind m-CRP through Fc specificity are not, these results suggest that the particular plasmapheresis fluid used did not contain autoantibodies to m-CRP. This is of particular interest in that certain autoimmune diseases such as systemic lupus erythematosis have been postulated to have autoantibodies to CRP as a contributing factor to disease. While not every plasmapheresis fluid tested produced acid eluates from m-CRP-immobilized surfaces, in most cases additional protein identified as immunoglobulin of both IgG and IgM classes was recovered from such affinity surface:5 using chaotrope solutions. The fact that the affinity matrices were able to bind immunoglobulin which includes IgM would support the findings of Gupta et a1.“9 who isolated immune complexes from the sera of patients with acute rheumatic fever using polyethyleneglycol precipitation. These authors reported that CRP precipitated with recovered ICs and that IgM was the predominant class of Ig present in such complexes. Another report4’ and the data presented here refutes the finding that native, pentameric CRP may bind to immunoglobulin or ICs. However, our data indicate that the modified can bind ICs. Part of the reason behind conflicting reports in defining the activities of CRP may in fact be due to the uncertainty of which CRP form is actually being used and/or measured in experimental assays. We have found that isolated native-CRP can spontaneously convert to m-CRP with storage or when native-CRP is immobilized on particular surfaces.” We have also

protein

to afinity

membranes

115

found that every anti-‘native ‘CRP antisera we have tested has some specificity to m-CRP.38 Without differentiating these two forms of the molecule, any test using ‘CRP’ or “anti-CRP’ may incorrectly assign particular activities to either form of the molecule. To illustrate the importance in differentiating the effects of native-CRP and mCRP, we have reported that m-CRP and not nativeCRP or native-CRP complexes is the active protein in various in-vitro assays of platelet, PMNL and monocyte function.27 We found that protein A could be immobilized on polyamide using glutaraldehyd’e and cyanoborohydride reagents (reductive am.ination chemistry). IgG from plasmapheresis fluid could be bound to this protein A immobilized surface and eluted with solutions of pFI < 39. We did find other proteins adsorbed on to the polyamide fibers which resisted acid elution, being recovered more readily with thiocyanate salts. Such binding to polyamide may reflect the ion exchange characteristic of this surface. When we tried to immobilize m-CRP onto polyamide using reductive amination procedures, although protein was bound to the fibers, we were unable to verify that functional protein was immobilized based on the inability to get specific antibody bound and eluted. We interpret this to indicate that treatment on m-C P with glutaraldehyde and cyanoborohydride adversely affects epitopes and binding sites critical for the binding of immunoglobulin by m-C We were able to immobilize m-CRP to polyamide fibers using cyanuric chloride as a linking agent. The m-CRP affinity matrix so prepared was different from all other surfaces so far tested in that the principal eluate recovered after perfusion of patient plasmapheresis fluid was by acid wash. Recovered material contained some immunoglobulin, fibronectin, vitronectin and albumin. We are continuing to investigate the utility of such a surface for extracorporeal adsorption. Overall, we report herein that m-CRP can be immobilized on a variety of experimental surfaces to bind immunoglobulins from plasma. Most generally, we were able to isolate an acid-elutable IgG from antiserum known to have specific antibody to m-CRP and we were further able to isolate another population of immunoglobulin from both goat antiserum and human plasmapheresis fluid which resisted acid elution. We interpret these results to indicate that m-CRP can bind immune complexes from plasma and that such binding is of relatively high avidity, requiring strong elution 9-2

116

Lawrence

A. Potempa

conditions for dissociation from m-CRP. The observation that some chaotrope isolates have a propensity to aggregate when they are removed from chaotrope solution suggests to us that the recovered protein, which includes immunoglobulin as well as some adhesion glycoproteins, may indeed be from a pool of soluble, circulating immune complexes. The general dogma in the pathophysiology of IC diseases is that BCs of sufficient size are generally not considered to they can form physica antibody molecules. are easily recognized by the m~~o~yte/~hago~yte effector systems of the body and are remove Soluble ICs that are not large enough toi precipitate are considered more pathological in that they can inappropriately deposit in tissues and lead to activation of both acute and responses We suggest that mprotein that contributes to the mechanism by which soluble ICs are made large enough to be removed from solution and thus reduce their pathogenic potential. The removal of such complexes from plasmapheresis fluid by extraco tion conditions coupled with exposure to harsh could have resulted in the protein ag~re~a~iQ~ we noted. In addition to irnrn~~~~~~~b~li~~we found the adhesive glycoproteins fibronectin and vitronectin strongly associated with most of the experimental surfaces tested. However, we are unsure whether these proteins adsorb more selectively to the solid support or whether they show a specificity for m-CRP. Since CRP has been reported to bind fibronectin35 and laminin,“’ we are continuing to investigate whether our results do represent a significant interaction. Our observa well as IgG are isolated on m-CRP and that fibronectin is also some gestion that immune eluted further supports the

some of our &rates is an intere all that unexpected in the association of vitronectin with SC%-9 d glomerulonephritie lesions. Such SC.%are often associated with deposits of HCs.

immobilized

onto

beaded

and

fibrous

and that this binding is off apparently high avidity.

et ai. km§

d a

of

Immobil-

varietyof surfaces

that

duri

analyses

e ac~?lc phase respmse

1

N.Y. ACULL Sri. 3 2

3

4

Siegel,J. $I Fieilel,a., C-rearrive M. L., Acute phase proteins with -&active protein and related proteins (Pentraxins) and Serum Amyloid A component. Ah ImFPlunoi., 34 (1983)141-212. Williams Jr, R. C. 6% Quie, P. G., Studies of human C’. tro phagocytic system. .B.

5 PT.,Interaction of C-reactive protein with lymphocytes and monocytes : complement-de~e~d~~t adherence and phago. cyhosis. J. hinmulzol., 117 (1976) 77481. 6 Kindmark, C-O., Stimulating effect of C-reactive proreiE on phagocytosis of various species of pathogenic bacteria. Cllh. .E.X~. Ii?ziKuPlzol., 7 Kaplan, M. W. & Vohakis, J. E., interactior; of Creactive protein complexes with the ~:~rn~~~me~t system. i. Consumption of human complement associated with the reaction of C-reactive protein with pneumococca? Cpolysaccbaside and with choli hosphatides. lecithin and sphingomyelin. .J. Ih~rn 8. Mold, C. & Gewurz, aceivaiion by liposomes protein on alternative C-iathway and Strq~~ococcus ~~~~~~n~~. J. hnnzunni.. 127 (1981) 2089-92. 9. 1 C., Nakayama, S., Holzer, 7’. J., Gewurz, H. & against OS, T. W., C-reactive protein is protective Stwptococcus pmwnonia infection in mice. J. E.x~. IMeLrS.. 154 (1981) 1703SlO. , D. W., E-Pumarz C10. Uother, J , Volanakis, i. E. & fatal Strq&KXIccus reactive protein is protective a 12 pneumonhz4 infection in mice. 3~ h~uno?., 23754 11. Nakayama, S., DuClos, T. “, Gewurz, H” & Mold, C., Inhibition of antibody responses to ~~os~~or~~c~~o~~~e by C-reactive protein. J. ~~~~nol,~ 132 (I 12. DuCIos, T. W., Bock, L. T., Hicks, toantibody levels and en Fl mice treated with C-reactive protein. (199l) 633a. 13. Deodhar, S. D., James, I., Chiang, ‘ii., Edinger, M. & Barna, B. P., Inhibition ~oflutlg metastases in mice bearing a malignant fibrosarcoma by treatment with liposomes containing human @-reactive protein. Carac~r &~errrc,k, 42 (1982) 5084-8. 14. r, S. D., Gautam, S., Yen-Lieberman, acrophage activation and generation B. 8r Roberts, D., of tumoricidal activity by liposome-associated human G reactive protein. CLUIC~Y Resea~h, 4’4 (1984) 305~~10.

Conjugation

of human C-reactive protein

15. Barna, B., Singh, S., Yen-Lieberman, B., Thomassen, M. J., Maier, M., Pettay, J., Eppstein, D. & Deodhar, S. D., Treatment with a synthetic peptide of human C-reactive protein (CRP) primes lung macrophages for enhanced production of tumor necrosis factor. FASEB J., 5 (1991) 1463a. 16. Osmand, A. P., Friederson, B., Gewurz, H., Painter, R. H., Hofmann, T. & Shelton, E., Characterization of Creactive protein and the complement subcomponent C 1t as homologous proteins displaying cyclic pentameric symmetry (pentraxins). Proc. Nat. Acad. Sci. USA, 74 (1977)

73943. 17. Volanakis,

J. E. & Kaplan, M. H., Specificity of C-reactive protein for choline phosphate residues of pneumococcal Cpolysaccharide. Proc. Sot. Exp. Biol. Med., 136 (1971)

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X2&9.