A microtiter plate enzyme-linked immunosorbent assay for measuring C3b receptors on human erythrocytes

Journal of Immunologwal Methods, 81 (1985) 259-269 Elsewer

259

JIM 03562

A Microtiter Plate Enzyme-Linked Immunosorbent Assay for Measuring C3b Receptors on Human Erythrocytes B S Thomsen l, H N i e l s e n a n d G B e n d l x e n Laboratory of Medtcal Immunology and Department of Internal Medtcme TA, Rtgshospttalet, Copenhagen, Denmark (Received 20 January 1985, accepted 5 Aprd 1985)

An enzyme-hnked lmmunosorbent assay was developed for quant~tatlon of complement C3b receptors (C3bR) on h u m a n erythrocytes fixed m monolayer to mlcroUter plates The &sadvantages of macro test tube systems (large consumption of sample material and reagents, te&ous washing procedures, cell loss and hemolysis) were avotded, and the fixed cells could be stored In return a modest reductxon m ant~gemc~ty reduced by glutaraldehyde was inevitable The cahbratlon curve expressed a detecUon hmlt of about 10% and d~scnmmatlon between levels of C3bR m the interval of about 10-120% of a standard erythrocyte w~th a high uptake of antl-C3b-receptor antibo&es The determinations of C3bR in normals as well as m pauents varied within these limits, and showed a preponderance of low levels m the patients The between-day coefficient of varmt~on was 10 3%, the within-assay lnterplate and lntraplate determinations respectively gave coefficients of variation of 9 7% and 6 2% The method ~s statable for further development and use for investigation of erythrocyte-bound immune complexes

Key words

C3b receptor - human er~throcyte - ELISA

Introduction The C3b receptor (C3bR) on human erythrocytes first described by Nelson (1953) and known as the immune adherence receptor is a glycoprotem with a molecular weight of 160,000-260,000 (Fearon, 1980, 1984, Dykman et al, 1984) Recent evidence indicates that the C3bR on erythrocytes may serve as a binding

1 Correspondence to B S Thomsen, Laboratory of Me&cal Immunology TA 7544, Rlgshospltalet, Tagensvej 18, DK-2200 Copenhagen, Denmark Abbrevtanons ELISA, enzyme-hnked lmmunosorbent assay, C3bR, complement C3b receptor, IC, ~mmune complexes, AML, acute myelmd leukemaa, SLE, systemac lupus erythematosus, RA, rheumatoid arthritis, PLL, poly-L-lysm, PBS, 0 01 M phosphate-buffered sahne, pH 7 2, PBS-T, PBS with 0 05% Tween 20, PNPP, p-mtrophenyl phosphate, DTT, &thlothreltol, BSA, bowne serum albumin, RBC, red blood cells 0022-1759/85/$03 30 © 1985 Elsevier Sctence Pubhshers B V (Biomedical Division)

260 capacity for complement-fixing immune complexes (IC) and may facilitate processing and clearance of IC (Siegel et al, 1981, Medof et al, 1982, Cornacoff et al, 1983) This would reduce the potential for deposition of IC in tissues and avoid acnvatlon of other cell types bearing C3bR Defective reacnvlty and low number of C3bR on erythrocytes has been described in patients with systemic lupus erythematosus (MIyakawa et al, 1981, hda et al 1982, Inada et al, 1982, Wilson et al, 1982, Taylor et al, 1983, Horgan and Taylor, 1984, Takemura et al, 1984), rheumatoid arthritis (Ilda et al, 1982, Taylor et al 1983) and acute myelold leukemia (Takemura et al, 1984) Conventional methods for measuring C3b receptors on erythrocytes include rosettlng (Fearon, 1980) or haemagglutmatlon (Mlyakawa et al, 1981, lnada et al 1982, Takemura et al, 1984) techniques with binding of indicator particles coated with C3b, measurement of the binding of preformed radlolabeled immune complexes (Medof et al, 1982, Taylor et al, 1983, Horgan and Taylor, 1984) or of antl-C3b-receptor antibody (Fearon, 1980, Mlyakawa et al, 1981, hda et al, 1982, Wilson et al, 1982) In addition measurement of the binding of dlmerlc C3b to the receptor (Wilson et al, 1982) and an assay based on C3bRs cofactor function for factor I has been used (Slm and Slm, 1983) Enzyme-hnked lmmunosorbent methods represent an attractive alternative to radlolmmunoassays especially because of the potentml medical hazards and short shelf-hfe of isotope labels (Voller et al, 1976) Confluent monolayers of erythrocytes are efficiently formed on poly-L-lysm (PLL)-treated plastic plates (Stultmg and Berke, 1973), and subsequent fixanon with glutaraldehyde results in a cell layer, which remains attached during washing and incubation procedures (Landsdorp et al, 1980) In this paper we describe an enzyme-linked lmmunosorbent assay for measuring C3bR on human erythrocytes fixed in monolayer to mlcrotlter plates The technical variables are systematically evaluated and a recommended procedure selected and described Materials and Methods

Sohd-phase matertal The polystyrene 96-well n~crotlter plate Nunc-Immunoplate I ~ with flat bottom (Nunc, Roskflde) was used as standard

Erythrocytes Whole blood was drawn into glass tubes with cttrate-phosphate-dextrose-adenm (CPDA) solution and stored at 4°C for less than 1 week Before use erythrocytes were washed 3 times with PBS, carefully removing the buffy coat after each centrlfugatlon Blood from the same healthy person of blood group AB Rh pos was used throughout the standardization

Albumin 1% bovine albumin (Sigma no A-4503, Sigma Chemical C o , St Louis, MO) in PBS

261

Poly-L-lysm The hydrobromlde (Sigma no P-2636) with molecular weight 30,000-?0,000 was used in PBS (0 1 m g / m l )

Glutaraldehvde 25% stock solution (Sigma no G-6257) diluted to 0 1% in PBS immediately before use

Dtthtothrettol Washed RBC (2 5% suspension) were incubated for 30 mln at 37°C with D T T (Sigma no D-0632) at final concentrations of 1 25 m M - 1 0 mM in PBS (Dierlch et al, 1974) The cells were washed again and added to the wells as usually

Monoclonal anttbodtes Monoclonal mouse antibody to human C3b receptor (DAKO-C3bR, code no M710, clone To 5) was obtained from Dakopatts (Copenhagen) According to the manufacturer the antibody is of subclass IgG 1, contains 28/~g mouse IgG per ml and shows characteristic distribution of the antigen among human cells together with inhibiting binding of erythrocyte-antlbody-complement complexes (EAC3b) to the receptor as described by Gerdes et al (1982) A monoclonal mouse antibody of identical IgG subclass against human IgM (Dakopatts, code no M702) was used as control

Enzyme-labeled antlbodtes The enzyme alkaline phosphatase was preferred for peroxMase, since alkahne phosphatase in the erythrocyte membrane has been found neghgible (Leikola and Perkins, 1980), while endogenous erythrocyte peroxldase must be destroyed (Lansdorp et al, 1980) Alkaline phosphatase-conjugated, affinity-purified rabbit immunoglobullns (Dakopatts, code no D314) and an alkaline phosphatase-conjugated F(ab')2 fragment preparation of sheep antibody (Sigma no A-0532) to mouse IgG were used

Enzyme substrate Disodium p-nltrophenyl phosphate (Sigma no 104-0) was dissolved in 0 1 M sodium carbonate-bicarbonate buffer, pH 9 6, with 1 mM MgC12

Spectrophotometer Tltertek ~ multiscan (Teknunc, Roskalde) measuring absorption directly in each well was used

Standard procedure The optimtzed procedure is described here The various factors optimizing the assay are described in the results section

(I) Coating of the mtcrottter plates (1) 100/~l PLL (0 1 m g / m l ) is placed in each well with manual multlchannel pipette

262

(2)

(3) (4)

(5) (6)

and incubated for 30 mln at 22°C The plate is emptied by turning it upside down and repeated blotting on suction-paper 100 /~1 erythrocyte suspension in PBS (5 × 106 cells/well) is added simultaneously to four wells (quadruplicate measurements) and allowed to settle for 30 m m at room temperature 100/~1 glutaraldehyde 0 1% in PBS is gently added and left for 30 mln at 22°C The glutaraldehyde is discarded and the plate washed with PBS-T each well is filled with 200 ~1, emptied after 3 mln and blotted on suction-paper, repeated 3 times Residual binding capacity of the polystyrene surface is occupied by incubation with 250 txl 1% BSA for 60 min at 22°C Unbound albumin is removed by 3 washings Same washing procedure as described above

(II) Incubating the erythrocvte coated plates wltt, antibodies (1) 100 /~1 monoclonal mouse antibody to human C3bR ( D A K O - C 3 b R ) diluted 1 100 in PBS is added to the wells and incubated for 60 min at 22°C (2) Wells are emptied and washed as described m I4 (3) Alkahne phosphatase-conjugated rabbit anti-mouse I g G is diluted 1 100 in PBS, and 100 ~1 added to each well and incubated for 60 rain at 22°C (4) Wells are emptied and washed as described in I4

(Ili) Colorlrnetrl~ reactton (1) 200 ~1 freshly prepared substrate solution (&sodmm p-mtrophenyl phosphate) is added to each well The enzymatic reaction is allowed to take place for 60 min (2) 150/~1 of the reaction product of individual wells is transferred to corresponding wells of another mlcronter plate, each well prefilled with 100 ~1 1 N N a O H The absorbance at 405 nm is measured

Results

(I) Coating of mlcrottter plates The binding of erythrocytes to the rmcrotiter plates was evaluated by the hemoglobin absorbance at 405 nm and by microscopy To determine the relation between the degree of fixation and hemolysis (or release) of erythrocytes reduced by the substrate solution, the colonmetrIc reaction was carried out on plates coated at different concentrations of glutaraldehyde, and without preceding incubation with antibodies It appears from Fig 1, that a final concentration of glutaraldehyde of 0 05% wa~ within the plateau of binding and prevented hemolysis The PLL concentration was varied between the limits of 0-1 m g / m l As shown (Fig 1) treatment of the plates with PLL (0 01 m g / m l ) increased the amount of RBC bound, increasing the absorbance from 0 4 to 1 0 PLL concentrations of 0 01-1 m g / m l gave almost identical results (not shown) To secure binding and fixation concentrations of 0 1 m g / m l of PLL and 0 05% of glutaraldehyde were selected These concentranons allowed binding and fixation of RBC m the range of 0 5 - 8 × 1 0 6 per well, and

263

°t as

°t 01

=

,\;-"'---.---"--T

d

0

0

Final conc of glutaraldehyde (wlv %) Fig ] Coating of the mlcrotHer plate The blndmg of RBC (4 × ]0 6 added/well) to the mlcrotlter plate at different final concentrations of glutaraldehyde and PLL at sero (©) and 0 1 m g / m l (e) was evaluated by the absorbance due to hemoglobin as measured at the end of the coating procedure (see Matermls and Methods) The degree of fixation appears from the hemolysxs reduced during a standard colonmetnc reaction at PLL 0 1 m g / m l ( I ) Mean and range of quadruplicate determinations

~20- 1.612-

¢/

i _

,/'/

°t/

2

,- 081

~ 0.4

051

3

4

5 6 7 8 RBC x lO'6per well

Fig 2 Coating of the microtiter plate at &fferent RBC numbers per well at conditions according to the standard procedure described m Materials and Methods The binding of RBC was evaluated by the absorbance due to hemoglobin (*) and by microscopy at the end of the coating Fixation of the cells appears from the lack of hemolysis during a standard colonmetrlc reaction (11) Mean and range of quadruphcate determinations

264

A4o5 0604-

+\

+\

02-

(~ 1:25 2'5 A 18 mM D'FT (final conc) Fig 3 Erythrocyies were treated with dithiothreilol (DTT) at concentrations as indicated along ihe abscissa (37°C 30 min) before examination of the antl-C3bR antibody binding (I) A control with a monoclonal antibody at equivalent concentration and with specificity against human IgM was performed m parallel (11) In this experiment the enzyme-labeled antibody Sigma A-0532 diluted 1 100 was used and the RBC number per well was 7 × 1 0 6 Other variables as described for the standard procedure in Materials and Methods Mean and range of quadruplicate determinations microscopy revealed, that a confluent monolayer was formed at 3 0 - 5 5 × 106 cells per well (Fig 2)

(II) Incubatton with anttbodles Speclftctt)' The reformation given by the manufacturer concerning specificity O! the monoclonal a n t i b o d y to the C 3 b R was supplemented by examining the antibody binding after removal of the C 3 b R by D T T (see Methods) The expected dose-dep e n d e n t loss of receptor ( D l e n c h et al. 1974, H o r g a n and Taylor, 1984) resulted m a gradual loss of binding of the antibody (Fig 3) In addition the monoclonal mouse a n t i b o d y against h u m a n I g M used as control at equivalent concentration showed no binding (Fig 3) Dose response The monoclonal mouse a n t l - C 3 b R antibody was added m increasing concentrations, and the enzymatic activity at different dilutions of the enzyme-labeled a n t i b o d y was measured (Fig 4) The binding of the monoclonal mouse a n t i b o d y against C 3 b R demonstrated saturation, and the absorbance levels varied proportional to the concentration of the enzyme-labeled antibody N o n specific absorption of the labeled anti-mouse a n t i b o d y and increasing variation was apparent at concentrations of 1 50 or higher The alkaline phosphatase-conjugated, affinity-purified rabbit antibody to mouse I g G showed less non-specific binding than the alkaline phosphatase-conjugated F(ab')2 fragment preparation of sheep a n t i b o d y to mouse IgG (not shown) Quantttauon of C3bR With a view to obtain a standard curve, tltratlons of the R B C used for standardization were made at antibody concentrations showing optimal dose responses Because (a) the n u m b e r of receptors varies by 8-fold a m o n g normal individuals, and the deficiency of the receptor m patients is within this range (Fearon, 1984), (b) preliminary measurements indicated, that the erythrocytes used for standardization had a high uptake of antx-C3b-receptor antibodies, and (c) a cell n u m b e r of about

,o]

265

A4o5

08

O6 0402-

Odut,onof DAKO-C3bR

Fig 4 Dose response of the monoclonal antl-C3bR antibody (DAKO-C3bR) and alkaline phosphataseconjugated rabbit antibody to mouse IgG Determmauons were performed m accordance with the standard procedure described m Matermls and Methods Absorbance is plotted against concentration of DAKO-C3bR • denotes 1 50, @, 1 100, D, 1 200 and O, mfimte ddut~on of the enzyme-labeled antibody Mean and range of quadruphcate determmatxons 5 × 106 p e r well was desired, the d e m a n d s on the assay was a t h r e s h o l d at a b o u t 0 5 x 106 cells per well a n d d i s c r i m i n a t i o n p o w e r in the range of a b o u t 0 5 - 6 x 106 R B C p e r well A s shown m F~g 5 a detecUon hrmt of a b o u t 10% a n d a dlSCrlmlnat~on a b o u t 10-120% of C 3 b R on 5 × 106 s t a n d a r d R B C c o u l d be o b t a i n e d b y using b o t h a n t l b o & e s at a d d u t l o n of 1 100 This d i l u t i o n was selected, a n d the s t a n d a r d was i n c l u d e d at deternunat~on of C 3 b R on test cells T h e i n c u b a t i o n t~me was v a n e d from 30 m l n to 24 h for b o t h a n t i b o d i e s F o r p r a c t i c a l reasons a n d b e c a u s e of a g o o d s i g n a l / n o i s e ratio i n c u b a t i o n for 1 h was chosen A l t h o u g h g l u t a r a l d e h y d e r e d u c e d the a n t l g e m c l t y of the r e c e p t o r at the selected c o n c e n t r a t i o n of 0 05% (Fsg 6), it a p p e a r s f r o m the a b o v e m e n t i o n e d , that the c o n c e n t r a t t o n c o u l d be k e p t at 0 05%, which gave safe b i n d i n g a n d ftxatlon of the R B C (Figs 1 a n d 2)

(111) Colortmetrtc reaction P N P P was tested at c o n c e n t r a t i o n s from 0 5 to 5 m g / m l C o n c e n t r a t i o n s at 2 m g / m l a n d higher c a u s e d hemolysis, whtch was unaffected b y r e n d e r i n g the b u f f e r i s o t o n i c with NaC1 A f t e r transfer the color p r o d u c t was stable for at least 1 h

(IV) Vartabthty of the test and calculatton of results T h e b e t w e e n - d a y d e t e r m i n a t i o n s gave a coefficient of v a r i a t i o n of 10 3%, a n d the w l t l u n - a s s a y l n t e r p l a t e a n d l n t r a - p l a t e coefficients of vartatlon were 9 7% a n d 6 2% respectively T h e s t a n d a r d was i n c l u d e d m each run, a n d the relative n u m b e r of C 3 b R on test

266

A4o5 09" 07°

05-

°'1 0!

RBC x10 -6 per w e l l Fig 5 Standard curve for C3bR obtained by tltratmg the erythrocytes used for standar&zat]on The assays were performed by the standard procedure as descnbed m the Matermls and Methods section Mean and range of quadruphcate determmattons

RBC expressed in percentages by calculatmg the absorbance at 405 nm ol each test sample (5 × 10 6 RBC/well) as a percentage of the absorbance of the standard sample at this RBC number Whole blood could be stored in C P D A solution at 4°C for at least 1 week wtthout change m antibody binding Sodium azlde is known to be without acute effects on the C3bR (D~erlch et al, 1974), and coated plates could be stored at 4°C for at least 2 months without loss of the receptor, tf 0 1% sodmm azlde was added to the BSA solution, which was left m the wells Coated plates with BSA left m the wells and kept at - 2 0 ° C , showed a moderate loss of receptor beyond one month

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~)0

0

0

F:ool eonc of glutoroldehyde (w/v °/.) Fig 6 Effect of fixation of RBC at &fferent concentrations of glutaraldehyde on subsequent binding of the antl-C3bR-antlbody The standard procedure descnbed un Materials and Methods was followed except that the enzyme-labeled antibody Sigma A-0532 diluted 1 100 was used Mean and range of quadruphcate determinations

267

110-

i

90-

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.,.

0

ID

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50-

!

'°1

•

~

1

; •

Is 'r •

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Normols

SLE

RA

AML

Fig 7 Erythrocytes from 15 normal individuals, 10 patients with systemic lupus erythematosus (SLE) 12 patnents wnth rheumatoid arthritis (RA) and 12 patients with acute myelold leukemm (AML) were tested by the standard procedure, and the results expressed m percentages of the standard RBC = median for each group The C3bR levels of the SLE patients are slgmficantly dnmxmshed ( P < 0 01)

(V) Values tn normal individuals and patients The assay was performed on erythrocytes from 15 healthy volunteers, 10 patients with at least 4 of the revised criteria for the diagnosis of SLE (Tan et al, 1982), 12 patients that satisfied the American Rheumatism Association criteria for classic or definite RA (Ropes et al, 1958), and 12 patients with AML according to the criteria defined by the French-American-British cooperative group (Bennett et al, 1976) (Fig 7) By application of the Mann-Whltneys test, the receptor levels were found significantly lower in SLE patients than those of normals ( P < 0 01) Corresponding figures for RA and AML were 0 05 < P < 0 10 and 0 01 < P < 0 05

Discussion

The purpose of the present study was to develop a specific, sensitive, reproducible and preferably non-radioactive procedure for determination of C3bR In addition the method should be suitable for further development with respect to investigation of experimentally or in VlVO C3bR-bound immune complexes The macro test tube ELISA system implies large consumption of sample material and reagents, loss of cells and tlme-consunung centrlfugation and resuspendlng of cells dunng washing procedures In addition erythrocytes are hemolysed by the substrate solution (Bruner and Klsshng, 1979, Lelkola and Perkins, 1980) These drawbacks are avoided by the microtlter plate system with fixed cells, and the fixed cells are stable to storage A particular disadvantage connected with fixed cells is the dose-dependent destruction of antigens induced by glutaraldehyde (Baron et al, 1977) However, the low concentration of glutaraldehyde used here (0 05%) was not prohibitive

268 According to the manufacturer's information and our control studies the monoclonal antibody D A K O - C 3 b R had specific reactivity to the C3bR (Gerdes et al, 1982, Fig 3) The monoclonal antl-C3bR antibody showed a saturating dose response (Fig 4) As in this assay, it may be impossible to achieve saturation with the second (enzyme-labeled) antibody, because enzyme-conjugated antibodies show non-specific absorption in low dilution (Fig 4), contrasting with a decrease in specific bmdlng efficiency as bmdmg sites become saturated (Masseyeff and Ferrua, 1980) In addition monoclonal antibodies are difficult to saturate or may be eluted at high concentrations of the second antibody (Lansdorp et al, 1980, Merry et al, 1984) We found absorbance levels proportional to the concentration of enzymelabeled antibody (F~g 4), and prozone phenomena could not be demonstrated (data not shown) The C3b receptor level is inherited as an autosomal codommant trait and vanes by about 8-fold m normals as well as in patients (Wilson et al, 1982, Fearon, 1984) A preponderance of low levels has been found among pattents with SLE (Mlyakawa et al, 1981, hda et al, 1982, Inada et al, 1982, Wilson et al, 1982, Taylor et al, 1983, Horgan and Taylor, 1984, Takemura et al, 1984), RA (hda et al, 1982, Taylor et al, 1983) and A M L (Takemura et al, 1984) As expressed by titration of the standard erythrocyte (Fig 6) and the determinations of C3bR in normals and patients (Fig 7), the selected concentrations of antlbodtes persued adequate sensitivity wtth respect to threshold and dlscrlmmatlon between erythrocytes wtth receptor numbers wtthm the relevant range Further, a high signal-to-norse ratio was achieved, and the varlabihty of the assay was acceptable It ~s concluded, that the method is statable for research purposes in chmcal s~tuat~ons Investigations of experimentally or tn VlVO C3bR-bound immune complexes are in progress (B S Thomsen and H Nielsen, in preparation) In princtple, the assay could be adapted to measurement of surface antigens of any nature

Acknowledgements The skllful techmcal assistance of Aase Bendlxen and Jette Bendtsen xs gratefully acknowledged This work was supported by grants from the National Danish Association against Rheumatic Dtseases

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269 Dlench, M P, S Ferrone, M A Pellegrlno and R A Relsfeld, 1974, J Immunol 113, 940 Dykman, T R , J A Hatch and J P Atkmson, 1984, J Exp Med 159, 691 Fearon, D T, 1980, J Exp Med 152 20 Fearon, D T, 1984, Fed Proc 43, 2553 Gerdes, J, M Nalem, D Y Mason and H Stem, 1982, Immunology 45,645 Horgan, C and R P Taylor, 1984, Arthritis Rheum 27, 320 hda, K , R Mornaghl and V Nussenzwelg, 1982, J Exp Med 155, 1427 Inada Y, M Kamlyama, T Kanemltsu, C L Hyman and W S Clark, 1982, Chn Exp Immunol 50 189 Lansdorp P M, G C B Astaldl, F Oosterhof, M C Janssen and W P Zeulemaker, 1980, J Immunol Methods 39 393 Lelkola, J and H A Perkins, 1980, Transfusion 20 138 Masseyeff, R and B Ferrua, 1980, in Immunoenzymatlc Assay Techniques, ed R Malvano (Martmus Nijhoff, The Hague) p 28 Medof, M E, G M Prince and J J - F Oger, 1982, Chn Exp lmmunol 48 715 Merry, A H, E E Thomson, B J Anstee and F Stratton 1984, Immunology 51,793 Mlyakawa Y, A Yamada, K Kosaka, F Tsuda, E Kosugl and M Mayuml, 1981, Lancet n, 493 Nelson, R A, 1953, Science 118, 733 Ropes, M W, G A Bennett, S Cobb, R Jacox and R A Jessor, 1958, Bull Rheum Dis 9, 175 Siegel, I, T L Lm and N Glelcher, 1981, Lancet n, 556 Slm E and R B Slm, 1983, Blochem J 210, 567 Stultmg, R D and G Berke, 1973, J Exp Med 137, 932 Takemura, S, M Deguchl, M Ueda, N Yoshlda, H Kato, T Yoshlkawa, S Sugmo and M Kondo, 1984, Immunol Lett 7, 325 Tan, E M, A S Cohen, J R Fries, A T Masl, D J McShane, N F Rothfield J G Schaller, N Talal and R J Winchester, 1982, Arthrms Rheum 25, 1271 Taylor, R P, C Horgan, R Buschbacher, C M Brunner, C E Hess W M O'Bnen and H J Wanebo, 1983, Arthritis Rheum 26, 736 Voller, A , D E Bldwell and A Bartlett, 1976, Bull WHO 53, 55 Wilson, J G , W W Wong, P H Schur and D T Fearon, 1982, N Engl J Med 307, 981