Langmuir 1995,11, 1822-1826
1822
Real-Space Differentiation of IgG and IgM Antibodies Deposited on Microtiter Wells by Scanning Force Microscopy C. J. Roberts,*tt P. M. Williams," J. Davies,# A. C. Dawkes,# J. Sefton,# J. C. Edwards,# A. G. Haymes,# C. Bestwick,? M. C. Davies,*st and S. J. B. Tendler*$t Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, Nottingham NG7 2RD, U.K., and Kodak Clinical Diagnostics Ltd., Pollards Wood Laboratories, Nightingales &ane, Chalfont St. Giles, Buckinghamshire HP8 4SP, U.K. Received December 15, 1994. I n Final Form: February 6, 1995@ Enzyme-linkedimmunosorbent assay as carried out in microtiter wells is a common method of identifying the presence of specific biomolecules in human samples to allow diagnosis of medical conditions. The presence, in a minority of samples,ofhuman antibodies reactive with the immunoassay-specificantibodies is a well-known phenomenon (Maxey, K. M.; Maddipati, K. R.; Birkmeir, B. J. Clin. Immunoassay 1992, 15, 116). These antibodies are often referred to as heterophilic antibodies or rogue samples and are commonly of the IgM isotype (Levinson, S. S. Clin. Biochem. 1992,25,77). The presence of such antibody species within a sample can result in incorrect analyte determination and clinical misclassification. We have employed scanning force microscopy (SFM)to study microtiter wells coated with a passively adsorbed IgG antibody that is specific for human IgM in order to determine if SFM is an appropriate technology to discriminate between these two biomolecular types and hence useful in the evaluation of interactions between IgG coated on microtiter wells and IgM in human sample fluids. It should be noted that in this model system, IgG that specifically binds human IgM has been employed. In a true immunoassay system where interference occurs, the IgM normally binds to IgG specific for the analyte being measured. SFM data clearly differentiate between IgM bound by IgG and any free IgG. The observed molecular diameter for the IgG antibody was approximately 33 nm, and that for the IgG-IgM antibody complex was approximately 53 nm. While these observations are made from traditional raw SFM data images,we also demonstrate here the potential value of removing tip profile information from the data to allow more accurate determination of molecular dimensions (28 nm for the IgG and 47 nm for the IgG-IgM complex).
Introduction The use of polystyrene microtiter wells for enzymelinked immunosorbent assay (ELISA) has become commonplace in recent years1to test for the presence of specific biomolecules in human body fluids, in order to expedite the diagnosis of patient conditions. Occasionally, however, such tests give a falsely elevated or depressed reading due to the presence of human anti-mouse antibodies which can interfere in the immunoassay. In this study, we have developeda model system using scanning force microscopy (SFM) to determine if a n interaction between IgM and IgG can be visualized and if this technique has sufficient sensitivity to discriminate between these two biomolecules. While we shall show that these observations are easily made from traditional raw SFM data, we also demonstrate the value of removing tip profile information from the data to allow more accurate determination of molecular dimensions. From X-ray crystallographic data, it is known that a n IgG antibody (MW 146 000) is arranged in three discrete domains, two Fab fragments and one F c . ~ -It~ is also known that the hinge region between the Fc and two Fab domains is extremely flexible; as a consequence, it is
' The University of Nottingham.
* Kodak Clinical Diagnostics, Ltd.
Abstract published in Advance A C S Abstracts, April 1, 1995. (1)Kenny, D. M.; Chantler, S. InAn introduction toELISA inELISA and other solidphase immunoassays. Theoretical andpractical aspects; Kenny, D. M., Challacanbe, S. J., Eds.; John Wiley: Chichester, U.K., 1988. (2) Silverton, E. W.; Navia, M. A.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A., 1977,74,5140. (3)Amzel, L. M.; Poljak, R. J . Annu. Reu. Biochem. 1979,48,961. (4) Harris, L. J.; Larson, S. B.; Hasel, K. W.; Day, J.; Greenwod, A.; McPherson, A.Nature 1992,360,369. @
0743-746319512411-1822$09.00/0
difficult to predict the exact conformation a n IgG would adopt when adsorbed to a surface and hence its size when adsorbed. An estimate of the dimensions as would be observed by SFM can be made fromX-ray crystallographic work on isolated Fab fragments5and electron microscopy images6,' giving an expected maximum molecular dimension on the surface of -16- 19 nm. Previously, using SFM and surface plasmon resonance, we demonstrated that the proportion of an antibody-coated surface that is functional (i.e., able to bind antigen) can be determined.8,9 IgM antibodies are present in blood serum a t concentrations from 0.5 to 2.0 mg/mL. A small proportion of IgM molecules in some sera can cause interference in immunoassay by binding to the assay-specific immunoglobulins. The IgM antibody is a large pentameric molecule (MW r~ lo6) composed of 21 distinct peptide chains linked by 99 disulfide bridges with expected molecular dimensions as observed by electron microscopy of -30-39 nm diameterloand -35-37 nm height by X-ray solution scattering with molecular mode1ing.'lJ2 In an (5) Brady, R. L.; Hubbard, R. E.; King, D. J.; Low, D. C.; Roberts S. M; Todd, R. J . J. Mol. Biol. 1991,219, 603. (GIValentine, R. C.; Green, N. M. J. Mol. Biol. 1967,27,615. (7)Wrigley, N. G.; Brown E. B.; Skehel, J. J. J . Mol. Biol. 1983,169, -7, I I I .
(8)Davies, J.; Dawkes A. C.; Haymes A. G.; Roberts C. J.;Wilkins, M. J.; Davies, M. C.; Tendler, S. J. B.; Jackson, D. E.; Edwards, J . C. J . Immunol. 1994,167,263. (9) Davies, J.; Roberts C. J.; Dawkes A. C.; Sefton, J.; Edwards, J. C.; Glasbey, T. 0.; Haymes A. G.; Davies, M. C.; Jackson, D. E.; Lomas, M.; Shakesheff, K. M.; Tendler, S. J. B.; Wilkins, M. J.; Williams, P. M. Langmuir 1994,10,2654-2661. (10)Parkhouse, R. M. E.; Askonas, B. A,; Dourmashkin, R. Immunology 1970,18,575. (11)Wilhelm, P.; Pilz, I.; Schwarz, E.; Mihaesco, C.; Mihaesco, E. Int. J. Biol. Macromol. 1984,6, 273.
0 1995 American Chemical Society
Differentiationof IgG and IgM on Microtiter Wells
Langmuir, Vol. 11, No. 5, 1995 1823
Figure 1. SFM scan of a naked microtiter well showing large granular features due to impregnated white pigment particles and the ribbed appearance resulting from the polystyrene. w
immunoassay where heterophilic species are present, the IgM often binds to the heavy chain region of the immunoassay-specific IgG and thus may add to the apparent size of any IgM features as observed by SFM. In this model system, we have employed a n IgG molecule that specifically binds to a heavy chain region in a n IgM such that the overall molecular dimensions of the complex will be similar but the orientations of the individual immunoglobulins may differ. Probe microscopes such as the SFM represent ideal tools for molecular resolution studies since they allow threedimensional surface topographical information at high resolution to be obtained. However, the successful application of SFM to biological problems requires a number of critical issues to be addressed. Most importantly, molecular movement caused by tip-sample interactions15-17needs to be overcome by the development of appropriate methods of sample preparation. In this study, we have employed a physically robust coating to immobilize the antibodies. Such a coating precludes the possibilty of obtaining images with detailed submolecular features but was required to avoid excessive sample damage during imaging. l8 Thus, the coating procedure facilitated the imaging process and allowed the successful differentiation of antibody types as was required in this study. Since SFM data inevitably contain information from both the probe and the sample, surfacefeatureswill always be overestimated in dimensions. Recently, several techniques for modeling probe profiles and then estimating the increase in feature size resulting from such a probe have been suggested. Keller and Frankelg and other ~ o r k e r s ~ Ohave - ~ ~shown that the effects of finite tip geometry can be removed from a n image surface to
*+-
.I* s.
13914
(12)Perkins, S.J.; Nealis, A. S.; Sutton, B. J.; Feinstein, A.J. Mol. Biol. 1991,221,1345. (13)Engel, A. Annu. Rev. Biophys. Biophys. Chem. 1991,20,79. (14)Marti, 0.; Amrein, M., Eds. STM and SFMin Bio1ogy;Academic Press: San Diego, 1993. (15)Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B.; Kreusel, K. M.; Wilkins, M.J.;Williams, P. M. In Polymer Surfaces and Interfaces II; Feast, W.J., Munro, H. S., Richards, R. W., Eds.; John Wiley: Chichester, U.K., 1993;pp 227-245. (16)Wilson, T. E.;Murray, M. N.; Ogletree, D. F.; Bednarski, M. D.; Cantor, C. R.; Salmeron, M. B. J. Vac. Sci. Technol. 1991,B9, 1171. (17)Roberts, C. J.; Wilkins, M. J.; Beamson, G.; Davies, M. C.; Jackson, D. E.; Scholes, P. D.; Tendler, S. J. B.; Williams, P. M. Nanotechnology 1992,3,98. (18)Wilkins, M. J.; Davies, M. C.; Jackson, D. E.; Mitchell, J. R.; Roberts,C. J.; Stokke, B. T.; Tendler, S. J. B. Ultramicroscopy 1993, 48, 197. (19)Keller, D. J.; Franke, F. S. Surf Sci. 1993,294,409. (20)Markiewicz, P.;Goh, M. C. Langmuir 1994,10, 5. (21)Williams, P.M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B.; Wilkins, M. J. Nanotechnology 1991,2,172.
Figure 2. (a) SFM image showing a microtiter well surface after passive adsorption of IgG antibodies and contact with IgM antibodies. The circular featuresdispersed over the surface correspond to the antibodies, the larger mounds (diameter 53 nm, height = 15 nm) equate to the IgM molecules and the smaller ones to the IgG (diameter = 33 nm, height = 10 nm). (b, c) Higher resolution scans again showing the antibodies. Some distortion in the shape of the antibodies is apparent in the scan direction (left to right) due to frictional effects.
reconstruct a more accurate representation of the sample. These techniques rely on a prior knowledge of the tip geometry before a reconstruction can be attempted, and commonly, tip geometry is assumed from the manufacturer’s data, derived from the imaging of “standard samples” or inferred from electron microscopy. Here we investigate the employement of novel algorithms within G e n e s i ~ I that I ~ ~ derive the bluntest possible tip profile from each scan by finding the common volume of sample (22)Glaseby, T. 0.; Batts, G. N.; Davies, M. C.; Jackson, D. E.; Nicholas, C. V.; Roberts C. J.; Tendler, S. J. B.; Williams, P. M. Surf Sci. Lett. 1994,318,L1219-1224. (23)Williams, P.M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Shakesheff, K. M.; Tendler, S. J. B., submitted to Langmuir. (24)Williams, P. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B. J. Vac. Sci. Technol. 1994,B12,1456.
1824 Langmuir, Vol. 11, No.5, 1995
Roberts et al.
Figure 3. (a-c, left, top to bottom) Contour plots of probable probe profiles derived respectively from the SFM images in Figure 2a-c (scalebars 30,18, and 15 nm, respectively).Having obtained a probe profile, the original image may be deconvoluted to remove information due solely to the probe. Such images are shown in the right column (d-f, top to bottom). A comparison of these scans with the originals in Figure 2 shows that while the distribution of the features remains the same, some reduction in size has taken place.
below each maxima in the image data.23g25This probe profile is then used to reconstruct a n image.lg The reconstructed data are then analyzed in the same way as the raw data.
mode under ambient conditions with a Polaron SP300 (VG Microtech, Uckfield, U.K.), using forces of approximately 1nNor less and probe cantilevers with spring constantsbetween 0.032 and 0.064 N m-l (Park Scientific, Sunnyvale, CAI. Sample Preparation. The microwells used in this study were white Immulon microtiter wells from Dynatech (Billingshurst, U.K.). Wells were coated with streptavidin
by Kodak Clinical Diagnostics Ltd. (KCDL). Murine IgG, anti-IgM monoclonal antibody, was raised and prepared at KCDL. Purified human IgM was obtained from Calbiochem (Nottingham, U.K.). The monoclonal IgG antibody was biotinylated using standard procedures.26 The biotinylated IgG antibody-coated wells were prepared by adding 250pL of a 100mM potassium phosphate buffer solution containing 1pg/mL biotinylated antibody to streptavidin-coated wells. Following a 30 min incubation at 37 "C on a n Amerlite shaker incubator, the wells were washed with deionized water. After the washing, 250 pL of a 100 mM potassium phosphate buffer solution containing IgM at 1pg/mL and bovine serum albumin (2% w/v) was added to the wells, and a second incubation at 37 "C for 30 min was carried out on a n Amerlite shaker
(25) The probe derivationroutinesillustrated here and other routines within GenesisI121 are available on World-Wide Web server URL http://pharrnl.nott.ac.uk.
(26)Bayer, E. A.; Wilchek, M. In Methods in Enzymology. Protein BiotirzyZation;Wilchek, M., Bayer, E. A., Eds.; Academic Press: New York, 1990; pp 138-159.
Materials and Methods
SFM Analysis. SFM data were recorded in the contact
Langmuir, Vol. 11, No. 5, 1995 1825
Differentiationof IgG and IgM on Microtiter Wells
incubator. The concentration of IgM was chosen so as to saturate all possible IgG binding sites upon the well surface and not necessarily to mimic clinical ELISA conditions. The bovine serum albumin was required only as an IgM conformational stabilizer and does not interact with the bound IgG. After the second incubation, the wells were washed again with deionized water, and the bottom section was cut from each for imaging. For SFM analysis, the wells were coated with a 4-5 nm layer of Pt/C using a Polaron E6000 electron beam rotary evaporator at a Torr. The average Pt/C grain size was pressure of found to be 3-4 nm. SFM Data Analysis. The dimensions and coverages for the antibody and antibody complexeswere determined from data sets containing dispersed molecules using the GenesisI1computational graphics e n v i r ~ n m e n tData . ~ ~ ~ ~ ~Figure ~ ~ 4. Three-dimensional image of a derived probe profile (from the data set in Figure 2b), showing an approximately sets were passed through a Kirsch edge detector filter pyramidal shape with some distortion in the scanning direction matrix operator which is sensitive to gradients in data. due to friction effects. Enclosed areas were then filled to identify molecular features, and the areas were determined. A plot of feature size distributions was then made to distinguish features with different size ranges. In addition to analyzing the raw data, we have employed novel algorithms within Genesis I1 to derive a probable tip profile from each image without prior assumptions and then use this to reconstruct an image.23*25The reconstruction algorithm uses a method similar to that describedby Keller and Franke,lgwhere the reconstructed surface is calculated as being equal to the surface (or envelope)of the three-dimensional space not occupied by the probe as it formed the image. These newly reconstructed image data are then treated in the same way as 0 the raw data to derive size distributions.
Results and Discussions Controls. Surface features relating solely to the microtiter wells were identified by extensive imaging of the wells using SFM. Figure 1 shows a representative SFM image of the well surface. The two most prominent types of structures observed were large particulate matter and long parallel ridges. The particles, which are up to several hundreds of nanometers in diameter, are features of the polystyrene surface and originate from particles of pigment used in the formulation of the wells. The ridges observed probably result from the manufacturing mould process used to fabricate the microtiter wells. Images detailing the appearance of the well surfaces after being coated with streptavidin and IgG antibodies have been shown e l s e ~ h e r e . ~These .~ data allow the confident assignment of features within the data presented here as being microtiter well, IgG, or IgM related. IgGDgM-CoatedWells. The SFM images in Figure 2 are a typical set of scans recorded from a well after deposition of the IgG and introduction of the IgM. The images show background features similar to those in Figure 1; however, there are also distinct spherical features evenly spread over the well surface. Visual inspection of the scans in Figure 2 suggests that there are two distinct sizes of the surface features, with average diameters of approximately 33 and 53 nm and respective heights of around 10 and 15 nm. Such images were obtained for a number of samples, all of which showed uniform surface coverage of similar spherical features. The smaller features observed in Figure 3 are believed to result from the IgG-type antibody and the larger from the IgM antibody bound to the IgG. While these raw SFM data clearly differentiate the antibody types, they are nevertheless somewhat distorted by the inclusion of probe profile data. The finite size of the probe inevitably broadens the feature sizes. In a n
Feature diameter (nm) 40
,
1
Feature diameter (nm)
Figure 5. Feature size distribution plots from (a) raw SFM data and (b) reconstructed SFM data. The measurements have been made from a number of data sets containing over 300 molecular features. Both plots show two size ranges related to the IgG and the larger IgG-IgM complexedmolecules.However, in the case of the reconstructed data, the molecular sizes have been down-shifted due to the removal of some probe effects. attempt to reduce this effect, but not to reveal additional topographic detail, the data shown in Figure 2 were processed to yield probable probe profiles from each image (Figure 3a-c), which were then used to deconvolute the raw images (Figures 3d-0. The predicted probe profiles are approximately pyramidal, as would be expected for a silicon nitride probe (Figure 4). A visual comparison of the raw and reconstructed data shows the features in the reconstructed image to be smaller and more circular. This can confirmed by size distribution plots for each data set
Roberts et al.
1826 Langmuir, Vol. 11, No. 5, 1995 Table 1. Comparison of the Molecular Dimensions of IgG and IgM as Esimated from Electron Microscopy, X-ray Crystallography and X-ray Solution Scattering with SFM Data antibody t.ne
estd size from literature (nm)
av size from raw SFM data (nm)
av size from reconstructed d a t a (nm)
33 28 16-19 IgG 53a 47a 35-39 IgM a IgM is bound to IgG, which may affect the observed dimensions.
(plus several others), as shown in Figure 5. Both of the histograms in Figure 5, which are built up from measurements from over 300 molecules, distinguish two populations of features, but with the reconstructed data yielding smaller diameters at the maxima, e.g., 28 and 47 nm compared to 33 and 53 nm for the raw data. These data confirm the earlier assertion of two distinct size ranges of surface features and their identification as the IgGtype antibody for the smaller and the IgM antibody bound to the IgG for the larger features. When the SFM data are compared with molecular dimensions predicted from other biophysical techniques, there appears to be some overestimate in sizes (Table 1). While the reconstructed data are somewhat closer to the expected values, there is still some overestimate. We believe that this residual increase is most probably due to the metallic overlayer, which will also increase the size of the surface features.
Conclusions Scanning force microscopy has been employed to give a real-space differentiation of two antibody types on a microtiter well surface, specifically IgG and IgM. Previously, using SFM and surface plasmon resonance, we have demonstrated that the proportion of a n antibody-coated surface that is functional can be d e t e ~ m i n e d .Raw ~ SFM data were processed to give probable SFM probe profiles and reconstructed data sets. This novel data treatment reduced distortions due to the probe effects and gave a diameter of 28 nm for the IgG antibody and 47 nm for the IgG-IgM antibody complex. Application of the methodologies described here to immunoassays where sera-coatinghuman anti-mouse IgG activity have been used will permit visualization of IgMmediated surface IgG interference. This, along with previously described methods for determination of surface functionality: will allow quantification of the proportion of functional surface antibody no longer available to bind antigen. The potential impact of these sera on clinical assessments by immunoassay and the likelihood of clinical misclassification can than be evaluated. Acknowledgment. We would like to acknowledgethe support of the SERC/DTI Nanotechnology LINK Programme with Kodak Ltd., Oxford Molecular Group plc, and VG Microtech, as well as the SERC/DTI Link Protein Engineering Programme with Glaxo Group Research and VG Microtech Ltd. LA941009F
