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Loss of ELISA specificity due to biotinylation of monoclonal antibodies
Journal of Immunological Methods 235 Ž2000. 91–99 www.elsevier.nlrlocaterjim
Loss of ELISA specificity due to biotinylation of monoclonal antibodies Gunilla Høyer-Hansen a,) , Maria J.A.G. Hamers a , Anders N. Pedersen a , a , Keld Danø a , Ross W. Stephens a Hans Jørgen Nielsen b, Nils Brunner ¨ a
The Finsen Laboratory, Copenhagen UniÕersity Hospital, StrandbouleÕarden 49, DK-2100 Copenhagent Ø, Denmark b Department of Surgical Gastroenterology, HÕidoÕre Hospital, Denmark Received 2 June 1999; received in revised form 27 September 1999; accepted 25 October 1999
Abstract A significant degree of nonspecificity was found in ELISA determinations of soluble urokinase receptor ŽsuPAR. in human blood plasma when biotinylated monoclonal antibodies ŽMabs. were used for the detection layer. Surface plasmon resonance studies using both nonbiotinylated and biotinylated antibodies demonstrated that biotinylation reduced specific binding of the antibodies to their target antigen, suPAR. Furthermore, biotinylation produced a new interaction with unknown human plasma proteinŽs., unrelated to suPAR. Nonspecific interaction with plasma proteinŽs. was also observed after biotinylation of a Mab having no specific target antigen in human plasma and, in both cases, the level of nonspecific interaction was directly related to the degree of antibody biotinylation. These results reinforce earlier observations that biotinylation of antibodies can reduce the affinity of antibodies, but also indicate that, in addition, biotinylation can reduce the specificity of immunoassays for plasma proteins. q 2000 Elsevier Science B.V. All rights reserved. Keywords: ELISA specificity; Biotinylated antibodies ; Biotinylation ratio; Surface plasmon resonance
1. Introduction Biotinylation of antibodies and many other biological probes has been of great value, enabling sensitive and quantitative detection of specific antigens and other target molecules in a wide variety of analytical procedures such as immunohistochemistry, immunocytochemistry, immunoblotting, and ELISA,
AbbreÕiations: Mab, monoclonal antibody; PBS, phosphate buffered saline; RAM, rabbit anti-mouse antibodies; RU, resonance unit; suPAR, soluble form of urokinase receptor; uPAR, urokinase receptor; TNP, trinitrophenyl hapten ) Corresponding author. Tel.: q45-35-45-5874; fax: q45-3538-5450; e-mail:[email protected]
as well as nucleic acid analysis in Northern blotting, PCR techniques, chromosome painting and gene linkage studies. There have been widespread applications also in the investigation of ligand interactions with cell surface receptors, and particularly in screening for antagonists of such interactions, in order to discover new molecules for development as potential drugs. In all these techniques, specific binding of the biotinylated probe can be detected by high affinity binding of appropriately labeled conjugates of avidin or streptavidin. Implicit in these diverse applications of biotinylated probes is the assumption that biotin substitution of the specific probe does not significantly alter its specificity or affinity for the target molecule, and especially that it does not in
0022-1759r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 2 2 2 - 7
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itself generate new interactions with other nontarget components of the system. However, the question whether these ideal properties of biotinylated reagents can be relied upon in all applications is often not considered, and particularly in relation to the effect that the degree of biotin substitution may have on the specificity of a given probe. It has been common practice not to report the conditions employed for biotinylation of immunoassay reagents, such as, for example, the molar ratio of reactive biotin to IgG in the case of antibodies. Authoritative laboratory handbooks Že.g., Harlow and Lane, 1988. recommend that biotin ester is added to antibody at a molar ratio of from 10 to 100 Ži.e., 25–250 mg of ester per milligram of antibody protein.. However, the use of higher molar ratios, even over 1000:1, in order to enhance sensitivity, may be widespread. The increase in biotin substitution of the antibody obtained with increasing molar ratios of reagent is not routinely monitored or reported, and the actual final ratio of biotin substitution obtained is unknown in virtually all cases. The discussion of problems arising from biotinylation of antibodies has so far mainly been confined to loss of affinity ŽGhose et al., 1983; Pearson et al., 1998.. However, there has been at least one report suggesting that biotinylation of antibodies, or even gelatin, can produce interactions with unknown human plasma proteins, leading to loss of specificity in plasma immunoassays employing biotinylated reagents ŽDale et al., 1994.. This finding indicates that the use of biotinylated reagents for analysis of human blood components may give rise to specificity problems. We report here loss of specificity due to biotinylation of a monoclonal antibody ŽMab. in ELISA determinations of soluble urokinase receptor ŽsuPAR; see review by Behrendt and Stephens, 1998. in human plasma. suPAR occurs in the plasma of healthy individuals ŽRønne et al., 1995. at a concentration of only approximately 1 mgrl ŽStephens et al., 1997., but it may be elevated in patients with several types of cancer ŽStephens et al., 1997, 1999; Sier et al., 1998; Mustjoki et al., 1999., as well as patients with rheumatoid arthritis ŽSlot et al., 1999.. The issue of specificity in the assay of such a low concentration plasma component becomes crucial when it is evaluated for prognostic significance
ŽStephens et al., 1999., and even more so when contemplating the application of this parameter in cancer patient management, particularly in identifying high-risk patients who may most benefit from adjuvant chemotherapy. It was found that an ELISA developed for measurement of uPAR in detergent extracts of tumor tissue, which employs biotinylated antibodies, was unsuitable for ELISA of suPAR in blood samples. The plasma suPAR measurements made using biotinylated antibodies were distorted by degeneration of antibody specificity. We also report surface plasmon resonance analysis of biotinylation-dependent interactions of Mabs with human plasma proteins. The results indicate that the use of biotinylated Mabs for sensitive immunoassays of proteins which occur at only low concentrations in blood give rise to false readings due to nonspecific interactions.
2. Materials and methods 2.1. Blood collections and plasma separation Blood samples from 44 healthy donors were obtained through the co-operation of the blood bank at the Hvidovre University Hospital, Copenhagen. Peripheral venous blood was drawn into pre-chilled EDTA collection tubes ŽBecton-Dickinson, Mountain View, CA. and quickly mixed by inversion. The plasma was separated from blood cells within 1.5 h by centrifugation at 48C at 1200 = g for 30 min, and stored frozen at y808C prior to assay. A plasma pool was made with freshly collected samples from ten donors, aliquoted and stored frozen at y808C. For ELISA measurements plasma was diluted 1r10, and for real-time biomolecular interaction analyses using Biacore, plasma was diluted 1r100. 2.2. suPAR ELISA Immunoassay plates ŽMaxisorp, Nunc, Roskilde, Denmark. were coated for 16 h at 48C with 100 mlrwell of purified rabbit anti-human uPAR Ž0.5 mg IgGrl. in 0.1 molrl carbonate buffer, pH 9.5. This capture antibody was previously absorbed on a column of mouse IgG in order to reduce the ELISA background. Prior to use, the assay wells were rinsed
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twice with 200 mlrwell of SuperBlocke solution ŽPierce Chemicals, Rockford, IL. diluted 1r2 with phosphate-buffered saline ŽPBS., followed by three washes with PBS containing 1 grl Tween 20. Wells were then treated for 1 h at 378C with 100 mlrwell of triplicate or duplicate 1r10 dilutions of plasma made in a sample dilution buffer of 50 mmolrl phosphate buffer, pH 7.2, 0.1 molrl NaCl, 10 grl bovine serum albumin ŽFraction V, Boehringer– Mannheim, Penzberg, Germany. and 1 grl Tween 20. On every assay plate, a series of standards was included, which consisted of seven serial dilutions in triplicate of purified recombinant suPAR Ži.e., uPAR lacking the glycolipid anchor; see Rønne et al., 1994., starting from 1 mgrl, then 0.5, 0.25, 0.125, 0.0625, 0.0313 and 0.0156 mgrl. Also included on each plate were triplicate blank wells containing only sample dilution buffer, and triplicate wells of a 1r10 dilution of a control citrate plasma pool. After suPAR binding the wells were washed six times, then treated for 1 h at 378C with 100 mlrwell of a mixture of three murine monoclonal anti-human uPAR antibodies wR2 Ž23 mgrl., R3 Ž281 mgrl., and R5 Ž70 mgrl. ŽRønne et al., 1995.x in sample dilution buffer. These antibodies were used either after biotinylation or without any biotinylation as described below. After six washes, the wells were then incubated for 1 h at 378C with 100 mlrwell of either rabbit anti-mouse immunoglobulinsralkaline phosphatase conjugate ŽDako, Glostrup, Denmark. diluted 1r1000 Žfor nonbiotinylated Mabs., or streptavidin conjugated alkaline phosphatase ŽDako. diluted 1r1000 Žfor biotinylated Mabs.. After six washes with washing solution, and three washes with pure water, 100 ml of freshly made p-nitrophenyl phosphate ŽSigma, St. Louis, MO. substrate solution Ž1.7 grl in 0.1 molrl Tris–HCl, pH 9.5, 0.1 molrl NaCl, 5 mmolrl MgCl 2 . was added to each well and the plate was placed in a Ceres 900J plate reader ŽBioTek Instruments, Winooski, VT.. The yellow color development at 238C was monitored automatically, with readings taken at 405 nm against an air blank every 10 min for 60 min. KinetiCalc II software was used to manage the data, calculate the rate of color change for each well Žlinear regression analysis. and compute from the rates for the suPAR standards a four-parameter fitted standard curve, from which the suPAR concentration of each plasma sample was
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calculated. Development of color in each well was a linear function of time for all concentrations of suPAR measured in these experiments, with correlation coefficients for the automatically fitted lines typically better than 0.97. The standard curve of the rates plotted against the suPAR concentration was slightly sigmoidal from 0 to 1.0 mgrl and the correlation coefficient for the four-parameter fit was typically better than 0.999. 2.3. Biotinylation of antibodies The Mabs were biotinylated using a modification of a widely used method first described by Guesdon et al. Ž1979.. The biotin was supplied as biotinamidocaproate N-hydroxy-succinimide ester ŽSigma.. The final concentration of the purified antibodies in the biotinylation mixture was 1 grl, and the molar ratios between biotin and Mab was 11.6:1, 116:1 and 342:1 for anti-TNP, and 116:1 and 342:1 for antiuPAR clone R5. Biotinylated antibodies were diluted 1r2 with pure glycerol ŽSigma G-5516. and stored at y208C. The change in biotin substitution of antibody obtained using the different molar ratios of biotin ester was monitored by ELISA. In brief immunoassay plates were coated for 20 h at 48C with 100 mlrwell of 10 mgrl of affinity-purified rabbit antimouse immunoglobulins ŽDako.. After blocking for 1 h with 10 grl skimmed milk powder in PBS, the plates were incubated with samples of biotinylated antibody in serial dilutions from 5 to 0.04 mgrl. Bound antibody was detected with peroxidase coupled streptavidin ŽDako., and development with ophenylenediamine as substrate. 2.4. Surface plasmon resonance analyses All binding measurements were performed employing the BIAcoree 2000 instrument ŽBIACORE, Uppsala, Sweden.. Rabbit anti-mouse antibodies ŽRAM Fc g, BIACOREe. were immobilized on the sensor chip CM 5 ŽBIACOREe. at a concentration of 30 mgrl using the amine coupling method according to the procedure provided by the manufacturer. In the experiments, Mabs Ž0.2 mmolrl. were bound to the immobilized RAM Fc g. Then either suPAR Ž0.2 mmolrl. or diluted plasma Ž1r100. was added
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and interaction with the antibodies was measured. The plasma dilution was chosen such that the signal representing the nonspecific binding from the blank channel on the sensor chip was below 20 resonance units ŽRU.. The amount of protein bound to the anchored antibodies was calculated using Biaevaluation 2.1 software. The chip was regenerated with 1 molrl formic acid.
3. Results When suPAR was measured in EDTA plasma from healthy donors using an ELISA with biotinylated detection Mabs, a positively skewed distribution of the suPAR signal was found, with several patient samples contributing to a high-end tail Žsee Fig. 1a.. This distribution was studied further by examining the contribution of individual detection Mabs to the total signal. It was found ŽFig. 1a. that the skewness was primarily due to the contribution of one antiuPAR Mab, biotinylated R5, which recognizes an epitope on the N-terminal domain 1 of suPAR. The contribution of another Mab, biotinylated R2, which recognizes an epitope on domain 3, was noticeably disproportionate to R5 across the spectrum of samples. A simple regression plot revealed that no real correlation existed between the two antibody signals ŽFig. 1b.. This result was unexpected, since each uPAR molecule should contain one binding site for each of the antibodies. A set of plasma samples from healthy individuals was then assayed twice with the same basic ELISA protocol, but using biotinylated and nonbiotinylated detection antibodies in parallel. In the latter case, bound nonbiotinylated Mab was quantified using an alkaline phosphatase conjugate of a RAM. Both ELISAs were calibrated with a reference standard of purified recombinant suPAR. As shown in Fig. 2a, the distribution of uPAR levels differed considerably, depending on the detection antibody. With biotinylated Mabs the levels of uPAR in plasma extended over five times the range of that obtained with nonbiotinylated detection Mabs. Furthermore, there was no correlation between the levels determined using biotinylated and nonbiotinylated Mabs, as shown in Fig. 2b.
Fig. 1. Ža. ELISA of suPAR in 36 EDTA plasma samples, measured using a combination of two biotinylated monoclonal detection antibodies, R2 and R5 ŽI., with biotinylated R2 alone Ž=., and with biotinylated R5 alone Ž'.. The signals are expressed as rates Žmilliabsorbance units at 405 nmrmin. obtained for the alkaline phosphatase reaction, and plotted here as percentiles. Note the pronounced skewness of the distribution, due to the contribution of the biotinylated R5 antibody, which is not found when biotinylated R2 is used alone. Žb. Correlation plot for the data shown in Fig. 1a. The signal obtained in suPAR ELISA with biotinylated R2 detection antibody is plotted against the signal obtained from the same sample using biotinylated R5 as the detection antibody. No correlation was found using regression analysis.
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Mab was used in a more heavily biotinylated form than R2, it was suspected that biotinylated Mabs could be giving rise to a false signal, and experiments were designed to test the specificity of the Mabs before and after biotinylation. In order to verify that increasingly large molar ratios of a biotinylation reagent do actually produce an increase in the amount of biotin molecules covalently attached to Mabs, we used a simple ELISA. The capture antibody consisted of affinity-purified rabbit anti-mouse IgG. A dilution series of the substituted Mab obtained after biotinylation using different molar ratios was then applied. The detector was peroxidase coupled streptavidin, which binds very strongly Ž K D s 10y1 5 molrl; Guesdon et al., 1979. to biotin molecules, so that the more biotin covalently attached to the Mab the higher the ELISA signal obtained. In addition to the molar ratio of biotinylation reagent employed, the degree of biotinylation obtained with a given Mab is also dependent on the number of accessible free amino groups of lysines in the Mab, which clearly differs between different Mabs.
Fig. 2. Ža. Comparison of plasma suPAR levels determined by ELISA with biotinylated and nonbiotinylated detection antibodies. Each of 44 EDTA plasma samples was assayed using ELISA with a mixture of three biotinylated detection antibodies ŽR2qR3qR5; v ., and ELISA with the same detection antibodies, but unbiotinylated Ž`.. In the latter case detection of bound monoclonals then required an antibody conjugate, rabbit anti-mouse immunoglobulinsralkaline phosphatase. Each assay format was calibrated with a standard of purified recombinant suPAR and the units on both axes are micrograms of suPAR per litre. Žb. Correlation plot for the same data shown in Fig. 2a. The signal obtained for each sample in suPAR ELISA with biotinylated detection antibodies is plotted against the signal obtained from the same sample using nonbiotinylated detection antibodies. No correlation was found using regression analysis.
Since the distortion of the results above appeared to be related to biotinylated R5 ŽFig. 1., and this
Fig. 3. Monitoring the change in biotinylation of a Mab by ELISA. Affinity-purified RAM was used as the capture layer in a sandwich ELISA format. A dilution series of the substituted Mab obtained after biotinylation using different molar ratios of biotinylation reagent was then allowed to bind. A streptavidin conjugate with peroxidase was used for detection of biotin, so that the more biotin covalently attached to the Mab the higher the ELISA signal that was obtained. The lines shown are for Mab applied at 0.63 ŽB., 1.25 Žv . and 2.5 mgrl Ž'., respectively.
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When a model Mab, anti-trinitrophenyl hapten ŽTNP., was treated with increasing molar ratios of a biotinylation reagent, covering the range 11.6:1– 342:1, there was a readily measurable increase in the biotinylation of the Mab ŽFig. 3.. Surface plasmon resonance analyses showed, firstly, that biotinylation of Mabs modified their binding to RAMs, so that their binding to an antibody coated sensor chip was impaired ŽFigs. 4 and 5.. Secondly, biotinylation of the R5 Mab reduced the specific interaction of this antibody with purified suPAR, from 440 Žunbiotinylated. to 77 RU for R5 biotinylated using the molar ratio of 342:1, which
was the ratio employed for the ELISA reagent ŽFig. 4a.. R5 which was biotinylated employing the molar ratio of 116:1, bound 220 RU of suPAR, suggesting that the size of the reduction in specific affinity for suPAR was dependent on the ratio of biotinylation. However, biotinylation of R5 also affected its binding to RAMs on the sensor chip. If the data from Fig. 4a were corrected for the reduced amount of Mab bound, it was found that biotinylation leads to a 25% decrease in specific signal when R5 is biotinylated using the ratio 116:1, while the signal was reduced by 70% when R5 was biotinylated with the ratio 342:1.
Fig. 4. Surface plasmon resonance analysis of interactions between biotinylated anti-uPAR R5 and suPAR or plasma. The binding of suPAR and plasma proteins were tested using sensor chips on which Mab R5 or biotinylated R5 were anchored with RAMs. Arrows indicate start and stop of injection of 0.2 mmolrl R5, and start and stop, respectively, of 0.2 mmolrl suPAR Ža. or a 1r100 dilution of EDTA plasma Žb.. The flow rate was 10 mlrmin. Each injection lasted for 3 min after which the chip was flushed with buffer to allow dissociation for 3 min. The steep increase in the curves upon completion of the injection of biotinylated antibody is due to buffer flow removal of the interference in the light path produced by biotinylated protein. The encircled letter B denotes biotin labeling.
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Fig. 5. Surface plasmon resonance analysis of interactions between biotinylated anti-TNP and suPAR or plasma proteins. The binding of suPAR and plasma proteins were tested using sensor chips on which anti-TNP or biotinylated anti-TNP were anchored with RAMs. Arrows indicate start and stop of injection of 0.2 mmolrl anti-TNP, and start and stop, respectively, of injection of 0.2 mmolrl suPAR Ža. or a 1r100 dilution of EDTA-plasma Žb.. The flow rate was 10 mlrmin. Each injection lasted for 3 min after which the chip was flushed with buffer to allow dissociation for 3 min. The encircled letter B denotes biotin labeling.
Further plasmon resonance analysis showed, that while the R5 Mab itself had a negligible interaction with diluted plasma proteins, biotinylation leads to a stronger interaction with plasma proteinŽs. and, further, the magnitude of this interaction increased with increasing biotinylation ŽFig. 4b.. After 3 min dissociation, 38 RU of plasma protein were bound to nonbiotinylated R5, while 88 RU became bound to R5 which had been biotinylated using the ratio 342:1. In this experiment, it should be noted that we used plasma at a dilution of 1r100, and yet the nonspecific signal was still clearly visible. By contrast, ELISA measurements of suPAR are usually performed with plasma samples diluted 1r10, so that larger nonspecific binding and considerable distortion of specific signal can be expected. To test whether this plasma protein binding phenomenon was related only to the Mab R5, the same
experiments were repeated with the Mab anti-TNP, which is of the same sub-class as R5 ŽIgG1.. It was clear that while this Mab itself had no interaction with suPAR ŽFig. 5a. or plasma proteins ŽFig. 5b., nevertheless after biotinylation at a molar ratio of 342:1 it gave a signal and bound, after the 3-min dissociation period, 30 RU of protein from human plasma ŽFig. 5b.. As with R5, the amount of plasma protein which became bound to the biotinylated Mab was related to the degree of biotinylation.
4. Discussion Biotinylated antibodies are used as detection reagents in a multitude of immunoassays and receptor antagonist screening assays, and casual reading of the immunoassay literature could lead one to believe
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that antigen-binding capacity and specificity of antibodies are not significantly affected by biotinylation. The prevalence of this assumption is evident from the fact that in reports describing immunoassays, the molar ratio of reactive biotin used to prepare labeled antibody reagents is rarely deemed worthy of mention. However, reports of partial or even total loss of affinity in binding to antigens after derivatization of Mabs have been published previously ŽGhose et al., 1983; Pearson et al., 1998.. The results presented here provide another instance where the specific interaction of a Mab ŽR5. with its antigen ŽsuPAR. is progressively reduced with increasing biotinylation. This effect was also observed, although to a lesser extent, with two other Mabs ŽR2 and R3. to suPAR Ždata not shown.. It was also found that the interaction of the Mabs with the immobilized anti-mouse antibody used in the surface plasmon resonance experiments was progressively reduced after biotinylation of the Mabs. One can speculate that the reason for the decrease in specific binding is the biotinylation of one or more lysine amino groups in the combining site of the R5 antibody, impairing binding to the antigen. Clearly, from one Mab to another, there will be variations depending upon whether there happens to be a lysine or not in such a sensitive location. Another possibility is that biotinylation causes a conformational change of the Mab, resulting in a lower affinity for the epitope. Reduction in recognition by an anti-mouse antibody is similarly explained as a result of biotinylation of lysineŽs. in the recognition sites. Since ELISAs are usually designed so that Mabs with high affinity for the target antigen are employed in the detection layer, a modest drop in affinity may in many cases be tolerated, and not invalidate an ELISA. It has been suggested that loss of the specific binding properties of antibodies may be avoided altogether by biotinylation of the carbohydrate chains on immunoglobulins ŽO’Shannesy and Quarles, 1987., rather than the polypeptide moiety. However, to our knowledge this method has not been used to make biotinylated Mabs for quantitative ELISA applications, probably because the degree of biotin substitution is dependent on the outcome of an oxidation step which is difficult to reproduce. The most disturbing finding in our study was that biotinylation of the R5 monoclonal, as well as a
model Mab, led to interactions with unknown human plasma proteins. This more serious problem is not readily explained, and just how biotinylation produces interactions with human plasma proteins remains unclear. However, by extending the work of Dale et al. Ž1994., our results provide further evidence that biotinylation can degrade the specificity of immunoassays to such an extent that the measured signals may be at least misleading, if not invalid. In our study, this problem was particularly related to the need to assay suPAR at low levels in human plasma, while the nonspecific reaction was not seen when cytosol preparations from human tumors were tested Ždata not shown.. This could indicate that the biotinylated Mab is recognized by a protein, possibly an antibody, which is only found in significant amounts in blood. It could be argued that in a sandwich ELISA nonspecific binding of a biotinylated Mab to plasma proteins should not matter when the Mab is used in the detection layer. Indeed, the plasma sample is first allowed to bind to the capture layer, and then the surface is extensively washed before the detection antibody is added. The detection antibody should then only be able to react with the specific antigen which has been captured by the immobilized first antibody layer. However, in very sensitive ELISAs developed to measure low concentrations of antigens, despite the best attempts at blocking the plastic surface used for such assays, it is widely recognized that detection of binding of sample components other than the specific antigen may occur. This is especially likely for plasma samples, which contain a multitude of different proteins, many represented at extremely high concentrations relative to an antigen occurring at only 1 or 2 mgrl. Thus, it is imperative that reagents used for the detection antibody layer not only have a high specific affinity, but also that this specificity be virtually absolute. In conclusion, we have identified biotinylation of detection antibodies as a potential source of nonspecific signals when ELISAs are applied to blood plasma. This distortion of results is related to the particular antibody used, as well as the degree to which it is biotinylated. On the basis of these results, it is evident that biotinylation is not without effect on the specificity of reagent probes, and for some applications with blood analysis it may be preferable to
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avoid this potential problem by using a second Mab–enzyme conjugate ŽStephens et al., 1999.. However, the search for other labeling methods offering higher sensitivity with lower degrees of antibody substitution should be continued. Europium labeling, detected by time-resolved fluorescence ŽTRF., is one possible alternative which may solve the problem for analytes occurring at very low concentrations in blood ŽPiironen et al., 1996.. Acknowledgements The authors thank Pernille Møller and Anne Margrete Poulsen for excellent technical assistance and John Post for making the illustrations. This work was supported by the Danish Cancer Society and the Foundation of 17.12.1981. References Behrendt, N., Stephens, R.W., 1998. The urokinase receptor. Fibrinolysis Proteolysis 12, 191. Dale, G.L., Gaddy, P., Pikul, F.J., 1994. Antibodies against biotinylated proteins are present in normal human serum. J. Lab. Clin. Med. 123, 365. Ghose, T.L., Blair, A.H., Kulkarni, P.N., 1983. Preparation of antibody-linked cytotoxic agents. Methods Enzymol. 93, 280. Guesdon, J.L., Ternunck, T., Avrameas, S., 1979. The use of avidin–biotin interaction in immunoenzymatic techniques. J. Histochem. Cytochem. 27, 1131. Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, p. 341. Mustjoki, S., Alitalo, R., Stephens, R.W., Vaheri, A., 1999. Blast cell-surface and plasma soluble urokinase plasminogen activator receptor in acute leukemia patients: relationship to classification and response to therapy. Thromb. Haemostasis 81, 705.
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O’Shannesy, D.J., Quarles, R.H., 1987. Labeling of the oligosaccharide moieties of immunoglobulins. J. Immunol. Methods 99, 153. Pearson, J.E., Kane, J.W., Petraki-Kallioti, I., Gill, A., Vadgama, P., 1998. Surface plasmon resonance: a study of the effect of biotinylation on the selection of antibodies for use in immunoassays. J. Immunol. Methods 221, 87. Piironen, T., Løvgren, J., Karp, M., Eerola, R., Lundvall, A., Dowell, B., Løvgren, T., Lilja, M., Pettersson, K., 1996. Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular Kallekrein ŽhK2. in serum. Clin. Chem. 42, 1034. Rønne, E., Behrendt, N., Ploug, M., Nielsen, H.J., Wollisch, E., Weidle, U., Danø, K., Høyer-Hansen, G., 1994. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay. J. Immunol. Methods 167, 91. Rønne, E., Pappot, H., Grøndahl-Hansen, J., Høyer-Hansen, G., Plesner, T., Hansen, N.E., Danø, K., 1995. The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria. Br. J. Haematol. 89, 576. Sier, C.F.M., Stephens, R.W., Bizik, J., Mariani, A., Bassan, M., Pedersen, N., Frigerio, L., Ferrari, A., Danø, K., Brunner, N., ¨ Blasi, F., 1998. The level of urokinase plasminogen activator receptor ŽuPAR. is increased in serum of ovarian cancer patients. Cancer Res. 58, 1843. Slot, O., Brunner, N., Locht, H., Oxholm, P., Stephens, R.W., ¨ 1999. Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis. Ann. Rheum. Dis. 58, 488. Stephens, R.W., Nielsen, H.J., Christensen, I.J., Thorlacius-Ussing, O., Sørensen, S., Danø, K., Brunner, N., 1999. Plasma ¨ urokinase receptor levels in patients with colorectal cancer: relationship to prognosis. J. Natl. Cancer Inst. 91, 869. Stephens, R.W., Pedersen, A.N., Nielsen, H.J., Hamers, M.J., Høyer-Hansen, G., Rønne, E., Dybkjær, E., Danø, K., Brunner, ¨ N., 1997. ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer patients. Clin. Chem. 43, 1868.
Loss of ELISA specificity due to biotinylation of monoclonal antibodies Gunilla Høyer-Hansen a,) , Maria J.A.G. Hamers a , Anders N. Pedersen a , a , Keld Danø a , Ross W. Stephens a Hans Jørgen Nielsen b, Nils Brunner ¨ a
The Finsen Laboratory, Copenhagen UniÕersity Hospital, StrandbouleÕarden 49, DK-2100 Copenhagent Ø, Denmark b Department of Surgical Gastroenterology, HÕidoÕre Hospital, Denmark Received 2 June 1999; received in revised form 27 September 1999; accepted 25 October 1999
Abstract A significant degree of nonspecificity was found in ELISA determinations of soluble urokinase receptor ŽsuPAR. in human blood plasma when biotinylated monoclonal antibodies ŽMabs. were used for the detection layer. Surface plasmon resonance studies using both nonbiotinylated and biotinylated antibodies demonstrated that biotinylation reduced specific binding of the antibodies to their target antigen, suPAR. Furthermore, biotinylation produced a new interaction with unknown human plasma proteinŽs., unrelated to suPAR. Nonspecific interaction with plasma proteinŽs. was also observed after biotinylation of a Mab having no specific target antigen in human plasma and, in both cases, the level of nonspecific interaction was directly related to the degree of antibody biotinylation. These results reinforce earlier observations that biotinylation of antibodies can reduce the affinity of antibodies, but also indicate that, in addition, biotinylation can reduce the specificity of immunoassays for plasma proteins. q 2000 Elsevier Science B.V. All rights reserved. Keywords: ELISA specificity; Biotinylated antibodies ; Biotinylation ratio; Surface plasmon resonance
1. Introduction Biotinylation of antibodies and many other biological probes has been of great value, enabling sensitive and quantitative detection of specific antigens and other target molecules in a wide variety of analytical procedures such as immunohistochemistry, immunocytochemistry, immunoblotting, and ELISA,
AbbreÕiations: Mab, monoclonal antibody; PBS, phosphate buffered saline; RAM, rabbit anti-mouse antibodies; RU, resonance unit; suPAR, soluble form of urokinase receptor; uPAR, urokinase receptor; TNP, trinitrophenyl hapten ) Corresponding author. Tel.: q45-35-45-5874; fax: q45-3538-5450; e-mail:[email protected]
as well as nucleic acid analysis in Northern blotting, PCR techniques, chromosome painting and gene linkage studies. There have been widespread applications also in the investigation of ligand interactions with cell surface receptors, and particularly in screening for antagonists of such interactions, in order to discover new molecules for development as potential drugs. In all these techniques, specific binding of the biotinylated probe can be detected by high affinity binding of appropriately labeled conjugates of avidin or streptavidin. Implicit in these diverse applications of biotinylated probes is the assumption that biotin substitution of the specific probe does not significantly alter its specificity or affinity for the target molecule, and especially that it does not in
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itself generate new interactions with other nontarget components of the system. However, the question whether these ideal properties of biotinylated reagents can be relied upon in all applications is often not considered, and particularly in relation to the effect that the degree of biotin substitution may have on the specificity of a given probe. It has been common practice not to report the conditions employed for biotinylation of immunoassay reagents, such as, for example, the molar ratio of reactive biotin to IgG in the case of antibodies. Authoritative laboratory handbooks Že.g., Harlow and Lane, 1988. recommend that biotin ester is added to antibody at a molar ratio of from 10 to 100 Ži.e., 25–250 mg of ester per milligram of antibody protein.. However, the use of higher molar ratios, even over 1000:1, in order to enhance sensitivity, may be widespread. The increase in biotin substitution of the antibody obtained with increasing molar ratios of reagent is not routinely monitored or reported, and the actual final ratio of biotin substitution obtained is unknown in virtually all cases. The discussion of problems arising from biotinylation of antibodies has so far mainly been confined to loss of affinity ŽGhose et al., 1983; Pearson et al., 1998.. However, there has been at least one report suggesting that biotinylation of antibodies, or even gelatin, can produce interactions with unknown human plasma proteins, leading to loss of specificity in plasma immunoassays employing biotinylated reagents ŽDale et al., 1994.. This finding indicates that the use of biotinylated reagents for analysis of human blood components may give rise to specificity problems. We report here loss of specificity due to biotinylation of a monoclonal antibody ŽMab. in ELISA determinations of soluble urokinase receptor ŽsuPAR; see review by Behrendt and Stephens, 1998. in human plasma. suPAR occurs in the plasma of healthy individuals ŽRønne et al., 1995. at a concentration of only approximately 1 mgrl ŽStephens et al., 1997., but it may be elevated in patients with several types of cancer ŽStephens et al., 1997, 1999; Sier et al., 1998; Mustjoki et al., 1999., as well as patients with rheumatoid arthritis ŽSlot et al., 1999.. The issue of specificity in the assay of such a low concentration plasma component becomes crucial when it is evaluated for prognostic significance
ŽStephens et al., 1999., and even more so when contemplating the application of this parameter in cancer patient management, particularly in identifying high-risk patients who may most benefit from adjuvant chemotherapy. It was found that an ELISA developed for measurement of uPAR in detergent extracts of tumor tissue, which employs biotinylated antibodies, was unsuitable for ELISA of suPAR in blood samples. The plasma suPAR measurements made using biotinylated antibodies were distorted by degeneration of antibody specificity. We also report surface plasmon resonance analysis of biotinylation-dependent interactions of Mabs with human plasma proteins. The results indicate that the use of biotinylated Mabs for sensitive immunoassays of proteins which occur at only low concentrations in blood give rise to false readings due to nonspecific interactions.
2. Materials and methods 2.1. Blood collections and plasma separation Blood samples from 44 healthy donors were obtained through the co-operation of the blood bank at the Hvidovre University Hospital, Copenhagen. Peripheral venous blood was drawn into pre-chilled EDTA collection tubes ŽBecton-Dickinson, Mountain View, CA. and quickly mixed by inversion. The plasma was separated from blood cells within 1.5 h by centrifugation at 48C at 1200 = g for 30 min, and stored frozen at y808C prior to assay. A plasma pool was made with freshly collected samples from ten donors, aliquoted and stored frozen at y808C. For ELISA measurements plasma was diluted 1r10, and for real-time biomolecular interaction analyses using Biacore, plasma was diluted 1r100. 2.2. suPAR ELISA Immunoassay plates ŽMaxisorp, Nunc, Roskilde, Denmark. were coated for 16 h at 48C with 100 mlrwell of purified rabbit anti-human uPAR Ž0.5 mg IgGrl. in 0.1 molrl carbonate buffer, pH 9.5. This capture antibody was previously absorbed on a column of mouse IgG in order to reduce the ELISA background. Prior to use, the assay wells were rinsed
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twice with 200 mlrwell of SuperBlocke solution ŽPierce Chemicals, Rockford, IL. diluted 1r2 with phosphate-buffered saline ŽPBS., followed by three washes with PBS containing 1 grl Tween 20. Wells were then treated for 1 h at 378C with 100 mlrwell of triplicate or duplicate 1r10 dilutions of plasma made in a sample dilution buffer of 50 mmolrl phosphate buffer, pH 7.2, 0.1 molrl NaCl, 10 grl bovine serum albumin ŽFraction V, Boehringer– Mannheim, Penzberg, Germany. and 1 grl Tween 20. On every assay plate, a series of standards was included, which consisted of seven serial dilutions in triplicate of purified recombinant suPAR Ži.e., uPAR lacking the glycolipid anchor; see Rønne et al., 1994., starting from 1 mgrl, then 0.5, 0.25, 0.125, 0.0625, 0.0313 and 0.0156 mgrl. Also included on each plate were triplicate blank wells containing only sample dilution buffer, and triplicate wells of a 1r10 dilution of a control citrate plasma pool. After suPAR binding the wells were washed six times, then treated for 1 h at 378C with 100 mlrwell of a mixture of three murine monoclonal anti-human uPAR antibodies wR2 Ž23 mgrl., R3 Ž281 mgrl., and R5 Ž70 mgrl. ŽRønne et al., 1995.x in sample dilution buffer. These antibodies were used either after biotinylation or without any biotinylation as described below. After six washes, the wells were then incubated for 1 h at 378C with 100 mlrwell of either rabbit anti-mouse immunoglobulinsralkaline phosphatase conjugate ŽDako, Glostrup, Denmark. diluted 1r1000 Žfor nonbiotinylated Mabs., or streptavidin conjugated alkaline phosphatase ŽDako. diluted 1r1000 Žfor biotinylated Mabs.. After six washes with washing solution, and three washes with pure water, 100 ml of freshly made p-nitrophenyl phosphate ŽSigma, St. Louis, MO. substrate solution Ž1.7 grl in 0.1 molrl Tris–HCl, pH 9.5, 0.1 molrl NaCl, 5 mmolrl MgCl 2 . was added to each well and the plate was placed in a Ceres 900J plate reader ŽBioTek Instruments, Winooski, VT.. The yellow color development at 238C was monitored automatically, with readings taken at 405 nm against an air blank every 10 min for 60 min. KinetiCalc II software was used to manage the data, calculate the rate of color change for each well Žlinear regression analysis. and compute from the rates for the suPAR standards a four-parameter fitted standard curve, from which the suPAR concentration of each plasma sample was
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calculated. Development of color in each well was a linear function of time for all concentrations of suPAR measured in these experiments, with correlation coefficients for the automatically fitted lines typically better than 0.97. The standard curve of the rates plotted against the suPAR concentration was slightly sigmoidal from 0 to 1.0 mgrl and the correlation coefficient for the four-parameter fit was typically better than 0.999. 2.3. Biotinylation of antibodies The Mabs were biotinylated using a modification of a widely used method first described by Guesdon et al. Ž1979.. The biotin was supplied as biotinamidocaproate N-hydroxy-succinimide ester ŽSigma.. The final concentration of the purified antibodies in the biotinylation mixture was 1 grl, and the molar ratios between biotin and Mab was 11.6:1, 116:1 and 342:1 for anti-TNP, and 116:1 and 342:1 for antiuPAR clone R5. Biotinylated antibodies were diluted 1r2 with pure glycerol ŽSigma G-5516. and stored at y208C. The change in biotin substitution of antibody obtained using the different molar ratios of biotin ester was monitored by ELISA. In brief immunoassay plates were coated for 20 h at 48C with 100 mlrwell of 10 mgrl of affinity-purified rabbit antimouse immunoglobulins ŽDako.. After blocking for 1 h with 10 grl skimmed milk powder in PBS, the plates were incubated with samples of biotinylated antibody in serial dilutions from 5 to 0.04 mgrl. Bound antibody was detected with peroxidase coupled streptavidin ŽDako., and development with ophenylenediamine as substrate. 2.4. Surface plasmon resonance analyses All binding measurements were performed employing the BIAcoree 2000 instrument ŽBIACORE, Uppsala, Sweden.. Rabbit anti-mouse antibodies ŽRAM Fc g, BIACOREe. were immobilized on the sensor chip CM 5 ŽBIACOREe. at a concentration of 30 mgrl using the amine coupling method according to the procedure provided by the manufacturer. In the experiments, Mabs Ž0.2 mmolrl. were bound to the immobilized RAM Fc g. Then either suPAR Ž0.2 mmolrl. or diluted plasma Ž1r100. was added
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and interaction with the antibodies was measured. The plasma dilution was chosen such that the signal representing the nonspecific binding from the blank channel on the sensor chip was below 20 resonance units ŽRU.. The amount of protein bound to the anchored antibodies was calculated using Biaevaluation 2.1 software. The chip was regenerated with 1 molrl formic acid.
3. Results When suPAR was measured in EDTA plasma from healthy donors using an ELISA with biotinylated detection Mabs, a positively skewed distribution of the suPAR signal was found, with several patient samples contributing to a high-end tail Žsee Fig. 1a.. This distribution was studied further by examining the contribution of individual detection Mabs to the total signal. It was found ŽFig. 1a. that the skewness was primarily due to the contribution of one antiuPAR Mab, biotinylated R5, which recognizes an epitope on the N-terminal domain 1 of suPAR. The contribution of another Mab, biotinylated R2, which recognizes an epitope on domain 3, was noticeably disproportionate to R5 across the spectrum of samples. A simple regression plot revealed that no real correlation existed between the two antibody signals ŽFig. 1b.. This result was unexpected, since each uPAR molecule should contain one binding site for each of the antibodies. A set of plasma samples from healthy individuals was then assayed twice with the same basic ELISA protocol, but using biotinylated and nonbiotinylated detection antibodies in parallel. In the latter case, bound nonbiotinylated Mab was quantified using an alkaline phosphatase conjugate of a RAM. Both ELISAs were calibrated with a reference standard of purified recombinant suPAR. As shown in Fig. 2a, the distribution of uPAR levels differed considerably, depending on the detection antibody. With biotinylated Mabs the levels of uPAR in plasma extended over five times the range of that obtained with nonbiotinylated detection Mabs. Furthermore, there was no correlation between the levels determined using biotinylated and nonbiotinylated Mabs, as shown in Fig. 2b.
Fig. 1. Ža. ELISA of suPAR in 36 EDTA plasma samples, measured using a combination of two biotinylated monoclonal detection antibodies, R2 and R5 ŽI., with biotinylated R2 alone Ž=., and with biotinylated R5 alone Ž'.. The signals are expressed as rates Žmilliabsorbance units at 405 nmrmin. obtained for the alkaline phosphatase reaction, and plotted here as percentiles. Note the pronounced skewness of the distribution, due to the contribution of the biotinylated R5 antibody, which is not found when biotinylated R2 is used alone. Žb. Correlation plot for the data shown in Fig. 1a. The signal obtained in suPAR ELISA with biotinylated R2 detection antibody is plotted against the signal obtained from the same sample using biotinylated R5 as the detection antibody. No correlation was found using regression analysis.
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Mab was used in a more heavily biotinylated form than R2, it was suspected that biotinylated Mabs could be giving rise to a false signal, and experiments were designed to test the specificity of the Mabs before and after biotinylation. In order to verify that increasingly large molar ratios of a biotinylation reagent do actually produce an increase in the amount of biotin molecules covalently attached to Mabs, we used a simple ELISA. The capture antibody consisted of affinity-purified rabbit anti-mouse IgG. A dilution series of the substituted Mab obtained after biotinylation using different molar ratios was then applied. The detector was peroxidase coupled streptavidin, which binds very strongly Ž K D s 10y1 5 molrl; Guesdon et al., 1979. to biotin molecules, so that the more biotin covalently attached to the Mab the higher the ELISA signal obtained. In addition to the molar ratio of biotinylation reagent employed, the degree of biotinylation obtained with a given Mab is also dependent on the number of accessible free amino groups of lysines in the Mab, which clearly differs between different Mabs.
Fig. 2. Ža. Comparison of plasma suPAR levels determined by ELISA with biotinylated and nonbiotinylated detection antibodies. Each of 44 EDTA plasma samples was assayed using ELISA with a mixture of three biotinylated detection antibodies ŽR2qR3qR5; v ., and ELISA with the same detection antibodies, but unbiotinylated Ž`.. In the latter case detection of bound monoclonals then required an antibody conjugate, rabbit anti-mouse immunoglobulinsralkaline phosphatase. Each assay format was calibrated with a standard of purified recombinant suPAR and the units on both axes are micrograms of suPAR per litre. Žb. Correlation plot for the same data shown in Fig. 2a. The signal obtained for each sample in suPAR ELISA with biotinylated detection antibodies is plotted against the signal obtained from the same sample using nonbiotinylated detection antibodies. No correlation was found using regression analysis.
Since the distortion of the results above appeared to be related to biotinylated R5 ŽFig. 1., and this
Fig. 3. Monitoring the change in biotinylation of a Mab by ELISA. Affinity-purified RAM was used as the capture layer in a sandwich ELISA format. A dilution series of the substituted Mab obtained after biotinylation using different molar ratios of biotinylation reagent was then allowed to bind. A streptavidin conjugate with peroxidase was used for detection of biotin, so that the more biotin covalently attached to the Mab the higher the ELISA signal that was obtained. The lines shown are for Mab applied at 0.63 ŽB., 1.25 Žv . and 2.5 mgrl Ž'., respectively.
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When a model Mab, anti-trinitrophenyl hapten ŽTNP., was treated with increasing molar ratios of a biotinylation reagent, covering the range 11.6:1– 342:1, there was a readily measurable increase in the biotinylation of the Mab ŽFig. 3.. Surface plasmon resonance analyses showed, firstly, that biotinylation of Mabs modified their binding to RAMs, so that their binding to an antibody coated sensor chip was impaired ŽFigs. 4 and 5.. Secondly, biotinylation of the R5 Mab reduced the specific interaction of this antibody with purified suPAR, from 440 Žunbiotinylated. to 77 RU for R5 biotinylated using the molar ratio of 342:1, which
was the ratio employed for the ELISA reagent ŽFig. 4a.. R5 which was biotinylated employing the molar ratio of 116:1, bound 220 RU of suPAR, suggesting that the size of the reduction in specific affinity for suPAR was dependent on the ratio of biotinylation. However, biotinylation of R5 also affected its binding to RAMs on the sensor chip. If the data from Fig. 4a were corrected for the reduced amount of Mab bound, it was found that biotinylation leads to a 25% decrease in specific signal when R5 is biotinylated using the ratio 116:1, while the signal was reduced by 70% when R5 was biotinylated with the ratio 342:1.
Fig. 4. Surface plasmon resonance analysis of interactions between biotinylated anti-uPAR R5 and suPAR or plasma. The binding of suPAR and plasma proteins were tested using sensor chips on which Mab R5 or biotinylated R5 were anchored with RAMs. Arrows indicate start and stop of injection of 0.2 mmolrl R5, and start and stop, respectively, of 0.2 mmolrl suPAR Ža. or a 1r100 dilution of EDTA plasma Žb.. The flow rate was 10 mlrmin. Each injection lasted for 3 min after which the chip was flushed with buffer to allow dissociation for 3 min. The steep increase in the curves upon completion of the injection of biotinylated antibody is due to buffer flow removal of the interference in the light path produced by biotinylated protein. The encircled letter B denotes biotin labeling.
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Fig. 5. Surface plasmon resonance analysis of interactions between biotinylated anti-TNP and suPAR or plasma proteins. The binding of suPAR and plasma proteins were tested using sensor chips on which anti-TNP or biotinylated anti-TNP were anchored with RAMs. Arrows indicate start and stop of injection of 0.2 mmolrl anti-TNP, and start and stop, respectively, of injection of 0.2 mmolrl suPAR Ža. or a 1r100 dilution of EDTA-plasma Žb.. The flow rate was 10 mlrmin. Each injection lasted for 3 min after which the chip was flushed with buffer to allow dissociation for 3 min. The encircled letter B denotes biotin labeling.
Further plasmon resonance analysis showed, that while the R5 Mab itself had a negligible interaction with diluted plasma proteins, biotinylation leads to a stronger interaction with plasma proteinŽs. and, further, the magnitude of this interaction increased with increasing biotinylation ŽFig. 4b.. After 3 min dissociation, 38 RU of plasma protein were bound to nonbiotinylated R5, while 88 RU became bound to R5 which had been biotinylated using the ratio 342:1. In this experiment, it should be noted that we used plasma at a dilution of 1r100, and yet the nonspecific signal was still clearly visible. By contrast, ELISA measurements of suPAR are usually performed with plasma samples diluted 1r10, so that larger nonspecific binding and considerable distortion of specific signal can be expected. To test whether this plasma protein binding phenomenon was related only to the Mab R5, the same
experiments were repeated with the Mab anti-TNP, which is of the same sub-class as R5 ŽIgG1.. It was clear that while this Mab itself had no interaction with suPAR ŽFig. 5a. or plasma proteins ŽFig. 5b., nevertheless after biotinylation at a molar ratio of 342:1 it gave a signal and bound, after the 3-min dissociation period, 30 RU of protein from human plasma ŽFig. 5b.. As with R5, the amount of plasma protein which became bound to the biotinylated Mab was related to the degree of biotinylation.
4. Discussion Biotinylated antibodies are used as detection reagents in a multitude of immunoassays and receptor antagonist screening assays, and casual reading of the immunoassay literature could lead one to believe
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that antigen-binding capacity and specificity of antibodies are not significantly affected by biotinylation. The prevalence of this assumption is evident from the fact that in reports describing immunoassays, the molar ratio of reactive biotin used to prepare labeled antibody reagents is rarely deemed worthy of mention. However, reports of partial or even total loss of affinity in binding to antigens after derivatization of Mabs have been published previously ŽGhose et al., 1983; Pearson et al., 1998.. The results presented here provide another instance where the specific interaction of a Mab ŽR5. with its antigen ŽsuPAR. is progressively reduced with increasing biotinylation. This effect was also observed, although to a lesser extent, with two other Mabs ŽR2 and R3. to suPAR Ždata not shown.. It was also found that the interaction of the Mabs with the immobilized anti-mouse antibody used in the surface plasmon resonance experiments was progressively reduced after biotinylation of the Mabs. One can speculate that the reason for the decrease in specific binding is the biotinylation of one or more lysine amino groups in the combining site of the R5 antibody, impairing binding to the antigen. Clearly, from one Mab to another, there will be variations depending upon whether there happens to be a lysine or not in such a sensitive location. Another possibility is that biotinylation causes a conformational change of the Mab, resulting in a lower affinity for the epitope. Reduction in recognition by an anti-mouse antibody is similarly explained as a result of biotinylation of lysineŽs. in the recognition sites. Since ELISAs are usually designed so that Mabs with high affinity for the target antigen are employed in the detection layer, a modest drop in affinity may in many cases be tolerated, and not invalidate an ELISA. It has been suggested that loss of the specific binding properties of antibodies may be avoided altogether by biotinylation of the carbohydrate chains on immunoglobulins ŽO’Shannesy and Quarles, 1987., rather than the polypeptide moiety. However, to our knowledge this method has not been used to make biotinylated Mabs for quantitative ELISA applications, probably because the degree of biotin substitution is dependent on the outcome of an oxidation step which is difficult to reproduce. The most disturbing finding in our study was that biotinylation of the R5 monoclonal, as well as a
model Mab, led to interactions with unknown human plasma proteins. This more serious problem is not readily explained, and just how biotinylation produces interactions with human plasma proteins remains unclear. However, by extending the work of Dale et al. Ž1994., our results provide further evidence that biotinylation can degrade the specificity of immunoassays to such an extent that the measured signals may be at least misleading, if not invalid. In our study, this problem was particularly related to the need to assay suPAR at low levels in human plasma, while the nonspecific reaction was not seen when cytosol preparations from human tumors were tested Ždata not shown.. This could indicate that the biotinylated Mab is recognized by a protein, possibly an antibody, which is only found in significant amounts in blood. It could be argued that in a sandwich ELISA nonspecific binding of a biotinylated Mab to plasma proteins should not matter when the Mab is used in the detection layer. Indeed, the plasma sample is first allowed to bind to the capture layer, and then the surface is extensively washed before the detection antibody is added. The detection antibody should then only be able to react with the specific antigen which has been captured by the immobilized first antibody layer. However, in very sensitive ELISAs developed to measure low concentrations of antigens, despite the best attempts at blocking the plastic surface used for such assays, it is widely recognized that detection of binding of sample components other than the specific antigen may occur. This is especially likely for plasma samples, which contain a multitude of different proteins, many represented at extremely high concentrations relative to an antigen occurring at only 1 or 2 mgrl. Thus, it is imperative that reagents used for the detection antibody layer not only have a high specific affinity, but also that this specificity be virtually absolute. In conclusion, we have identified biotinylation of detection antibodies as a potential source of nonspecific signals when ELISAs are applied to blood plasma. This distortion of results is related to the particular antibody used, as well as the degree to which it is biotinylated. On the basis of these results, it is evident that biotinylation is not without effect on the specificity of reagent probes, and for some applications with blood analysis it may be preferable to
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avoid this potential problem by using a second Mab–enzyme conjugate ŽStephens et al., 1999.. However, the search for other labeling methods offering higher sensitivity with lower degrees of antibody substitution should be continued. Europium labeling, detected by time-resolved fluorescence ŽTRF., is one possible alternative which may solve the problem for analytes occurring at very low concentrations in blood ŽPiironen et al., 1996.. Acknowledgements The authors thank Pernille Møller and Anne Margrete Poulsen for excellent technical assistance and John Post for making the illustrations. This work was supported by the Danish Cancer Society and the Foundation of 17.12.1981. References Behrendt, N., Stephens, R.W., 1998. The urokinase receptor. Fibrinolysis Proteolysis 12, 191. Dale, G.L., Gaddy, P., Pikul, F.J., 1994. Antibodies against biotinylated proteins are present in normal human serum. J. Lab. Clin. Med. 123, 365. Ghose, T.L., Blair, A.H., Kulkarni, P.N., 1983. Preparation of antibody-linked cytotoxic agents. Methods Enzymol. 93, 280. Guesdon, J.L., Ternunck, T., Avrameas, S., 1979. The use of avidin–biotin interaction in immunoenzymatic techniques. J. Histochem. Cytochem. 27, 1131. Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, p. 341. Mustjoki, S., Alitalo, R., Stephens, R.W., Vaheri, A., 1999. Blast cell-surface and plasma soluble urokinase plasminogen activator receptor in acute leukemia patients: relationship to classification and response to therapy. Thromb. Haemostasis 81, 705.
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O’Shannesy, D.J., Quarles, R.H., 1987. Labeling of the oligosaccharide moieties of immunoglobulins. J. Immunol. Methods 99, 153. Pearson, J.E., Kane, J.W., Petraki-Kallioti, I., Gill, A., Vadgama, P., 1998. Surface plasmon resonance: a study of the effect of biotinylation on the selection of antibodies for use in immunoassays. J. Immunol. Methods 221, 87. Piironen, T., Løvgren, J., Karp, M., Eerola, R., Lundvall, A., Dowell, B., Løvgren, T., Lilja, M., Pettersson, K., 1996. Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular Kallekrein ŽhK2. in serum. Clin. Chem. 42, 1034. Rønne, E., Behrendt, N., Ploug, M., Nielsen, H.J., Wollisch, E., Weidle, U., Danø, K., Høyer-Hansen, G., 1994. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay. J. Immunol. Methods 167, 91. Rønne, E., Pappot, H., Grøndahl-Hansen, J., Høyer-Hansen, G., Plesner, T., Hansen, N.E., Danø, K., 1995. The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria. Br. J. Haematol. 89, 576. Sier, C.F.M., Stephens, R.W., Bizik, J., Mariani, A., Bassan, M., Pedersen, N., Frigerio, L., Ferrari, A., Danø, K., Brunner, N., ¨ Blasi, F., 1998. The level of urokinase plasminogen activator receptor ŽuPAR. is increased in serum of ovarian cancer patients. Cancer Res. 58, 1843. Slot, O., Brunner, N., Locht, H., Oxholm, P., Stephens, R.W., ¨ 1999. Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis. Ann. Rheum. Dis. 58, 488. Stephens, R.W., Nielsen, H.J., Christensen, I.J., Thorlacius-Ussing, O., Sørensen, S., Danø, K., Brunner, N., 1999. Plasma ¨ urokinase receptor levels in patients with colorectal cancer: relationship to prognosis. J. Natl. Cancer Inst. 91, 869. Stephens, R.W., Pedersen, A.N., Nielsen, H.J., Hamers, M.J., Høyer-Hansen, G., Rønne, E., Dybkjær, E., Danø, K., Brunner, ¨ N., 1997. ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer patients. Clin. Chem. 43, 1868.