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Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Microwells
Analytical Biochemistry 282, 232–238 (2000) doi:10.1006/abio.2000.4602, available online at http://www.idealibrary.com on
Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Microwells Michael Steinitz Experimental Pathology, The Hebrew University–Hadassah Medical School, P.O. Box 12272, Jerusalem, 91120, Israel
Received January 21, 2000
ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-L-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 g/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 g/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent. © 2000 Academic Press
Key Words: ELISA; Tween 20; blocking; alkaline phosphatase; poly-L-lysine; BSA; saturation.
Enzyme-linked immunosorbent assay (ELISA) 1 is a quantitative, sensitive, rapid, easy-to-perform assay which does not require the use of radioactive reagents 1
Abbreviations used: ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; IgG, human ␥-globulins; FCS, fetal calf serum; PBS, phosphate-buffered saline; PLL, poly-L-lysine. 232
(1). In the assay, a color reaction develops in a microwell as the direct result of an interaction between a ligand adhering to the solid phase and a specific molecule added in an aqueous sample. The intensity of the optical signal is proportional to the amount of that specific molecule. Generally, the ligands adhere spontaneously to ELISA microwells and a primary antibody is then added, followed by an enzyme-conjugated secondary antibody that is specific for the primary antibody. A substrate is then added that yields a color reaction, the magnitude of which correlates with the activity of the enzyme. The catalytic activity provides the chemical amplification that allows the detection of extremely low concentrations of specific molecules. Obviously, it is important to keep the background of this multistage assay as low as possible so that even weak optical signals can be distinguished. ELISA microplates possess a high capacity for binding proteins, peptides, and other molecules. It is therefore essential, following coating of the microwells with the appropriate ligand, to saturate the residual noncoated solidphase area with an inert reagent, such as albumin, gelatin, hemoglobin, or FCS. Although these reagents prevent background binding, their use is sometimes not desirable due to their interactions with the primary and/or secondary antibodies (2). In addition, saturation of microwells with large-size proteins may result in somewhat lower reactivity between primary antibodies and small, absorbed ligands, possibly due to steric hindrance. In the original ELISA, Engvall and Perlmann (1) blocked nonspecific binding with Tween 20 only. Today, proteins are generally used to saturate the ELISA microwells, whereas Tween 20 is very often added to the washing buffer to reduce nonspecific binding. Tween 20 is similarly used in Western blot analysis (2– 4). Recently, we described a new method for assessing the magnitude of binding of any ligand to ELISA mi0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
BLOCKING EFFECT OF TWEEN 20 AND BOVINE SERUM ALBUMIN IN ELISA
crowells without the need for a specific antibody and independently of any other ligand-specific binding molecule (5). This enables quantitation of adherence to the microwells of any type and size molecules such as proteins, peptides, sugars, DNA, and hormones. Moreover, the method provides a practical tool for defining the necessary conditions for satisfactory coating of molecules that normally do not readily adhere to ELISA microwells. We used this method in the present study to determine the amount of Tween 20 and BSA required to saturate nontreated and poly-L-lysine-coated ELISA microwells. This approach is widely applicable also to other candidate reagents for blocking the surface area of ELISA microwells. MATERIALS AND METHODS
Reagents ELISA immunoplates (MaxiSorp) were purchased from Nunc (Denmark). The following reagents were obtained from Sigma Chemical Company (St. Louis, MO): alkaline phosphatase derived from calf intestinal mucosa (5.9 mg protein/ml, 2290 units/mg protein); bovine serum albumin (BSA), fraction V; human ␥-globulins (IgG), Cohn fraction II; p-nitrophenyl phosphate; and poly-L-lysine, (hydrobromide) MW120,000. Tween 20 (polyoxyethylene-20-sorbitan monolaurate) was acquired from J. T. Baker (Phillipsburg, NJ). Fetal calf serum (FCS) was purchased from Biological Industries (Beit Haemek, Israel). Determination of Available Vacant Sites for Binding of Alkaline Phosphatase to ELISA Microwells Aliquots of 100 or 200 l containing phosphate-buffered saline (PBS)-diluted alkaline phosphatase are added to ELISA microwells, and the plates are incubated as indicated. Following five washings with PBS, p-nitrophenyl phosphate (1 mg/ml) in 1 M diethanolamine buffer, pH 9.8, containing 0.5 mM MgCl 2 is added and light absorbance at 405 nm is measured with an ELISA reader (Dynatech MR5000). ELISA for Estimation of the Blocking Effect of Tween 20 and BSA The procedure includes four steps: a. Pretreatment of microwells: ELISA microwells are incubated with buffer only or with poly-L-lysine (PLL) in 0.05 M carbonate buffer, pH 9.6, for 1 h at 37°C and then washed five times with PBS. b. Blocking of microwell surface area: Tween 20 or BSA in PBS is added, the plates are incubated at 37°C for the indicated intervals, and then washing is performed as above.
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c. Binding of alkaline phosphatase to residual vacant binding sites: 1/2000 PBS-diluted alkaline phosphatase (i.e., 2.95 g/ml) is added, the plates are incubated for 2 h at 37°C, and washing is performed as above. d. Color reaction: The reaction is performed with the specific substrate as described above. The extent to which Tween 20, BSA, or any other reagent blocks the microwell surface area is estimated according to the magnitude of inhibition of the color reaction, compared with that of nontreated microwells. Obviously, high values of light absorbance indicate the presence of vacant binding sites, whereas low values, due to reduced alkaline phosphatase activity, indicate proportionally less vacant available sites. In contrast to conventional ELISA, all washings in this assay are performed with PBS devoid of Tween 20 or any other background-reducing reagents (such as albumin, gelatin, or FCS). Since in the present study we measure the extent of binding of specific blocking molecules it would be senseless to supplement the washing buffers with any molecule that obscures it. In conventional ELISA Tween 20 is added to the washing buffer to reduce or avoid nonspecific background. In contrast, in our assay it is in fact that “nonspecific” binding which is measured and which resembles inversely the extent of binding of the corresponding blocking reagent. Binding of Alkaline Phosphatase to PLL-Treated Microwells Microwells in 96-well microplates are incubated for 1 h at 37°C with 200 l of carbonate buffer-diluted PLL. Following washing, PBS is added, and the microplates are incubated for 1 h. The plates are washed again, 1/2000 PBS-diluted alkaline phosphatase is added, and the color reaction is measured after the addition of substrate. The Combined Effect of Tween 20 and BSA on ELISA Microwell Saturation Microwells in two 96-well microplates are first incubated for 1 h at 37°C with 200 l of buffer only or with PLL, 20 g/ml in carbonate buffer. Microwells are washed; Tween 20, 0.05% in PBS, is added for 1 h at 37°C; and then washing is repeated, followed by 10-fold dilutions of BSA in PBS. Finally, 1/2000 PBS-diluted alkaline phosphatase followed by substrate is added and light absorbance at 405 nm is recorded. Due to the rapid color reaction that develops in the PLL-treated microwells compared with that in the nontreated microwells, the results are recorded in tandem.
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FIG. 1. Correlation between alkaline phosphatase binding and enzyme concentration. Aliquots containing 100 l with increasing concentrations of alkaline phosphatase in PBS were added to ELISA microwells, and the plates were incubated overnight at room temperature. Following washing, substrate was added and light absorbance at 405 nm was measured. The experiment was carried out in triplicate. The curve represents the average values obtained.
Second, binding of the alkaline phosphatase to the microwells was correlated with the specific activity of the enzyme (i.e., enzyme units/microgram protein). Aliquots containing an equal amount of alkaline phosphatase (i.e., 2.95 g/ml), in an admixture with increasing concentrations of a nonenzyme carrier protein (i.e., human IgG), were added to the microwells and the adsorbed enzymatic activity was determined. Figure 2 shows that the amount of alkaline phosphatase adsorbed to the microwells was inversely correlated with the concentration of human IgG. Obviously, the effect of IgG is not discernable if admixed at a low concentration, compared with that of alkaline phosphatase. The binding of alkaline phosphatase reaches the background value when the carrier protein is present at a 25 times higher concentration than the enzyme. We conclude that measuring the binding of alkaline phosphatase to microwells is indeed a reliable method for assessing the comparable binding of proteins. Tween 20 Saturates the Surface Area of ELISA Microwells
Correlation between Binding of Alkaline Phosphatase to ELISA Microwells and Enzyme Concentration
In ELISA, the nonspecific binding of primary and secondary antibodies to the residual ligand-free surface of the microwells is minimized by adding a nonrelevant protein that saturates any remaining nonoccupied area. We used our assay to assess in a dose– response experiment the blocking effect of Tween 20 on
In the present study, the binding of alkaline phosphatase to the surface of ELISA microwells was assumed to resemble in general the binding of any protein. This assumption was confirmed in two sets of experiments. First, we show that the amount of alkaline phosphatase that binds to the microwells is dependent on the concentration of enzyme. ELISA microwells were coated with increasing concentrations of alkaline phosphatase in PBS. The binding of the enzyme was determined by the intensity of the color reaction measured after the addition of substrate. Figure 1 shows that the light absorbance is in direct proportion to the concentration of the protein which means that the amount of this protein that adsorbs to the microwell surface area is proportional to the concentration of the enzyme. Under the conditions of the experiment shown in Fig. 1 and because of the limited range of the ELISA optical reader, the enzymatic reaction apparently reached a plateau when alkaline phosphatase was added at concentration higher than 738 g/ml. However, when the color reaction was measured at a time point closer to the addition of the substrate a similar direct correlation was also observed at higher concentrations whereas at low concentrations the color reaction was undetectable (results not presented). It is concluded that the diminished color reaction at higher dilutions of the enzyme resembles reduced adsorbance of the enzyme to the microwells surface area.
FIG. 2. Correlation between alkaline phosphatase binding and specific activity of the enzyme. Aliquots containing 100 l of 1/2000 PBSdiluted alkaline phosphatase (2.95 g/ml) mixed with increasing concentrations of human IgG were allowed to adhere to ELISA microwells for 2 h at 37°C. Following washing, substrate was added and light absorbance at 405 nm was measured. The experiment was carried out in triplicate and the curve represents the average values obtained.
RESULTS
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FIG. 3. Binding of alkaline phosphatase to Tween 20-pretreated ELISA microwells. Aliquots containing 200 l decreasing concentrations Tween 20 were added to ELISA microwells and the plates incubated overnight at 4°C. Following washing, 1/2000 PBS-diluted alkaline phosphatase and substrate were added and light absorbance at 405 nm was measured. The experiment was carried out in triplicate and the curve represents the average values obtained.
the residual surface area available for the adherence of alkaline phosphatase. The intensity of the optical signal induced by the enzymatic activity of alkaline phosphatase adsorbed onto the microwells thus resembles the nonsaturated surface of the microwell. Figure 3 shows the titration curve of an experiment in which microwells were incubated with decreasing concentrations of Tween 20 followed by alkaline phosphatase and substrate. The light absorbance at 405 nm resembles the activity of the solid-phase-adhered enzyme. In this experiment, binding of alkaline phosphatase was completely inhibited by ⬃2.4 g/ml, corresponding to 0.00024% Tween 20. Although Fig. 3 demonstrates that complete saturation was obtained in a series of Tween 20-treated microwells, it was crucial to prove that this was not merely related to the time point at which enzymatic activity was measured. Therefore, microwells were incubated with decreasing concentrations of Tween 20 and the kinetics of the color reaction were measured (Fig. 4). As can be seen, the minimal concentration of Tween 20 at the saturation plateau was the same regardless of the time point the color reaction was measured, although, as expected, the slopes of the curves and the magnitude of their top values differed. The results demonstrate clearly that at 2.4 g/ml of Tween 20 (corresponding to 0.00024%) no enzymatic activity was detected in the pretreated microwells, even if measured after 19 h. The light absorbance values, corresponding to alkaline phosphatase activity in these Tween 20-saturated microwells, were identical to those of control microwells saturated with 50% FCS. We conclude that the dose-dependent saturation curves
indeed show complete inhibition of alkaline phosphatase binding to the pretreated microwells. FCS, the reagent most often used to block ELISA microwells, was tested similarly for its comparable inhibition effect on the binding of alkaline phosphatase in a dose–response experiment. Complete blocking was attained when microwells were preincubated with FCS at 0.1% (results not presented). Binding of Alkaline Phosphatase to PLL-Treated Microwells Poly-L-lysine, a positively charged polypeptide, is sometimes used to increase the capacity of ELISA microwells to bind certain ligands. Figure 5 shows that in a dose–response experiment PLL-treated microwells bound about three times more alkaline phosphatase than wells not treated with the polypeptide (compare the intensity of the color reaction at the plateau level of the PLL-treated microwells with that of the nontreated microwells). Obviously, for a meaningful comparison, the light absorbance must be measured at the same time point when both values, that of the PLL-treated and that of the nontreated microwells, are still in the measurable range. As can be seen, the binding of alkaline phosphatase was totally inhibited when FCS was added to the PLL-treated microwells prior to the enzyme. The Combined Effect of Tween 20 and BSA on ELISA Microwell Saturation In the following set of experiments the blocking effect of Tween 20 and BSA was examined in PLL-coated
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FIG. 4. Binding of alkaline phosphatase to Tween 20-pretreated ELISA microwells: Kinetics of color development. Aliquots containing 200 l decreasing concentrations of Tween 20 in PBS were added to microwells and the plates were incubated for 1 h at 37°C. The microwells were supplemented with alkaline phosphatase and incubation was continued for 1 h. Substrate was then added and light absorbance was measured at different time points. The control microwells were saturated for 1 h with 50% FCS prior to addition of the enzyme. The experiment was carried out in duplicate and the curves represent the average values obtained.
ELISA microwells and in normal ELISA microwells. Microwells in a 96-well microplates were first coated with PLL and then Tween 20 was added to saturate residual protein binding sites. Tween 20 was likewise
added to microwells of a PLL-nontreated plate. Subsequently, 10-fold dilutions of BSA were added and nonsaturated surface area was then determined by adding alkaline phosphatase. Figure 6a shows, in compliance
FIG. 5. Binding of alkaline phosphatase to PLL-pretreated microwells. Aliquots of 200 l with decreasing concentrations of PLL in carbonate buffer were added to ELISA microwells and the plates were incubated for 1 h at 37°C. Following washing, PBS (F) or 50% FCS (E) was added for 1 h. After a second wash, 1/2000 PBS-diluted alkaline phosphatase was added and the color reaction was measured. The experiment was carried out in duplicate and the curves represent the mean values obtained.
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sites. Interestingly, when both blocking reagents were used to saturate the PLL-coated microwells a complete saturation was accomplished only if the concentration of BSA was the same as that required for BSA alone to reach complete saturation. DISCUSSION
FIG. 6. The combined effect of Tween 20 and BSA on saturation of ELISA microwell. Microwells in two 96-well microplates were first incubated for 1 h at 37°C with 200 l of buffer (a) or with PLL, 20 g/ml in carbonate buffer (b). Tween 20, 0.05% in PBS, was added for 1 h at 37°C, followed by decreasing concentrations of BSA in PBS, as indicated. Finally, 1/2000 PBS-diluted alkaline phosphatase followed by substrate was added and light absorbance at 405 nm was recorded. Microwells with and without Tween 20, saturated with 50% FCS, were included as controls in each of the two microplates. In addition, PLL-treated and PLL-nontreated control microwells were included in microplates b and a, respectively. Due to the rapid color reaction developing in the PLL-treated microwells versus that in the nontreated microwells, the color reactions were recorded in succession. The experiments were carried out in duplicate and the curves represent the average values obtained.
with the former results, that 0.05% Tween 20 completely saturated the PLL-nontreated ELISA microwells irrespective of the presence of BSA. BSA alone blocked the surface completely at a concentration of 5 g/ml and, as expected, this effect was also achieved with 50% FCS in the control wells. Obviously, in the experiment shown in Fig. 6a, PLL-coated control microwells displayed a much higher capacity for binding alkaline phosphatase. When the microwells were precoated with PLL, a different pattern of saturation was evident (Fig. 6b). Here, while BSA alone allowed complete inhibition of adherence of alkaline phosphatase, the detergent by itself was unable to fully saturate the available binding
ELISA is now one of the most commonly used methods for the detection and quantitation of antibodies and antigens. The sensitivity of the assay, which is normally very high, can be significantly increased by introducing additional amplification stages (7). The specificity of the assay depends on the quality of the two reagents used for the detection of the solid-phase-adhered ligand, namely, the specific primary antibody and the enzyme-conjugated secondary antibody. However, the sensitivity of the assay, i.e., the lower detection limit of the ligand, depends primarily on the affinity of the reagents and the color reaction background “noise” level. Background is due mainly to the nondesired interaction of the reagents with each other, and also to their unavoidable adherence to residual, nonsaturated sites in the solid-phase area of the microwells. The incorporation of various molecules as blocking reagents in ELISA offers an efficient strategy for minimizing nonspecific background binding. Generally, the reagent of choice is an inert protein(s), added after coating the microwells with the specific ligand which minimally interacts with the other reagents included in the assay. Beside BSA, gelatin, FCS, lowfat milk, and other proteins used for saturation in ELISA, Tween 20 has proven to be a good blocking reagent too, and its synergistic effect with protein has also been described (6, 8). We recently presented a rapid and facile method for evaluating the adherence of any ligand to ELISA microwells, without the need for a specific antibody (5). Accordingly, the binding of the ligand is assessed by measuring the residual surface area available for binding of alkaline phosphatase. Alkaline phosphatase, in turn, is quantitated by the color reaction it yields with its specific substrate. Thus, binding of the ligand to the surface area of the microwells is inversely proportional to the intensity of the enzymatic reaction. In the present study (Figs. 1 and 2) we show that binding of alkaline phosphatase to ELISA microwells, which correlates directly with the remaining vacant sites (5), indeed resembles the binding of protein in general to the nonsaturated surface area. In contrast to a previous study by Jitsukkawa et al. (9), we did not encounter enhanced enzyme binding as a result of the admixture of alkaline phosphatase with a nonrelevant protein, such as BSA or human IgG. Binding of alkaline phosphatase was then used as a criterion for quantitating the blocking effect of Tween
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20 in ELISA microwells. Our results clearly demonstrate (Fig. 3) that this nonionic detergent blocks the vacant binding sites of the microwells and, thus, prevents any residual nonspecific binding of the reagents used in ELISA. At concentrations higher than 2 g/ml (i.e., 0.0002%), Tween 20 provides optimal saturation. The experiment in Fig. 4 emphasizes that this value is not related to the limited time of incubation allowed for the enzyme to cleave the substrate, but that the binding of alkaline phosphatase to the precoated microwells was indeed completely inhibited at the corresponding concentrations of Tween 20. Poly-L-lysine is known to increase significantly the ability of ELISA microwells to bind protein (10, 11). Figs. 5 and 6 show that the amount of alkaline phosphatase that bound to PLL-coated microwells was about three times higher than that binding to nontreated microwells. Complete saturation of PLLtreated ELISA microwells required nearly the same concentration of BSA as that needed for PLL nontreated microwells (Figs. 6a and 6b). However, in contrast to PLL-nontreated microwells, Tween 20 was not adequate for complete saturation of PLL-treated microwells. We assume that Tween 20, which is a nonionic detergent, is not sufficient to saturate the positively charged sites of the polypeptide that varnishes the PLL-coated microwells. Complete saturation with Tween 20 was not attained in the absence of BSA. In PLL-treated microwells and after adding Tween 20, the concentration of BSA required to entirely block residual vacant sites was the same as that which afforded complete saturation when BSA was added alone. These results indicate that, in contrast to regular ELISA microwells, some sites in the PLL-treated surfaces are inaccessible to Tween 20. The finding that these sites can be completely blocked by only relatively high concentrations of BSA may be attributable to low binding affinity of the BSA epitope(s) to the corresponding PLL sites. Alternately, it is feasible that it is not the albumin, but rather a minor contamination, which actually blocks those PLL residual sites and, therefore, a diluted BSA preparation does not suffice. Earlier reports showed that Tween 20 is a satisfactory blocking reagent for ELISA microwells and for the nitrocellulose paper used in Western blots. Indeed, Tween 20 is very often added at a concentration of 0.05% to ELISA and Western blots washing buffers. Our data support these notions and show that Tween 20 saturates ELISA microwells even at much lower concentrations (Figs. 3 and 4). BSA (and obviously FCS as well) provides full saturation for both PLL-treated and -nontreated surfaces. There are clear advantages in using Tween 20 as the sole blocking reagent in
ELISA, with no need for an “alternative inert” protein. “Inert” proteins, such as albumin, FCS, and milk, might increase background “noise,” thereby reducing the sensitivity of the assay. Using a novel semiquantitative method we show in this study that the nonionic detergent Tween 20 is an efficient reagent for saturating the surface of ELISA microwells, although it only partially saturates positively charged PLL-coated surfaces. ACKNOWLEDGMENT This study was supported by a grant from the Israel Ministry of Health.
REFERENCES 1. Engvall, E., and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen coated tubes. J. Immunol. 109, 129 –135. 2. Wedege, E., and Svenneby, G. (1986) Effects of the blocking agents bovine serum albumin and Tween 20 in different buffers on immunoblotting of brain proteins and marker proteins. J. Immunol. Methods 88, 233–237. 3. Mohammed, K., and Esen, A. (1989) A blocking agent and a blocking step are not needed in ELISA, immunostaining dotblots and Western blots. J. Immunol. Methods 117, 141–145. 4. Batteiger, B., Newhall, V. W. J., and Jones, R. B. (1982) The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 55, 297–307. 5. Steinitz, M., and Baraz, L. (2000) A rapid method for estimating the binding of ligands to ELISA microwells. J. Immunol. Methods 238, 143–150. 6. Qu, Y., Bergham, L. R., and Vandesande, F.(1998) An electrochemical enzyme immunoassay for chicken luteinizing hormone: Extension of the detection limit by adequate control of the nonspecific adsorption. Anal. Biochem. 259, 167–175. 7. O’connor, E., Roberts, E. M., and Davies, J. D. (1999) Amplification of cytokine-specific ELISAs increases the sensitivity of detection to 5–20 picograms per milliliter. J. Immunol. Methods 229, 155–160. 8. Jenkins, S. H., Heineman, W. R., and Halsall, H. B. (1988) Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level. Anal. Biochem. 168, 292–299. 9. Jitsukkawa, T., Nakajima, S., Sugawara, I., and Watanabe, H. (1989) Increased coating efficiency of antigens and preservation of original antigenic structure after coating in ELISA. J. Immunol. Methods 116, 251–257. 10. Brennand, D. M., Danson, M. J., and Hough, D. W. (1986) A comparison of ELISA screening methods for the production of monoclonal antibodies against soluble protein antigens. J. Immunol. Methods 93, 9 –14. 11. Kenneth, C. M., Crowther, J. R., and Butcher, R. N. (1985) Alteration in antibody with foot-and mouse disease virus (FMDV) 146S antigen before and after binding to a solid phase or complexing with specific antibody. J. Immunol. Methods 82, 91–100.
Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Microwells Michael Steinitz Experimental Pathology, The Hebrew University–Hadassah Medical School, P.O. Box 12272, Jerusalem, 91120, Israel
Received January 21, 2000
ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-L-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 g/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 g/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent. © 2000 Academic Press
Key Words: ELISA; Tween 20; blocking; alkaline phosphatase; poly-L-lysine; BSA; saturation.
Enzyme-linked immunosorbent assay (ELISA) 1 is a quantitative, sensitive, rapid, easy-to-perform assay which does not require the use of radioactive reagents 1
Abbreviations used: ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; IgG, human ␥-globulins; FCS, fetal calf serum; PBS, phosphate-buffered saline; PLL, poly-L-lysine. 232
(1). In the assay, a color reaction develops in a microwell as the direct result of an interaction between a ligand adhering to the solid phase and a specific molecule added in an aqueous sample. The intensity of the optical signal is proportional to the amount of that specific molecule. Generally, the ligands adhere spontaneously to ELISA microwells and a primary antibody is then added, followed by an enzyme-conjugated secondary antibody that is specific for the primary antibody. A substrate is then added that yields a color reaction, the magnitude of which correlates with the activity of the enzyme. The catalytic activity provides the chemical amplification that allows the detection of extremely low concentrations of specific molecules. Obviously, it is important to keep the background of this multistage assay as low as possible so that even weak optical signals can be distinguished. ELISA microplates possess a high capacity for binding proteins, peptides, and other molecules. It is therefore essential, following coating of the microwells with the appropriate ligand, to saturate the residual noncoated solidphase area with an inert reagent, such as albumin, gelatin, hemoglobin, or FCS. Although these reagents prevent background binding, their use is sometimes not desirable due to their interactions with the primary and/or secondary antibodies (2). In addition, saturation of microwells with large-size proteins may result in somewhat lower reactivity between primary antibodies and small, absorbed ligands, possibly due to steric hindrance. In the original ELISA, Engvall and Perlmann (1) blocked nonspecific binding with Tween 20 only. Today, proteins are generally used to saturate the ELISA microwells, whereas Tween 20 is very often added to the washing buffer to reduce nonspecific binding. Tween 20 is similarly used in Western blot analysis (2– 4). Recently, we described a new method for assessing the magnitude of binding of any ligand to ELISA mi0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
BLOCKING EFFECT OF TWEEN 20 AND BOVINE SERUM ALBUMIN IN ELISA
crowells without the need for a specific antibody and independently of any other ligand-specific binding molecule (5). This enables quantitation of adherence to the microwells of any type and size molecules such as proteins, peptides, sugars, DNA, and hormones. Moreover, the method provides a practical tool for defining the necessary conditions for satisfactory coating of molecules that normally do not readily adhere to ELISA microwells. We used this method in the present study to determine the amount of Tween 20 and BSA required to saturate nontreated and poly-L-lysine-coated ELISA microwells. This approach is widely applicable also to other candidate reagents for blocking the surface area of ELISA microwells. MATERIALS AND METHODS
Reagents ELISA immunoplates (MaxiSorp) were purchased from Nunc (Denmark). The following reagents were obtained from Sigma Chemical Company (St. Louis, MO): alkaline phosphatase derived from calf intestinal mucosa (5.9 mg protein/ml, 2290 units/mg protein); bovine serum albumin (BSA), fraction V; human ␥-globulins (IgG), Cohn fraction II; p-nitrophenyl phosphate; and poly-L-lysine, (hydrobromide) MW120,000. Tween 20 (polyoxyethylene-20-sorbitan monolaurate) was acquired from J. T. Baker (Phillipsburg, NJ). Fetal calf serum (FCS) was purchased from Biological Industries (Beit Haemek, Israel). Determination of Available Vacant Sites for Binding of Alkaline Phosphatase to ELISA Microwells Aliquots of 100 or 200 l containing phosphate-buffered saline (PBS)-diluted alkaline phosphatase are added to ELISA microwells, and the plates are incubated as indicated. Following five washings with PBS, p-nitrophenyl phosphate (1 mg/ml) in 1 M diethanolamine buffer, pH 9.8, containing 0.5 mM MgCl 2 is added and light absorbance at 405 nm is measured with an ELISA reader (Dynatech MR5000). ELISA for Estimation of the Blocking Effect of Tween 20 and BSA The procedure includes four steps: a. Pretreatment of microwells: ELISA microwells are incubated with buffer only or with poly-L-lysine (PLL) in 0.05 M carbonate buffer, pH 9.6, for 1 h at 37°C and then washed five times with PBS. b. Blocking of microwell surface area: Tween 20 or BSA in PBS is added, the plates are incubated at 37°C for the indicated intervals, and then washing is performed as above.
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c. Binding of alkaline phosphatase to residual vacant binding sites: 1/2000 PBS-diluted alkaline phosphatase (i.e., 2.95 g/ml) is added, the plates are incubated for 2 h at 37°C, and washing is performed as above. d. Color reaction: The reaction is performed with the specific substrate as described above. The extent to which Tween 20, BSA, or any other reagent blocks the microwell surface area is estimated according to the magnitude of inhibition of the color reaction, compared with that of nontreated microwells. Obviously, high values of light absorbance indicate the presence of vacant binding sites, whereas low values, due to reduced alkaline phosphatase activity, indicate proportionally less vacant available sites. In contrast to conventional ELISA, all washings in this assay are performed with PBS devoid of Tween 20 or any other background-reducing reagents (such as albumin, gelatin, or FCS). Since in the present study we measure the extent of binding of specific blocking molecules it would be senseless to supplement the washing buffers with any molecule that obscures it. In conventional ELISA Tween 20 is added to the washing buffer to reduce or avoid nonspecific background. In contrast, in our assay it is in fact that “nonspecific” binding which is measured and which resembles inversely the extent of binding of the corresponding blocking reagent. Binding of Alkaline Phosphatase to PLL-Treated Microwells Microwells in 96-well microplates are incubated for 1 h at 37°C with 200 l of carbonate buffer-diluted PLL. Following washing, PBS is added, and the microplates are incubated for 1 h. The plates are washed again, 1/2000 PBS-diluted alkaline phosphatase is added, and the color reaction is measured after the addition of substrate. The Combined Effect of Tween 20 and BSA on ELISA Microwell Saturation Microwells in two 96-well microplates are first incubated for 1 h at 37°C with 200 l of buffer only or with PLL, 20 g/ml in carbonate buffer. Microwells are washed; Tween 20, 0.05% in PBS, is added for 1 h at 37°C; and then washing is repeated, followed by 10-fold dilutions of BSA in PBS. Finally, 1/2000 PBS-diluted alkaline phosphatase followed by substrate is added and light absorbance at 405 nm is recorded. Due to the rapid color reaction that develops in the PLL-treated microwells compared with that in the nontreated microwells, the results are recorded in tandem.
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FIG. 1. Correlation between alkaline phosphatase binding and enzyme concentration. Aliquots containing 100 l with increasing concentrations of alkaline phosphatase in PBS were added to ELISA microwells, and the plates were incubated overnight at room temperature. Following washing, substrate was added and light absorbance at 405 nm was measured. The experiment was carried out in triplicate. The curve represents the average values obtained.
Second, binding of the alkaline phosphatase to the microwells was correlated with the specific activity of the enzyme (i.e., enzyme units/microgram protein). Aliquots containing an equal amount of alkaline phosphatase (i.e., 2.95 g/ml), in an admixture with increasing concentrations of a nonenzyme carrier protein (i.e., human IgG), were added to the microwells and the adsorbed enzymatic activity was determined. Figure 2 shows that the amount of alkaline phosphatase adsorbed to the microwells was inversely correlated with the concentration of human IgG. Obviously, the effect of IgG is not discernable if admixed at a low concentration, compared with that of alkaline phosphatase. The binding of alkaline phosphatase reaches the background value when the carrier protein is present at a 25 times higher concentration than the enzyme. We conclude that measuring the binding of alkaline phosphatase to microwells is indeed a reliable method for assessing the comparable binding of proteins. Tween 20 Saturates the Surface Area of ELISA Microwells
Correlation between Binding of Alkaline Phosphatase to ELISA Microwells and Enzyme Concentration
In ELISA, the nonspecific binding of primary and secondary antibodies to the residual ligand-free surface of the microwells is minimized by adding a nonrelevant protein that saturates any remaining nonoccupied area. We used our assay to assess in a dose– response experiment the blocking effect of Tween 20 on
In the present study, the binding of alkaline phosphatase to the surface of ELISA microwells was assumed to resemble in general the binding of any protein. This assumption was confirmed in two sets of experiments. First, we show that the amount of alkaline phosphatase that binds to the microwells is dependent on the concentration of enzyme. ELISA microwells were coated with increasing concentrations of alkaline phosphatase in PBS. The binding of the enzyme was determined by the intensity of the color reaction measured after the addition of substrate. Figure 1 shows that the light absorbance is in direct proportion to the concentration of the protein which means that the amount of this protein that adsorbs to the microwell surface area is proportional to the concentration of the enzyme. Under the conditions of the experiment shown in Fig. 1 and because of the limited range of the ELISA optical reader, the enzymatic reaction apparently reached a plateau when alkaline phosphatase was added at concentration higher than 738 g/ml. However, when the color reaction was measured at a time point closer to the addition of the substrate a similar direct correlation was also observed at higher concentrations whereas at low concentrations the color reaction was undetectable (results not presented). It is concluded that the diminished color reaction at higher dilutions of the enzyme resembles reduced adsorbance of the enzyme to the microwells surface area.
FIG. 2. Correlation between alkaline phosphatase binding and specific activity of the enzyme. Aliquots containing 100 l of 1/2000 PBSdiluted alkaline phosphatase (2.95 g/ml) mixed with increasing concentrations of human IgG were allowed to adhere to ELISA microwells for 2 h at 37°C. Following washing, substrate was added and light absorbance at 405 nm was measured. The experiment was carried out in triplicate and the curve represents the average values obtained.
RESULTS
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FIG. 3. Binding of alkaline phosphatase to Tween 20-pretreated ELISA microwells. Aliquots containing 200 l decreasing concentrations Tween 20 were added to ELISA microwells and the plates incubated overnight at 4°C. Following washing, 1/2000 PBS-diluted alkaline phosphatase and substrate were added and light absorbance at 405 nm was measured. The experiment was carried out in triplicate and the curve represents the average values obtained.
the residual surface area available for the adherence of alkaline phosphatase. The intensity of the optical signal induced by the enzymatic activity of alkaline phosphatase adsorbed onto the microwells thus resembles the nonsaturated surface of the microwell. Figure 3 shows the titration curve of an experiment in which microwells were incubated with decreasing concentrations of Tween 20 followed by alkaline phosphatase and substrate. The light absorbance at 405 nm resembles the activity of the solid-phase-adhered enzyme. In this experiment, binding of alkaline phosphatase was completely inhibited by ⬃2.4 g/ml, corresponding to 0.00024% Tween 20. Although Fig. 3 demonstrates that complete saturation was obtained in a series of Tween 20-treated microwells, it was crucial to prove that this was not merely related to the time point at which enzymatic activity was measured. Therefore, microwells were incubated with decreasing concentrations of Tween 20 and the kinetics of the color reaction were measured (Fig. 4). As can be seen, the minimal concentration of Tween 20 at the saturation plateau was the same regardless of the time point the color reaction was measured, although, as expected, the slopes of the curves and the magnitude of their top values differed. The results demonstrate clearly that at 2.4 g/ml of Tween 20 (corresponding to 0.00024%) no enzymatic activity was detected in the pretreated microwells, even if measured after 19 h. The light absorbance values, corresponding to alkaline phosphatase activity in these Tween 20-saturated microwells, were identical to those of control microwells saturated with 50% FCS. We conclude that the dose-dependent saturation curves
indeed show complete inhibition of alkaline phosphatase binding to the pretreated microwells. FCS, the reagent most often used to block ELISA microwells, was tested similarly for its comparable inhibition effect on the binding of alkaline phosphatase in a dose–response experiment. Complete blocking was attained when microwells were preincubated with FCS at 0.1% (results not presented). Binding of Alkaline Phosphatase to PLL-Treated Microwells Poly-L-lysine, a positively charged polypeptide, is sometimes used to increase the capacity of ELISA microwells to bind certain ligands. Figure 5 shows that in a dose–response experiment PLL-treated microwells bound about three times more alkaline phosphatase than wells not treated with the polypeptide (compare the intensity of the color reaction at the plateau level of the PLL-treated microwells with that of the nontreated microwells). Obviously, for a meaningful comparison, the light absorbance must be measured at the same time point when both values, that of the PLL-treated and that of the nontreated microwells, are still in the measurable range. As can be seen, the binding of alkaline phosphatase was totally inhibited when FCS was added to the PLL-treated microwells prior to the enzyme. The Combined Effect of Tween 20 and BSA on ELISA Microwell Saturation In the following set of experiments the blocking effect of Tween 20 and BSA was examined in PLL-coated
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FIG. 4. Binding of alkaline phosphatase to Tween 20-pretreated ELISA microwells: Kinetics of color development. Aliquots containing 200 l decreasing concentrations of Tween 20 in PBS were added to microwells and the plates were incubated for 1 h at 37°C. The microwells were supplemented with alkaline phosphatase and incubation was continued for 1 h. Substrate was then added and light absorbance was measured at different time points. The control microwells were saturated for 1 h with 50% FCS prior to addition of the enzyme. The experiment was carried out in duplicate and the curves represent the average values obtained.
ELISA microwells and in normal ELISA microwells. Microwells in a 96-well microplates were first coated with PLL and then Tween 20 was added to saturate residual protein binding sites. Tween 20 was likewise
added to microwells of a PLL-nontreated plate. Subsequently, 10-fold dilutions of BSA were added and nonsaturated surface area was then determined by adding alkaline phosphatase. Figure 6a shows, in compliance
FIG. 5. Binding of alkaline phosphatase to PLL-pretreated microwells. Aliquots of 200 l with decreasing concentrations of PLL in carbonate buffer were added to ELISA microwells and the plates were incubated for 1 h at 37°C. Following washing, PBS (F) or 50% FCS (E) was added for 1 h. After a second wash, 1/2000 PBS-diluted alkaline phosphatase was added and the color reaction was measured. The experiment was carried out in duplicate and the curves represent the mean values obtained.
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sites. Interestingly, when both blocking reagents were used to saturate the PLL-coated microwells a complete saturation was accomplished only if the concentration of BSA was the same as that required for BSA alone to reach complete saturation. DISCUSSION
FIG. 6. The combined effect of Tween 20 and BSA on saturation of ELISA microwell. Microwells in two 96-well microplates were first incubated for 1 h at 37°C with 200 l of buffer (a) or with PLL, 20 g/ml in carbonate buffer (b). Tween 20, 0.05% in PBS, was added for 1 h at 37°C, followed by decreasing concentrations of BSA in PBS, as indicated. Finally, 1/2000 PBS-diluted alkaline phosphatase followed by substrate was added and light absorbance at 405 nm was recorded. Microwells with and without Tween 20, saturated with 50% FCS, were included as controls in each of the two microplates. In addition, PLL-treated and PLL-nontreated control microwells were included in microplates b and a, respectively. Due to the rapid color reaction developing in the PLL-treated microwells versus that in the nontreated microwells, the color reactions were recorded in succession. The experiments were carried out in duplicate and the curves represent the average values obtained.
with the former results, that 0.05% Tween 20 completely saturated the PLL-nontreated ELISA microwells irrespective of the presence of BSA. BSA alone blocked the surface completely at a concentration of 5 g/ml and, as expected, this effect was also achieved with 50% FCS in the control wells. Obviously, in the experiment shown in Fig. 6a, PLL-coated control microwells displayed a much higher capacity for binding alkaline phosphatase. When the microwells were precoated with PLL, a different pattern of saturation was evident (Fig. 6b). Here, while BSA alone allowed complete inhibition of adherence of alkaline phosphatase, the detergent by itself was unable to fully saturate the available binding
ELISA is now one of the most commonly used methods for the detection and quantitation of antibodies and antigens. The sensitivity of the assay, which is normally very high, can be significantly increased by introducing additional amplification stages (7). The specificity of the assay depends on the quality of the two reagents used for the detection of the solid-phase-adhered ligand, namely, the specific primary antibody and the enzyme-conjugated secondary antibody. However, the sensitivity of the assay, i.e., the lower detection limit of the ligand, depends primarily on the affinity of the reagents and the color reaction background “noise” level. Background is due mainly to the nondesired interaction of the reagents with each other, and also to their unavoidable adherence to residual, nonsaturated sites in the solid-phase area of the microwells. The incorporation of various molecules as blocking reagents in ELISA offers an efficient strategy for minimizing nonspecific background binding. Generally, the reagent of choice is an inert protein(s), added after coating the microwells with the specific ligand which minimally interacts with the other reagents included in the assay. Beside BSA, gelatin, FCS, lowfat milk, and other proteins used for saturation in ELISA, Tween 20 has proven to be a good blocking reagent too, and its synergistic effect with protein has also been described (6, 8). We recently presented a rapid and facile method for evaluating the adherence of any ligand to ELISA microwells, without the need for a specific antibody (5). Accordingly, the binding of the ligand is assessed by measuring the residual surface area available for binding of alkaline phosphatase. Alkaline phosphatase, in turn, is quantitated by the color reaction it yields with its specific substrate. Thus, binding of the ligand to the surface area of the microwells is inversely proportional to the intensity of the enzymatic reaction. In the present study (Figs. 1 and 2) we show that binding of alkaline phosphatase to ELISA microwells, which correlates directly with the remaining vacant sites (5), indeed resembles the binding of protein in general to the nonsaturated surface area. In contrast to a previous study by Jitsukkawa et al. (9), we did not encounter enhanced enzyme binding as a result of the admixture of alkaline phosphatase with a nonrelevant protein, such as BSA or human IgG. Binding of alkaline phosphatase was then used as a criterion for quantitating the blocking effect of Tween
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20 in ELISA microwells. Our results clearly demonstrate (Fig. 3) that this nonionic detergent blocks the vacant binding sites of the microwells and, thus, prevents any residual nonspecific binding of the reagents used in ELISA. At concentrations higher than 2 g/ml (i.e., 0.0002%), Tween 20 provides optimal saturation. The experiment in Fig. 4 emphasizes that this value is not related to the limited time of incubation allowed for the enzyme to cleave the substrate, but that the binding of alkaline phosphatase to the precoated microwells was indeed completely inhibited at the corresponding concentrations of Tween 20. Poly-L-lysine is known to increase significantly the ability of ELISA microwells to bind protein (10, 11). Figs. 5 and 6 show that the amount of alkaline phosphatase that bound to PLL-coated microwells was about three times higher than that binding to nontreated microwells. Complete saturation of PLLtreated ELISA microwells required nearly the same concentration of BSA as that needed for PLL nontreated microwells (Figs. 6a and 6b). However, in contrast to PLL-nontreated microwells, Tween 20 was not adequate for complete saturation of PLL-treated microwells. We assume that Tween 20, which is a nonionic detergent, is not sufficient to saturate the positively charged sites of the polypeptide that varnishes the PLL-coated microwells. Complete saturation with Tween 20 was not attained in the absence of BSA. In PLL-treated microwells and after adding Tween 20, the concentration of BSA required to entirely block residual vacant sites was the same as that which afforded complete saturation when BSA was added alone. These results indicate that, in contrast to regular ELISA microwells, some sites in the PLL-treated surfaces are inaccessible to Tween 20. The finding that these sites can be completely blocked by only relatively high concentrations of BSA may be attributable to low binding affinity of the BSA epitope(s) to the corresponding PLL sites. Alternately, it is feasible that it is not the albumin, but rather a minor contamination, which actually blocks those PLL residual sites and, therefore, a diluted BSA preparation does not suffice. Earlier reports showed that Tween 20 is a satisfactory blocking reagent for ELISA microwells and for the nitrocellulose paper used in Western blots. Indeed, Tween 20 is very often added at a concentration of 0.05% to ELISA and Western blots washing buffers. Our data support these notions and show that Tween 20 saturates ELISA microwells even at much lower concentrations (Figs. 3 and 4). BSA (and obviously FCS as well) provides full saturation for both PLL-treated and -nontreated surfaces. There are clear advantages in using Tween 20 as the sole blocking reagent in
ELISA, with no need for an “alternative inert” protein. “Inert” proteins, such as albumin, FCS, and milk, might increase background “noise,” thereby reducing the sensitivity of the assay. Using a novel semiquantitative method we show in this study that the nonionic detergent Tween 20 is an efficient reagent for saturating the surface of ELISA microwells, although it only partially saturates positively charged PLL-coated surfaces. ACKNOWLEDGMENT This study was supported by a grant from the Israel Ministry of Health.
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