A High-Capacity Streptavidin-Coated Microtitration Plate

Bioconjugate Chem. 2003, 14, 103−111

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A High-Capacity Streptavidin-Coated Microtitration Plate Lasse Va¨limaa,* Kim Pettersson, Markus Vehnia¨inen, Matti Karp, and Timo Lo¨vgren University of Turku, Department of Biotechnology, Tykisto¨katu 6 A, FIN-20520, Turku, Finland. Received June 14, 2002; Revised Manuscript Received October 30, 2002

A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). The maximum immobilization capacity of the modified plate was up to 2.5 times higher than that of the control plate. The higher binding capacity was especially emphasized with small-size molecules. The modified high capacity plate increased the linear ranges of the immunoassays and thus delayed the high-dose hook effect. At high antigen concentrations the signal increased up to 59%, and at the conventional linear ranges of the assays, the increase was up to 29%. We conclude that the modified coating method will be valuable for the future miniaturized systems, where high immobilization capacity is needed at limited areas.

INTRODUCTION

Most of the presently used immunoassays utilize binding of a specific antigen or antibody on a solid phase followed by the removal of nonbound analyte and the sample matrix by extensive washing procedure (1). Commonly used solid phases are 96-well microtitration plates or single wells prepared of polystyrene. For the immunoassay purposes, the microtitration well surfaces are precoated with capturing protein like antibody to allow analyte immobilization. However, direct adsorption of antibodies on the plastic surface may destroy the functional sites to even less than one-tenth of the original activity (2-4). These studies have shown also that the protein activity could be preserved when the capturing antibody was immobilized as a secondary layer over a primary coated layer such as via streptavidin-biotin linkage. Therefore, streptavidin-coated wells are preferred to antibody or antigen coated wells. In addition, streptavidin-coated wells provide a universal immobilization surface for any biotinylated molecule and therefore tedious optimization of adsorption conditions for a number of different antibodies or antigens could be avoided. Generally, the streptavidin-biotin technology provides a valuable tool in clinical diagnostics (5) like immobilization of the molecules on the solid phases or signal amplification (6, 7). Therapeutic applications of biotinstreptavidin use are, e.g., delivery of drug molecules to pretargeted cancer cells (8, 9). Streptavidin, like its counterpart hen egg-white avidin, is a tetrameric protein consisting of four identical subunits each about 14 kDa in size. These proteins are known for their ability to bind biotin very tightly (10, 11). Unlike avidin, streptavidin is from microbial source; * Corresponding author. Phone: +358-2-333 8089. Fax: +3582-333 8050. E-mail: [email protected].

it is produced by the bacteria Streptomyces avidinii. In diagnostic applications streptavidin is preferred to avidin due to reduced nonspecific binding which is because of lower isoelectric point and lack of carbohydrate moieties. Biotin, also known as vitamin H, is a small molecule (MW ) 244) with a double-ring structure and a carboxyl acid side chain. The side chain can be extended with different linker molecules and active groups enabling chemical coupling of biotin to other molecules. The coupling can be done under gentle conditions (12), and the biological activity of protein is thus well preserved. The biotinstreptavidin interaction is nearly irreversible since the binding affinity (Ka) in liquid phase is about 2.5 × 1013 M-1 (13). However, decreased affinity (around 108-1010 M-1) was shown between biotinylated macromolecule and solid-phase adsorbed streptavidin (14). This may be due to partial rupture of the critical binding sites and adoption of stiff conformation upon adsorption and attachment on the solid phase. Nevertheless, the binding tightness is still strong enough to separate the biotinylated molecules from nonbiotinylated ones in the immunoassays. The influence of the solid phase is significant in noncompetitive, two-site immunoassays (1). The binding capacity of the surface must be high enough to provide a real reagent excess, wide dynamic range without highdose hook effect, and rapid kinetics. Nonspecific binding must be negligible in order to acquire the highest possible assay sensitivity. It is also evident that correct orientation of the immobilized antibody has a significant role (15, 16). Besides immunoassays, streptavidin surfaces are used for nucleic acid assays to immobilize biotinylated oligonucleotide probes or biotinylated amplification products (17). The immobilization of oligonuclotides directly on the plastic surface is often complicated, since they are too small in size for proper passive adsorption to take

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104 Bioconjugate Chem., Vol. 14, No. 1, 2003

place. Nucleic acid amplifications usually utilize a large excess of biotinylated primers, and therefore detection of the products at streptavidin wells requires a large number of binding sites. Dissolving streptavidin in appropriate buffer and subsequent plain passive adsorption into the microtitration wells usually results in quite poor coating densities and low binding capacities. Therefore, the advantageous effect of streptavidin-biotin immobilization to the features of immunoassays may be abolished by the poor quality of the primary streptavidin coat. Some trials have been made to improve the coats. High-capacity plates have been prepared by coupling streptavidin to hydrophobic proteins prior to adsorption or by immobilizing streptavidin on biotinylated macromolecule coats (18, 19). We introduce here a simple method to prepare streptavidin-coated plates having improved binding capacities. The method relies on the chemical cross-linking of streptavidin prior to the routine coating procedure. The properties of the high-capacity plate were compared to the control plate coated without pretreatment of streptavidin. These types of the wells are widely used, and this is the way a number of commercial plates are still prepared. The properties of the plates were studied in different capacity tests and in noncompetitive, sandwichtype immunoassays. MATERIALS AND METHODS

Coating Procedure. Streptavidin was obtained from BioSpa, Societa` Prodotti Antibiotici (Milan, Italy). Lyophilized protein was dissolved in pure water (Milli-Q) 10 mg/mL and stored frozen in aliquots. MaxisorpTM microtitration plates in C12-strips or single well formats were from Nunc A/S (Roskilde, Denmark). The normal coating procedure for streptavidin, the preparation of the control plate, is briefly described below. Streptavidin was diluted in the coating buffer (100 mM Na2HPO4/50 mM citric acid, pH 5.0) to the final concentration 5.0 µg/mL. Then 200 µL of the coating solution was dispensed into each well, giving 1 µg streptavidin per well. The plates were closed in a humidified box and incubated overnight at +35 °C. Then the plates were washed in a DELFIA Platewash (Perkin-Elmer Life Sciences, Turku, Finland) with DELFIA Wash Solution supplemented with Tween 20 (Merck, Hohenbrunn, Germany) to the final concentration 0.05%. After washing, 250 µL of saturation solution (50 mM Tris-HCl, pH 7.0; 150 mM NaCl; 0.05% NaN3; 0.2% bovine serum albumin and 6% D-sorbitol) was added per well. The plates were saturated overnight at +25 °C. The saturation solution was aspirated and the plates were dried (+35 °C, relative humidity < 5%) for 2 h. Finally the plates were packed with moisture adsorbent and stored dry at +4 °C. The procedure leading to the high-capacity, modified plate included a pretreatment step prior to addition of streptavidin into the coating buffer. A reaction mixture was prepared, containing 2 mg/mL streptavidin, 10 mM NaH2PO4/Na2HPO4 (pH 7.0), 150 mM NaCl, and 1.0% glutaraldehyde (J. T. Baker, Deventer, The Netherlands). The mixture was incubated 2 h at +4 °C. The excess glutaraldehyde was removed by purifying the reaction in gel filtration through NAP-5 and NAP-10 columns or PD-10 column (Amersham Biosciences AB, Uppsala, Sweden). The cross-linked glutaraldehyde-streptavidin (GA-SAv)1 was eluted in TSA buffer (50 mM Tris-HCl, pH 7.75; 150 mM NaCl; 0.05% NaN3). The streptavidin concentration of the eluate was determined in a Bradford dye-binding assay following the instructions of the kit

Va¨limaa et al.

supplier (Bio-Rad Laboratories, Hercules, CA). Originally dissolved streptavidin was used as a reference standard. GA-SAv was then diluted in the coating buffer (5.0 µg/ mL), and the coating was proceeded according to the procedure described above. Size-Exclusion Chromatography of Cross-Linked Streptavidin. GA-SAv was analyzed and fractionated in a preparative size-exclusion chromatography through Superose 12 HR 10/30 column driven by A ¨ KTA explorer system (both from Amersham Biosciences AB). An aliquot of completed cross-linking reaction mixture (before purification through NAP-columns) was injected into the column through a 2.0 mL sample loop, followed by isocratic elution in buffer consisting of 50 mM Tris-HCl (pH 8.40) and 0.05% NaN3 with flow rate 0.5 mL/min. The purification was monitored at 280 nm, and 0.5 mL fractions were collected in an automatic fraction collector. Molecular weight calibration standards of Gel filtration calibration kits (Amersham Biosciences AB) were run separately in identical conditions. The protein concentrations of the fractions were determined with the Bradford method as above. On the basis of absorbance and protein concentration profiles, adjacent fractions from the appropriate sites were combined to give three pools representing different molecular sizes. An aliquot from each pool was coated on the microtitration plates as described above. MALDI-TOF Mass Spectrometric Analyses. Bound proteins were eluted from ZipTip C4 reverse-phase matrix (Millipore, Bedford, U.S.A.) in 2.5% formic acid (Rathburn Chemicals, Walkerburn, Scotland), 50% acetonitrile (Rathburn Chemicals), and 10 mg/mL sinapinic acid (SigmaAldrich, Steinheim, Germany) to the sample plate. Perseptive Biosystems (Framingham, MA) Voyager DEPRO mass spectrometer with a pulsed nitrogen laser at 337 nm was operated in a linear mode. The instrument operated at a 25 kV accelerating potential. At least 120 scans were collected for data acquisition. For calibration we used lysozyme (Sigma-Aldrich) and bovine serum albumin (Sigma-Aldrich). Biotinylation and Labeling. The immobilization capacities of the streptavidin-coated plates were measured with molecules bearing both biotin moieties and europium (Eu) chelates as measurable labels. A monoclonal intact antibody H117 (20) was biotinylated with an isothiocyanate derived active biotin, BITC (21). The biotinylation was performed with an 80-fold molar excess (500 µM) of BITC in 50 mM carbonate buffer (pH 9.8). After 4 h incubation at room temperature the biotinylated antibody was separated from noncoupled BITC through NAP-5 and NAP-10 columns. The biotinylated antibody was labeled with an isothiocyanate-activated fluorescent Eu chelate, 2,2′,2′′,2′′′-[[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetic acid) (22). The labeling was performed with a 2040-fold molar excess (125-250 µM) of activated Eu chelate in 50 mM carbonate buffer (pH 9.8). After overnight incubation at +25 °C the biotinylated and labeled antibody (Bio-H117-Eu) was separated from noncoupled chelate in a Superdex 200 HR 10/30 column (Amersham Biosciences AB). The antibody was eluted in TSA buffer, and the fractions containing the purified labeled antibody were pooled. The protein concentration 1 Abbreviations: BITC, biotin isothiocyanate; DELFIA, dissociation-enhanced lanthanide fluoroimmunoassay; GA-SAv, glutaraldehyde pretreated streptavidin; hCG, human chorionic gonadotropin; MS, mass spectrometry; PSA, prostate specific antigen; SAv, streptavidin.

High-Capacity Streptavidin-Coated Plate

of the pool was determined by measuring absorbance at 280 nm. Eu contents of the pool was measured by preparing a dilution series of the solution in DELFIA Enhancement Solution and measuring time-resolved fluorescence in a Victor 1420 multilabel counter (PerkinElmer Life Sciences) after 30 min shaking. A dilution series of europium standard calibrator was run together with the sample. The labeling degree (Eu chelates/ antibody molecule) was calculated on the basis of measured Eu and protein concentrations. We used also horse skeletal muscle myoglobin (SigmaAldrich) and biotinylated peptide for the capacity measurements. Those represented smaller molecular weights (myoglobin ≈ 17 000 Da and peptide ) 1672 Da) than the intact antibody (≈160 000 Da). Myoglobin (Mb) was biotinylated, labeled, and purified as described above. The protein concentration of the purified, pooled BioMb-Eu solution was determined in the Bradford assay using originally dissolved myoglobin as a reference standard. The labeling degree was calculated based on the measured Eu and protein concentrations. The biotinylated and Eu-labeled peptide (Bio-Eu-peptide) was purchased from Perkin-Elmer Life Sciences. The peptide consisted of 11 amino acids and its molecular weight was 1672. The peptide bore two Eu chelates ([2-(4-isothiocyanatobenzyl)]diethylenetriaminetetraacetic acid), described by Mukkala et al. (23). Production and Purification of Recombinant Fab Fragment. Hybridoma cell line H117 was previously (24) cloned as a recombinant Fab fragment. It contains an engineered five amino acid long Thr-Ser-Cys-Ala-Ala peptide at C-terminus of the Fd chain. Fab fragments were expressed to periplasmic space of Escherichia coli in a 5-L BioFlo3000 -fermentor (New Brunswick Scientific, New Jersey) and purified with osmotic shock, Streamline SP (75 mL in Streamline25 column) expanded bed adsorption chromatography and finally 5 mL HiTrap Protein G affinity column (all from Amersham Biosciences AB). Unpaired cysteine in C-terminal peptide was used for site-specific biotinylation with 3-(N-maleimido-propionyl) biocytin (Sigma-Aldrich), and the biotinylated fraction was collected using 5 mL monomeric avidin column (SoftLink avidin resin, Promega, Madison, USA). The Determination of the Binding Capacities of the Coated Wells. Dilution series of Bio-H117-Eu or Bio-Mb-Eu were prepared in Assay Buffer (PerkinElmer Life Sciences) shortly before use. One hundred microliters of each dilution was added into four replicate wells of streptavidin-coated microtitration plates, giving 0.125-12.5 pmol protein inputs per well. The plates were sealed with tape and shaken for 1 h in the DELFIA Plateshake and washed six times with DELFIA Wash solution (5 mM Tris-HCl, pH 7.75; 154 mM NaCl; 0.1% Germall II+; 0.005% Tween20) in the Platewash. The plates were dried at +65 °C air blow for 3 min and were cooled to the room temperature for 10 min. The timeresolved fluorescence signal was measured from the bottom of the dried wells in the Victor counter. For this surface readout measurement the measurement focus was custom-adjusted close to the bottom of the well. An area of a few square millimeters in the bottom of the well becomes excited and measured. After the surface readout measurement, the plates were passed to the conventional dissociation enhancement (DELFIA) measurement. Two hundred microliters of Enhancement Solution was added into each well, and the time-resolved fluorescence was measured in the Victor counter after 30 min shaking. Known amounts of europium calibrators were measured

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parallel. A standard curve was derived and the Eu concentrations of the wells containing immobilized BioEu-molecules were calculated. Since the label degrees of the molecules were known, the actual immobilized quantities of Bio-Eu-molecules (pmol/well) could be calculated. The capacity test utilizing successive incubations with free biotin and Bio-Eu-peptide was performed as follows. D-Biotin (Sigma-Aldrich) was first dissolved in 1 M NaOH and diluted in the Assay Buffer to give a 11.9 mM stock solution from which the work dilutions containing up to 2.5 × 1013 molecules (41.5 pmol) D-biotin per 200 µL were prepared. Two hundred microliters of each dilution was added into four replicate wells of the streptavidin-coated plates. The plates were shaken at room temperature for 1 h and washed twice in the Platewash. Bio-Eu-peptide was diluted in Assay Buffer and 200 µL of the dilution was added into each well giving 0.17 pmol peptide input per well. After 1 h shaking the wells were washed six times, and Enhancement Solution was added. Time-resolved fluorescence was measured in Victor counter after 5 min shaking. For the antigen-binding capacity test, biotinylated inatct antibody H117 or site-specifically biotinylated recombinant Fab fragments of that were diluted in Assay Buffer and immobilized in streptavidin-coated wells during a 30 min shaking. Four hundred nanograms of Bio-H117 and two hundred nanograms of Bio-Fab per well were used. Prostate-specific antigen (PSA) standard was a kind gift from Professor Hans Lilja (Lund University, Malmo¨, Sweden). PSA was labeled with Eu chelate (22) as described above. A dilution series of Eu-PSA was prepared in Assay Buffer, and 30 µL of each dilution (5500 ng Eu-PSA) was added into the wells harboring the immobilized Bio-H117 or Bio-Fab fragments. The reaction was incubated 40 min at +30 °C in iEMS incubator shaker (Labsystems, Helsinki, Finland). The plates were then washed six times, Enhancement Solution was added, and time-resolved fluorescence was measured in Victor after five minutes shaking. Immunoassays. Prostate Specific Antigen. Four hundred nanograms of Bio-H117 (in 50 µL Assay Buffer) were added into streptavidin coated wells. The plates were shaken for 1 h at the room temperature in the Plateshake and washed twice in the Platewash. A series of PSA antigen standard dilutions (0.19-5000 µg/L) were prepared in 7.5% TSA-BSA (50 mM Tris-HCl, pH 7.75; 150 mM NaCl; 0.05% NaN3; 7.5% bovine serum albumin). Ten microliters of each standard dilution was added into the wells containing immobilized Bio-H117. The tracer antibody 5F7 was produced at our department as characterized by Nurmikko et al. (25). The antibody 5F7 was labeled with the fluoresecent Eu chelate (22) as described above. Eu-5F7 was diluted in Assay Buffer and 20 µL of the dilution containing 200 ng antibody was added to each well shortly after the addition of the standard dilutions. The plates were shaken 30 min at room temperature, followed by washing six times. Enhancement Solution was added (200 µL), and the signal was measured in Victor after 30 min shaking. In the other PSA assay, recombinant Fab fragment of H117 antibody (produced as described above) was used as capturing antibody instead of intact Bio-H117. The assay was run as above, except that the amount of Bio-Fab used for the immobilization in the first step was 600 ng/well, and the tracer antibody Eu-5F7 was used 800 ng/well to provide sufficient reagent excess to satisfy increased binding capacity.


Figure 1. The capacities of the streptavidin-coated plates measured with a biotinylated, Eu-labeled antibody Bio-H117Eu. The signal was measured either with a surface readout method from the dried bottom of the well (A) or with dissociation enhancement (DELFIA) method (B). The filled symbols (b) denote the control plate and the open symbols (O) the GA-SAv plate. The error bars indicate the within-assay standard deviations of four replicate wells.

Human Chorionic Gonadotropin (hCG). Biotinylated monoclonal antibody Bio-E27, hCG standards, and tracer antibody Eu-8D10 were from Perkin-Elmer Life Sciences. Four hundred nanograms of Bio-E27 was used for immobilization into streptavidin-coated microtitration wells in 50 µL Assay Buffer. The hCG standard dilutions including 2-80 000 units per liter hCG (1 U/L corresponds to about 0.1 µg/L) were prepared in 7.5% BSATSA. Shortly after the addition of the standard dilution, 300 ng/well of Eu-8D10 was added in 20 µL Assay Buffer. The plates were shaken 30 min at +36 °C in iEMS incubator shaker and washed six times. The signal measurement was run as described above for the surface readout method. RESULTS

The Immobilization Capacities of the Streptavidin-Coated Plates. The binding capacities of the plates measured with Bio-H117-Eu are shown in Figure 1. The GA-SAv plate shows up to 1.9 times higher signal than the control plate when the surface readout mea-

Va¨limaa et al.

Figure 2. The capacities of the streptavidin-coated control plate (b) and GA-SAv plate (O) measured with a biotinylated Eu-labeled myoglobin, Bio-Mb-Eu. The signal was measured either with a surface readout method from the dried bottom of the well (A) or with dissociation enhancement (DELFIA) method (B). The error bars indicate the within-assay standard deviations of four replicate wells.

surement was used and up to 1.6 times higher signal with dissociation enhanced (DELFIA) method. The surface readout method measures only a certain area in the bottom of the well, while the latter method rather represents an integrated signal of the total coated area. The capacity curves of Bio-Mb-Eu measurement are shown in Figure 2. Compared to the control plate, the GA-SAv plate presented 2.3-fold increase in signal with surface readout and 1.8-fold increase in DELFIA method. It was noted that as the size of the immobilized detection molecule decreased the differences between the control and GA-SAv plates increased, which most probably indicates less steric hindrance in the case of Bio-MbEu. The actual immobilized quantities were calculated from the DELFIA signals and were found to be 2.2 and 3.6 pmol for Bio-H117-Eu and 2.8 and 5.2 pmol for BioMb-Eu at the control well and GA-SAv well, respectively. The results of the capacity test based on the successive incubations with D-biotin and Bio-Eu-peptide are shown in Figure 3. The measured fluorescence signals



Figure 3. The capacities of the plates as measured in the D-biotin test. D-Biotin was first immobilized in the wells, and the unoccupied sites were detected with biotinylated, Eu-labeled peptide (Bio-Eu-peptide). The capacities of the control plate (b) and GA-SAv plate (O) can be read at the lower turn-points of the curves. The error bars indicate the within-assay standard deviations of the replicate wells.

Figure 4. Antigen-binding capacities of the intact antibody (b and O) and Fab fragments (2 and 4) at the control plate (filled symbols) or GA-SAv-plate (open symbols). The error bars indicate the within-assay standard deviations of the replicate wells.

Table 1. The Capacities of the Control Plate and the GA-SAv Plate to Immobilize Different Biotinylated Molecules capacity (pmol/well) biotinylated molecule

MW

control plate

intact antibody (IgG) myoglobin peptide (direct immob.) D-biotin/peptide

160 000 17 000 1672 1672

2.2 2.8 3.8 13

GA-SAv plate 3.6 5.2 8.3 30

were compared to the maximum signal measured from the zero wells (no D-biotin input), and the percentage values were calculated. The capacities of the plates were estimated at the lower turn-points of the curves. The capacity of the control plate was 0.8 × 1013 molecules (13 pmol), that of the modified plate 1.8 × 1013 molecules (30 pmol), and the difference between the plates thus 2.3. It was noted that the signal level did not reach close to zero but stayed around 20% of the maximum signal despite large excess of D-biotin used. We assume that some of the bound D-biotin dissociated out of the surface and thus the method overestimates the binding capacities. The binding capacities of the wells in the case when BioEu-peptide was immobilized direct (like Bio-H117-Eu and Bio-Mb-Eu above) were 3.8 pmol for the control well and 8.3 pmol for the GA-SAv well. Table 1 summarizes the binding capacities of the control and GASAv plates as measured with different biotinylated molecules. Figure 4 compares the capacities of immobilized biotinylated intact antibody (Bio-H117) or biotinylated Fab fragment (Bio-Fab) to bind cognate Eu-labeled PSA antigen. Changing from Bio-H117 to Bio-Fab resulted in around 1.8-fold increase in the binding capacity. Immobilizing Bio-Fab in the GA-SAv well resulted in 1.9-fold more increase in the binding capacity. Thus, combining Bio-Fab with high capacity GA-SAv plate altogehter resulted in 3.5-fold increase compared to the case of intact antibody immobilized in the control well. Size-Exclusion Analysis and Fractionation of Cross-Linked Streptavidin. The elution profile of cross-linked streptavidin is shown in Figure 5. Under the

Figure 5. Elution profiles of cross-linked streptavidin (straight line) and BSA as a molecular weight marker (dashed line). The fractionation of GA-SAv was performed in a Superose 12-gel filtration column. The profile represents molecular weights extending from around 200 to 25 kDa. The locations of the adjacent fractions combined to three pools are shown with bars.

described conjugation conditions (+4 °C, 2 h, 2 mg/mL SAv), the reaction resulted in a mixture of products ranging in molecular weights from around 25 to around 200 kDa. The exact determination of the highest molecular weights turned out to be inaccurate, since those eluted quite close to the column void volume determined with Blue Dextran 2000 from the calibration kit. The peak representing free glutaraldehyde appeared at an elution volume of 21 mL. The first one of the pooled fractions represented molecular weights from 95 to 200 kDa, the second 65 to 85 kDa, and the third 45 to 55 kDa. Prolonged incubation of the conjugation reaction at the room temperature increased the proportion of larger molecular weight products slightly. A considerable change was noted when protein concentration in the conjugation reaction was increased from 2 to 4 mg/mL. This resulted in an almost complete cross-linking; i.e., only one peak


Figure 6. Mass spectra of streptavidin. Native streptavidin (A) is mainly found as monomeric form (m/z 13 140) while di-, tri-, and tetrameric forms are hardly present. Glutaraldehydetreated streptavidin (B) is stabilized over native streptavidin and up to octameric subunit compositions are found.

appeared representing the largest conjugate sizes (data not shown). The binding capacities of the wells coated from the three pools of cross-linked, fractionated streptavidin (see Figure 5) did not exceed that coated from nonfractionated mixture. The immobilization capacities of the pools 1 and 2 were 80% and pool 3 around 50% of a nonfractionated, total mixture. The capacity of the pool 3 was close to the control plate. Neither the coated wells prepared from the cross-linking reaction with 4 mg/mL protein concentration introduced improved capacity compared to the 2 mg/ mL reaction but the capacities were equal or slightly decreased (data not shown). Analysis of Mass Spectrometry Results. Streptavidin from Streptomyces avidinii is synthesized as a 183 amino acids long precursor. After cleavage of a 24 aa long signal sequence, mature 159 aa long streptavidin (calculated mass 16 490 Da) is released. It undergoes proteolytic digestions, and finally a core streptavidin of 127 aa is formed. Monomeric core streptavidin has an expected molecular mass of 13 270 daltons (10), and thus calculated mass for tetrameric core streptavidin (native existence) is 53 080 daltons. For non-glutaraldehydetreated streptavidin (Figure 6 A), we found four peaks

Va¨limaa et al.

Figure 7. The standard curves of the PSA-assays. Either biotinylated intact antibody (A) or a recombinant Fab fragment of the antibody (B) was used as the immobilized capturing agent. The filled symbols (b) denote the assays run at the control plate and open symbols (O) at the GA-SAv plate. The inset graphs show the standard curve ranges up to 500 µg/L. The error bars show the within-assay standard deviations of four replicate wells.

corresponding to mono- (13 140 Da) to tetrameric streptavidin masses (56 782 Da). Monomeric peak dominated in the results. Glutaraldehyde treatment increased di- to tetrameric streptavidin peak intensities 2- to 10-fold. Also, new peaks differing in size at average intervals of 14 880 Da appeared clearly up to size 118 152 Da, which presumably corresponds to a dimer of streptavidin (Figure 6 B). PSA Immunoassay. Figure 7 A shows the standard curves of PSA immunoassays with the intact Bio-H117 antibody immobilized as the capturing antibody. The figure shows that the assay performed at the highcapacity GA-SAv plate has improved high-dose hook efficiency; i.e., the hook is avoided, or it tends to appear at higher antigen concentrations. The background signals were 637 cps at the control plate and 937 at the GASAv plate. The detection limits of the assays (background + 3 × SD) were 0.191 µg/L at the control plate and 0.239 µg/L at the GA-SAv plate. Figure 7 B shows the standard curves of the assays utilizing recombinant BioFab immobilized as the capturing agent. This assay clearly shows the potential of the high capacity GA-SAv


Figure 8. The standard curves of the hCG-assays at the control plate (b) and GA-SAv plate (O). The inset graph shows the range of the assay up to 20 000 U/L. The error bars show the within-assay standard deviations of four replicate wells.

plate since excellent linearity of the assay extends at least up to 5000 µg/L, while the standard curve at the control plate declines earlier. Because nonoptimal excess amounts of the labeled tracer antibody had to be used to satisfy increased capacities, the background signals tended to increase and thus lower detection limits of these assays were 0.795 µg/L at the control plate and 0.717 µg/L at the GA-SAv plate. hCG Immunoassays. The standard curves of the hCG immunoassays, measured by surface readout method, are shown in Figure 8. The assay shows improved linearity and higher binding capacities of the GA-SAv plate. Since hCG concentration in pregnancy can be as high as 200 000 IU/L or above, it is essential to have high binding capacity and improved linearity to avoid the high-dose hook effect. The background signals were 1782 at the control plate and 1876 at the GA-SAv plate. The detection limits of the assays were 4.5 U/L at the control plate and 4.2 U/L at the GA-SAv plate. The hCG assay was also run at the GA-SAv plates coated from the pools of fractionated cross-linking reactions. No improvement in linearity or binding capacity was obtained compared to the nonfractionated mixture (data not shown), as would have been expected also on the basis of the BioMb-Eu capacity test. Reproducibility and Storage Stability. Thirteen separate batches of GA-SAv plates were coated independently of each other. The coated plates were collected together, and the capacities of the plates were measured with Bio-Mb-Eu. The CV% values within one coated batch varied from 0.6% to 4.3%. The batch to batch CV% values varied from 10.0% to 14.3%. The stability of one batch of GA-SAv-coated plates was followed several months. The plates were stored in separate sealed boxes with moisture adsorbent at +4 °C after coating. One plate at a time was taken out and the capacity was measured with Bio-Mb-Eu using low input (1.25 pmol/well) and high input (12.5 pmol/well). Figure 9 shows the results of the assays measured during those 225 days of storage. A slight increase (19%) in the signal level was noted when measured with low amount of BioMb-Eu but a slight drop (6%) when measured with 12.5 pmol Bio-Mb-Eu.


Figure 9. The storage stability of the GA-SAv plate. BioMb-Eu was used to measure the capacities of GA-SAv plates after different storage periods. The measurements were performed either with 1.25 (b) or 12.5 pmol/well (O) inputs. The storage stability was followed up to 225 days. The error bars indicate the standard deviations of the replicate wells. DISCUSSION

The streptavidin-coated plates allow an immobilization of any biotinylated molecule and thus provide flexibility for the solid phase assays compared to the antibody- or antigen-coated plates. Streptavidin-coated plates are widely used in clinical diagnostics and there are numbers of manufacturers. Though the plates are widely used, the optimal coating conditions are based more or less on experimental optimization and the adsorption mechanisms are not well understood, as is the case for proteins in general. Also, the influence of the solid phase on the immunoassay performance is often underestimated. We presented a preparation of high-capacity streptavidin-coated microtitration plate with a convenient and cost-effective method. The properties of the plates were characterized in terms of binding capacities and immunoassay performance. The capacity increased considerably compared to the control plate but the size of the immobilized biotinylated molecule affected the difference, a result that is quite obvious from steric point of view. Furthermore, surface readout measurement gave larger differences between the plates than dissociation enhancement method. Probably adsorption to the bottom of the well was thus favored in GA-SAv coating, which is promising for the future surface measurement applications. Taking into account the different molecular sizes and measurement methods, we can deduce that the maximum binding capacity of the GA-SAv plate is at least 2-2.5-fold higher than that of the control plate. Fractionation of GA-SAv by gel filtration and coating from pooled peaks showed that the largest molecular weight products are mainly responsible for the increased capacities, but none of the fractions alone exceeded the capacities of a nonfractionated mixture. It shows that a range of molecular sizes is needed in order to achieve the highest performance. The fractionation also showed products that were smaller in size than a native streptavidin consisting of four identical subunits. A reason may be that some element in conjugation process had partially dissociated tetrameric structure to smaller units from which the cross-linking and building of the larger species was started. On the other hand, our gel filtration runs with native, nontreated streptavidin showed that some


of the original protein had dissociated to subunit dimers and thus peaked at around 25-30 kDa. The MALDI-TOF mass spectrometry results of nontreated streptavidin showed a dominance of the peak corresponding to one subunit of streptavidin. Most probably the tetrameric structure, held together by noncovalent interactions, was disrupted to subunits by intensive laser radiation. The MALDI-TOF results of GA-SAv showed peaks representing larger sizes, which were multiples of one subunit size. The existence of the peak corresponding to one subunit confirmed that the conjugation did not go to completion in the conditions we used for cross-linking. Noncompetitive reagent excess immunoassays are widely used to assay clinical analytes demanding high sensitivity, wide dynamic range, speed, and robustness. Minimal nonspecific binding, even background signal and high slope of the standard curve result in sensitive assays having low detection limits. The high-dose hook effect, caused by blocking of all antibody binding sites before sandwich formation, can be avoided or shifted to higher concentrations by increasing the number of binding sites for antigen. Our immunoassay results showed that the hook performance could be improved with high capacity solid phases. The influence of the high capacity plates to the assay sensitivities was insignificant, since the standard curves worked quite equally in the “conventional” linear ranges of the assays at both types of plates. In that area, the capacity of the control plate was high enough to immobilize the required quantity of the biotinylated antibody to give optimal binding of the antigen. It must be considered that the actual quantities of the antigens we used were quite low since small sample volumes (10 µL per well) were used. The GA-SAv coating protocol is likely to be important when the solid phases are reduced in area as in spots or in chip technologies. In these cases a considerable high density of the coat at the unit area is required to give enough binding capacity. A large increase in antigen binding capacity was obtained when Fab fragments were immobilized in GASAv plates (Figure 4). This shows clearly how dramatic the influence is of steric hindrance caused by the large size of the intact antibody. Large antibody molecules mask the streptavidin binding sites on the surface and therefore full potential of the GA-SAv plates is not available. In the Bio-Fab-based PSA-assay (Figure 7 B), we did not reach such improvement in assay performance as would have been expected on the basis of labeled antigen-binding assay. There are at least two reasons for that. First, the actual antigen amounts were quite small as discussed above. Second, we had to use intact whole size antibody as the tracer and this rises again the influence of steric factors. There was not a proper recombinant Fab fragment of any appropriate tracer antibody available. Nonspecific binding of the labeled antibody causes increase in the assay background signal (wells with zero standard), and especially background variation impairs the lower detection limit. The immunoassays run at the GA-SAv plate showed none or up to some 1.5-2-fold increase in background signals, but as the variation kept quite constant, the detection limits were only slightly affected. A higher degree of nonspecific binding is usually obvious when there are larger amounts of coated proteins or immobilized antibodies present. It must be anyhow considered that the background values and the effects of the solid phase may vary between the assays of different analytes, and even within one analyte if different batches of antibodies are used.

Va¨limaa et al.

In principle, the assay equilibrium should be reached faster (kinetics improved) as more antigen binding sites are introduced and therefore high-capacity plates provide potential for faster kinetics. Our hCG and PSA assays had quite fast kinetics (equilibration at e 30 min) in the optimized assay conditions, but no substantial improvement in kinetics beyond that point was noted when control plates were replaced to GA-SAv plates. The assays utilized well-selected antibody-antigen pairs that allowed fast kinetics as such. The effect of the solid-phase capacity may have become more remarkable in the case of slower-reacting antibodies. CONCLUSIONS

We have developed a modified streptavidin coating method, which leads to surfaces having high binding capacity for immobilization of biotinylated molecules. The method relies on a chemical cross-linking of streptavidin prior to the routine coating procedure. The treatment was also upscaled and tested in pilot production facilities. The maximum binding capacity of the modified plate increased up to 2.5 times compared to the control plate. The high-capacity surface increased the linear ranges of the sandwich-type immunoassays and improved the high-dose hook performance. Especially biotinylated Fab fragments immobilized in GA-SAv plate resulted in considerable increase in antigen binding capacities. Some of the potential of the high-capacity plates was masked by steric factors caused by the large size of the immobilized antibody. Furthermore, the coated area of a microtitration well is quite large, and the total binding capacity of low capacity plates is considered adequate for many purposes. We anticipate that the benefits of the GA-SAv coatings will be more evident when reduced surface areas are combined with small biotinylated antibody fragments. ACKNOWLEDGMENT

The study was financially supported by the National Technology Agency of Finland (TEKES) and InnoTrac Diagnostics Oy. We thank Mrs Pirjo Laaksonen for technical assistance. LITERATURE CITED (1) Gosling, J. P. (2000) Analysis by specific binding. In Immunoassays. A Practical Approach (J. P. Gosling, Ed.) pp 1-15, Oxford University Press, Oxford. (2) Butler, J. E., Ni, L., Nessler, R., Joshi, K. S., Suter, M., Rosenberg, B., Chang, J., Brown, W. R., and Cantarero, L. A. (1992) The physical and functional behavior of capture antibodies adsorbed on polystyrene. J. Immunol. Methods 150, 77-90. (3) Davies, J., Dawkes, A. C., Haymes, A. G., Roberts, C. J., Sunderland, R. F., Wilkins, M. J., Davies, M. C., Tendler, S. J., Jackson, D. E., and Edwards, J. C. (1994) A scanning tunnelling microscopy comparison of passive antibody adsorption and biotinylated antibody linkage to streptavidin on microtiter wells. J. Immunol. Methods 167, 263-269. (4) Butler, J. E. (2000) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. Methods 22, 4-23. (5) Schetters, H. (1999) Avidin and streptavidin in clinical diagnostics. Biomol. Eng. 16, 73-78. (6) Scorilas, A., Bjartell, A., Lilja, H., Moller, C., and Diamandis, E. P. (2000) Streptavidin-polyvinylamine conjugates labeled with a europium chelate: applications in immunoassay, immunohistochemistry, and microarrays. Clin. Chem. 46, 1450-1455.

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BC020058Y

A High-Capacity Streptavidin-Coated Microtitration Plate

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