Biomacromolecules 2003, 4, 1848-1855
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Poly(styrene/r-tert-butoxy-ω-vinylbenzylpolyglycidol) Microspheres for Immunodiagnostics. Principle of a Novel Latex Test Based on Combined Electrophoretic Mobility and Particle Aggregation Measurements Izabela Radomska-Galant† and Teresa Basinska*,‡ Department of Biotechnology and Food Science, Institute of Technical Biochemistry Technical University of Lodz, ul. Stefanowskiego 4/10, 90-924 Lodz, Poland, and Center of Molecular and Macromolecular Studies, Polish Academy of Sciences ul. Sienkiewicza 112, 90-363 Lodz, Poland Received August 7, 2003
The principle of a novel latex agglutination test based on combined results of electrophoretic mobility and particle aggregation measurements is described. Poly(styrene/R-tert-butoxy-ω-vinylbenzylpolyglycidol) (P(S/ PGL)) microspheres were synthesized by a one step soap-free emulsion copolymerization of styrene and R-tert-butoxy-ω-vinylbenzylpolyglycidol macromonomer with number average molecular weight M hn ) 2700 (polydispersity {M h w}/{M h n} ) 1.10). Particles with monomodal size distribution (number average diameter D h n ) 220 nm) and surface fraction of polyglycidol equal to f ) 0.42 mol % were obtained. Human serum albumin (HSA) was covalently bound onto the surface of P(S/PGL) microspheres activated with 1,3,5-trichlorotriazine. In a model immunodiagnostic assay for anti-HSA, in which P(S/PGL) particles with covalently bound HSA have been used, the electrophoretic mobility and aggregation of microspheres were measured simultaneously. This approach allowed detection of anti-HSA in the serum in the range of antiHSA concentrations from 0.1 to 150 µg/mL. The highest changes in electrophoretic mobility were registered for microspheres with surface concentration of immobilized HSA equal to Γ ) 9.2 × 10-4 g/m2. Introduction Latex particles with bound proteins have been used in many medical diagnostic tests, usually as agents magnifying effects of antigen-antibody interactions. The simplest slide latex test consists of mixing a drop of suspension of latex particles with bound antibodies (or antigens) and a drop of a liquid to be analyzed. The presence of a complementary antigen (or antibody) in an analyte results in aggregation of microspheres (latex particles) because antigens binding antibodies act as cross-linking agents. In the simplest tests, the above-mentioned aggregation, the visualizing effect of the immunoreaction between antigen and antibody, can be detected with the naked eye. However, such tests could indicate only whether the searched component is present in an analyzed liquid (usually in a broad range of concentrations). Quantitative determination of the analyzed compound is possible in tests, in which kinetics of aggregation and/or size of microsphere aggregates is monitored. In past years, many latex diagnostic tests, for detection of viruses, bacteria, parasitic infections, hormones, drugs, and other biologically active agents, were elaborated.1-12 In these tests, a variety of analytical instruments based on light scattering, ultrasound measurements, and measurements of rates of sedimentation were used with purpose to improve the sensitivity or to * To whom correspondence should be addressed. Phone: +48 (42) 6826537. Fax: +48 (42) 6847126. E-mail:
[email protected]. † Department of Biotechnology and Food Science. ‡ Center of Molecular and Macromolecular Studies.
extend concentration range in which a given compound could be detected. For example, the progress of aggregation of latex particles caused by antigen-antibody immunoreaction has been measured by light scattering,13,14 by Coulter Counter,15 nephelometrically,15-19 or turbidimetrically.20 It has to be noted that even the simple turbidimetric test is very sensitive. For example, the turbidimetric test based on microspheres with immobilized anti-plasminogen was suitable for detection of plasminogen down to 250 ng in a 0.2 mL sample of blood plasma.20 The size of aggregates formed during immunoreaction usually depends on the concentration of the analyte in the investigated sample. Thomas et al. used an image analysis technique for monitoring progress of aggregation of microparticles coated with antibodies against C-reactive protein (CRP) induced by CRP.21 It has been found that the size of the aggregates increases with increasing concentration of CRP and that a relationship between the concentration of the analyte and the size of aggregates (over a 100-fold dilution range of CRP) was almost linear. Recently, a variety of new latexes potentially suitable for latex diagnostic tests were synthesized. It is well-known that for diagnostic tests there are needed microspheres with special properties. Desirably, the microspheres should be monodisperse and their diameters should be in the range appropriate for the selected detection system, typically in the range from 100 nm to 50 µm.22,23 Chemical and colloidal stability of microspheres within required ranges of pH, ionic strength, and temperature is demanded. The surface of microspheres should be equipped with chemical groups suitable for
10.1021/bm0342887 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/10/2003
P(S/PGL) Microspheres for Immunodiagnostics
covalent binding of proteins (antibodies or antigens) in amounts allowing detection of chosen complementary analytes in the required range of concentration.24 An adventitious adsorption of proteins onto microspheres should be significantly reduced or eliminated. There are many ways to prepare latex particles with covalently bound proteins, but in many systems, not all proteins are bound covalently. Some are attached only by a simple reversible adsorption. For example, for the poly(styrene/acrolein) latex particles (with the surface concentration of polyacrolein in the range 0-80 mol %), the covalent immobilization of proteins (via Schiff-base formation) was always accompanied with protein adsorption.25 Recently, we found a method allowing for syntheses of latex particles with polystyrene cores and surface layers enriched in polyglycidol in a controlled manner.26 The main chain of polyglycidol is similar to the chain of poly(ethylene oxide), a compound known as a very efficient protein adsorption reducer.27,28 Indeed, it was found that adsorption of proteins onto microspheres with a molar surface fraction of polyglycidol exceeding 0.3 was almost completely eliminated.29 It is worth noting that polyglycidol comprises -CH2OH in each repeating unit. These groups, when activated with 1,3,5-trichlorotriazine (TCT), allow for efficient covalent immobilization of biomolecules with aminogroups.30,31 In this paper, we describe a new type of diagnostic model test based on changes of the ζ-potential and in effect on changes of electrophoretic mobility of the microspheres. In this model test, we used microspheres with immobilized human serum albumin (an antigen) for detection of anti-HSA (antibody against human albumin). It is well-known that antibodies and many antigens are polyelectrolytes. Therefore, their attachment onto microspheres with complementary antibodies (or antigens) leads to changes of ζ-potential of those particles. Such changes resemble changes of electrokinetic mobility of polystyrene microspheres after adsorption of lipopolysaccharides (LPS).2 Experimental Section (P(S/PGL) Microspheres. Microspheres used in our studies were synthesized in the following way. Styrene (Aldrich) was purified from a stabilizer by distillation under reduced pressure just before use. R-tert-Butoxy-ω-vinylbenzylpolyglycidol (PGL) macromonomer with M h n ) 2700 and M h w/M h n ) 1.10 (determined by MALDI-TOF) was synthesized and characterized as it was described earlier.32 Preparation of P(S/PGL) microspheres, as well as polystyrene latex particles (free from PGL), was described in details in our previous paper.26 Here is given only a short recipe. A soap-free emulsion copolymerization of styrene (10 g) and PGL macromonomer (1.0 g) initiated with K2S2O8 (0.2 g) was carried out in water (125 mL, three times distilled with pH adjusted to 6.5 by addition of K2CO3) under nitrogen, with stirring at 60 rpm, at 65 °C, for 24 h. Traces of unreacted styrene were removed by steam stripping. Then, the suspension of microspheres was purified by 4 times repeated isolation by centrifugation and redispersion in new portions of distilled water.
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The number average diameter (D h n) and polydispersity factor ({D h w}/{D h n}) for synthesized P(S/PGL) and PS microspheres determined from scanning electron microphotographs (registered using a JEOL 35C microscope) were equal 0.22 µm (1.02) and 0.52 µm (1.005), respectively. The concentration of anionic groups on the surface of the microspheres was determined by conductometric titration of particle suspension (4 wt %/v) with 10-2 mol/l KOH. A molar fraction of polyglycidol in the surface layer of the microspheres, equal to 0.426, was determined by XPS, according to the procedure described previously.26 Human Serum Albumin, Anti-HSA and Human AntiAmylase Serum. Human serum albumin (Sigma, Cohn fraction V) was used as received. The stock solution of the goat anti-HSA serum (Sigma, A-1151, cprotein ) 43 mg/mL, titer 1:16) was prepared by adding 2 mL of deionized water to the vial containing the lyophilized and fractionated serum. The anti-human R-amylase fractionated antiserum human serum developed in rabbit was used as received (no. A8273, Sigma). Attachment of HSA onto P(S/PGL) Microspheres. Adsorption of HSA was investigated in experiments consisting of incubation of 3 mL of HSA dissolved in the phosphate buffered saline (PBS, 0.2 M, pH ) 7.4; concentration of HSA from 3 × 10-5 to 4.0 × 10-4 g/mL) with 2 mL of P(S/PGL) microspheres in PBS buffer for 20 h at room temperature (r.t.). The amount of attached HSA was evaluated from the difference of the initial concentration of HSA in solution and concentration of HSA remaining in the supernatant after incubation with microspheres. Covalent immobilization of HSA onto P(S/PGL) microspheres was achieved using microspheres with surface hydroxyl groups (from polyglycidol) activated with 1,3,5trichlorotriazine (TCT) as it was described earlier.30 A typical recipe for immobilization is given below. TCT (0.18 g) was added to 5 mL of aqueous suspension containing 0.4 g of P(S/PGL) particles. The sample was incubated overnight at r.t. Then, microspheres were isolated from the unreacted TCT by centrifugation and resuspended in a new portion of water. Centrifugation and resuspension was repeated three times more. In the next step, HSA was covalently immobilized onto TCT activated microspheres by mixing the appropriate volume of protein solution with the chosen amount of suspension of microspheres in PBS. Scheme 1 illustrates the process of activation and covalent binding of protein. Samples were incubated overnight at r.t. and unbound protein was removed by centrifugation. The nonoccupied binding sites on activated particles were blocked in reaction with 1-aminoethanol (0.25 mL added to the sample) with the purpose of eliminating covalent binding of any serum proteins and increasing the stability of P(S/PGL)-HSA conjugates. After centrifugation, the microspheres with immobilized HSA were resuspended in phosphate buffer (pH ) 7.2, I ) 2 × 10-3 M) and kept for further studies. Determination of HSA Adsorbed and/or Covalently Bound onto P(S/PGL) Microspheres. The amounts of HSA adsorbed and/or covalently immobilized at the surface of the microspheres were determined by measuring the difference of protein concentrations before and after immobilization
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Scheme 1. Covalent Immobilization of HSA with the Surface of P(S/PGL) Latex Particlesa
a The unmodified -Cl groups of cyanuric chloride (TCT) were blocked with 1-aminoethanol.
and/or directly on particles by using the modified Lowry method.33,34 In both cases, proteins (in solution and/or attached to microspheres) reacted with analytical reagents in solution, and soluble products of these reaction were determined by UV spectroscopy. UV spectra were registered using a Hewlett-Packard 4852A diode array spectrophotometer. Determination of Size and Electrophoretic Mobility of P(S/PGL)-HSA Microspheres. Photon correlation spectroscopy (PCS) measurements were performed at 25 °C using a 3000 HSa Zeta-Sizer (Malvern) apparatus. The suspension of the investigated microspheres was illuminated with a laser at 633 nm, and the scattering angle was equal 90°. The diameter of the particles was evaluated by taking the average of 30 subsequent measurements. Calculations of size and size distribution of microspheres and microspheres aggregates for the P(S/PGL)-HSA-antiHSA systems were performed using a cumulants model suitable for suspensions of particles with multimodal size distribution. Electrophoretic mobilities and ζ-potential of P(S/PGL) microspheres and microsphere aggregates formed in the presence of anti-HSA were determined also using the 3000 HSa Zeta-Sizer (Malvern) apparatus. The electrophoretic mobility of particles (u) was measured in a cell to which a potential equal 400 V has been applied. The ζ-potential was calculated from Henry’s equation with the Smoluchowski approximation (f(Ka) ) 1.5), suitable for particles in polar media (1) ζ)
3uη 2f(Ka)
(1)
in which u, , and η denote the electrophoretic mobility of microspheres, dielectric constant, and viscosity of the medium, correspondingly. Results and Discussion 1. Studies of Adsorption and Covalent Immobilization of HSA onto P(S/PGL) Microspheres. The dependence of
Figure 1. a,b. Dependence of surface concentration of attached HSA on P(S/PGL) and polystyrene (PS) latex particles versus concentration of HSA (mg/mL) in solution: (a) HSA adsorbed, (b) HSA covalently immobilized onto P(S/PGL) microspheres activated with cyanuric chloride (TCT) (includes data from refs 29 and 35).
the surface concentration of HSA adsorbed onto P(S/PGL) microspheres and HSA covalently immobilized onto microspheres activated with TCT on the concentration of HSA in solution during incubation is shown in Figure 1a,b. According to Figure 1a,b, the surface concentrations of adsorbed/or covalently immobilized HSA increased with the increasing protein concentration in solution, and then at concentrations higher than ca. 0.2 mg/mL, it reached plateau. It has to be noted, however, that the surface concentration of adsorbed HSA on hydrophilic P(S/PGL) microspheres (at plateau) is much lower than the surface concentration of HSA immobilized onto P(S/PGL) microspheres activated with TCT. Plots in Figure 1a clearly support the hypothesis that adsorption of protein onto surfaces covered with hydrophilic and highly mobile polyglycidol chains is much lower than adsorption onto polystyrene microspheres prepared similarly as P(S/PGL) particles.29,36 On the other hand, interactions of HSA with TCT activated polyglycidol chains in the surface layer of P(S/PGL) results in covalent immobilization of the protein (Figure 1b). We found that surface concentration of HSA attached onto TCT activated P(S/PGL) microspheres did not change after “washing” (incubation) with a 2% solution of SDS what indicated that HSA has been covalently immobilized onto these particles.25
P(S/PGL) Microspheres for Immunodiagnostics
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Scheme 2. Change of the Charge of Latex Particles with Immobilized HSA (as Antigens) in the Presence of Anti-HSA (as Antibodies) in Solution with pH ) 7.2
It is not clear whether adsorption precedes the covalent immobilization or the immobilization occurs during collisions of protein molecules with TCT activated chains in the surface layer of the microspheres. However, it is important that finally all HSA bound on particles is immobilized covalently. Covalent immobilization eliminates detachment of and/or its adventitious exchange with other proteins, for example, proteins present in the analyzed liquid. On the other hand, it has to be stressed that microspheres with -Cl groups blocked with 1-aminoethanol are able only to adsorb HSA. We observed that 2% SDS removed essentially 100% of HSA adsorbed onto P(S/PGL) microspheres activated with TCT and deactivated with 1-aminoethanol. 2. Electrophoretic Mobility of P(S/PGL) Microspheress Effect of Immobilization of HSA and Interactions of P(S/ PGL)-HSA with anti-HSA. The polymer chains in P(S/ PGL) microspheres have negatively charged sulfate end groups (-SO4-) formed during initiation with •SO4 - from the K2S2O8 initiator. The negative charge of the ionic groups in the surface layer of particles (equals 4.1 × 10-7 mol/m2) is only partially compensated with cations of a diffuse cloud loosely bound to microspheres. In effect, the microspheres are electrically charged and, thus, migrate in an electric field. The mobility of microspheres induced by the external field (electrophoretic mobility) depends on the effective charge of particles (sum of charges of ions within a microsphere and charges of the fraction of counterions that are located outside of microsphere but move together with it). Charged particles are characterized also by their ζ-potential, i.e., by a potential at the surface of a sphere comprising a microsphere and bound counterions. Proteins contain ionogenic amino and carboxyl groups and are electrically charged at every pH different from their isoelectric points (pH at which carboxyl anions and ammonium cations in proteins compensate each other). Thus, the attachment of proteins onto particles results in a change of latex particles overall charges and, in effect, in a change of their ζ-potential and electrophoretic mobility.2,37 One could expect that particles with immobilized protein (e.g., HSA) will bind antibodies against this protein. In effect, the
ζ-potential and electrophoretic mobility of microspheres should change too. The above-mentioned effects, superimposed on aggregation of microspheres resulting from interactions of antibodies with immobilized antigens, in principle, could be used for quantitative determination of antibodies. Interactions of anti-HSA antibodies with particles with immobilized HSA are illustrated in Scheme 2. Figure 2 shows the dependence of the electrophoretic mobility of P(S/PGL) particles with covalently immobilized HSA on the concentration of anti-HSA in solution. According to Figure 2, the electrophoretic mobility of P(S/PGL) microspheres with covalently immobilized HSA (P(S/PGL)HSA) is negative. The particles migrate toward the anode. This is due to the negative charge of P(S/PGL) microspheres and the negative charge of HSA (isoelectric point for HSA is equal to 4.8) in phosphate buffer (pH ) 7.2, I ) 2 × 10-3 M). Addition of anti-HSA to a suspension of P(S/PGL)-HSA leads to a significant decrease of the absolute value of the electrophoretic mobility of microspheres (electrophoretic mobility becomes less negative). An extent of this change depends on the concentration of anti-HSA. Electrophoretic mobility of P(S/PGL)-HSA is very sensitive to the presence of anti-HSA up to the serum protein concentration equal to 30 µg/mL. Such dependence indicates a possibility of application of P(S/PGL) microspheres for an immunoassay based on electrokinetic measurements. Above concentrations of anti-HSA equal to 30 µg/mL, the changes of electrophoretic mobility of P(S/PGL)-HSA particles are smaller and eventually, when the concentration of anti-HSA exceeds 60 µg/mL, the electrophoretic mobility of microspheres reaches a plateau. In principle, any ionic species, including polyelectrolytes, could interact with charged particles via electrostatic interactions. It is possible also that some proteins from an analyzed serum may be adsorbed not specifically onto sites not occupied with HSA. Thus, it was important to check whether P(S/PGL)-HSA microspheres interact similarly with all kinds of sera or their interactions with anti-HSA serum are specific. Measurements of the electrophoretic mobility
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Figure 2. Dependence of the electrophoretic mobility of P(S/PGL) particles with covalently immobilized HSA on the concentration of anti-HSA containing serum. Surface concentration of HSA on microspheres Gcov ) 9.22 × 10-4 g/m2, concentration of microspheres equal to 1.83 × 10-4 g/mL, phosphate buffer, PB, pH ) 7.2, I ) 2 × 10-3 M).
of P(S/PGL)-HSA microspheres in the presence of various amounts of sera free from anti-HSA indicated (cf. Figure 2) that these not specific interactions are much weaker. In particular, for serum protein concentrations below 30 µg/ mL, the changes of electrophoretic mobility of P(S/PGL)HSA microspheres caused by not specific interactions did not exceed 21% of the electrophoretic mobility changes observed in the presence of anti-HSA (measurements at similar serum concentrations). Specific interactions between anti-HSA and HSA immobilized covalently on P(S/PGL) microspheres indicated that even after immobilization the particular epitopes of HSA are recognized by the corresponding anti-HSA antibodies. It is obvious that changes of electrophoretic mobility of P(S/PGL)-HSA microspheres should depend not only on the concentration of anti-HSA but also on the surface concentration of HSA immobilized on particles. Thus, we measured maximal changes of electrophoretic mobility (∆u) of P(S/PGL)-HSA microspheres (having various surface concentrations of HSA) induced by the addition of anti-HSA serum. It has been established that the concentration of antiHSA serum equal to 43 µg/mL was sufficient to trigger a maximal change of electrophoretic mobility of microspheres. Obviously, the higher ∆u is, the more sensitive the electrophoretic mobility of microspheres to the presence of antiHSA is. Figure 3 illustrates a growing trend in ∆u for increased ΓHSA. However, after reaching a maximum for ΓHSA)0.92 mg/m2, ∆u begins to decrease. Remembering that a specific surface of P(S/PGL) microspheres with a diameter of 220 nm equals 25.4 m2/g and that the molecular weight of HSA is 69 000 Da we were able to do the following calculations. For P(S/PGL)-HSA
Figure 3. Dependence of maximal changes in the electrophoretic mobility of P(S/PGL)-HSA microspheres (Du) induced by anti-HSA serum versus surface concentration of covalently immobilized HSA (Γcov).
particles with ΓHSA ) 0.92 mg/m2, the average number of HSA molecules immobilized on each particle is ca. 1200, and the surface of the microsphere per one immobilized macromolecule of HSA equals 125 nm2. The macromolecules of HSA in crystal form have a heart-like shape with the sides 8.3 × 7.0 × 8.2 nm and thickness of 3.0 nm.38,39 Thus, in a monolayer of crystalline HSA, each macromolecule occupies ca. 32 nm2. On the basis of the above considerations, we concluded that the optimal sensitivity (for ΓHSA ) 0.92 mg/ m2) was noticed for P(S/PGL)-HSA microspheres with immobilized HSA packed less densely than in the HSA crystal. Apparently, too dense of a packing of HSA on
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P(S/PGL) Microspheres for Immunodiagnostics Table 1. Average Size (Measured by PCS) after Addition of Anti-HSA to P(S/PGL)-HSA, (Γcov ) 0.92 mg/m2, conc. of Microspheres ) 1.83 × 10-4 g/mL) P(S/PGL)-HSA with anti-HSA serum free conc. of serum proteins, µg/mL
average size, nm
0.0
386.1
21.5
378.4
43
407.4
64.5
393.1
537.5
398.7
P(S/PGL)-HSA with anti-HSA serum conc. of serum proteins, µg/mL
average size, nm
fraction of “large” particles,a %
0.0 0.107 1.075 2.15 2.66 4.3 6.45 7.74 10.75
387.8 435.5 428.4 438.7 451.6 438.0 436.7 468.5 449.6
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
32.25 43 53.75 64.5 75.25 107.5 129 172
522.7 774.6 740.5 690.6 683.2 715.4 758.2 878.7
Figure 4. Dependence of the electrostatic charge of particles (Q) on the concentration of anti-HSA serum in solution. Surface concentration of HSA on P(S/PGL) microspheres ΓHSA ) 9.22 × 10-4 g/m2. Concentration of P(S/PGL)-HSA particles c ) 1.83 × 10-4 g/mL, experiments in PB, pH ) 7.2, I ) 2 × 10-3 M.
71 40 46 46 40 44 74 66
a Particles formed by aggregation of the primary P(S/PGL)-HSA microspheres.
microspheres interferes with access of anti-HSA to all immobilized HSA molecules. Changes in electrophoretic mobility of P(S/PGL)-HSA microspheres exposed to anti-HSA serum may be due not only to the attachment of electrically charged anti-HSA globulins (due to antibody-antigen interactions) but also to the aggregation of microspheres. Such aggregation induced by bridging P(S/PGL)-HSA microspheres with anti-HSA antibodies is often observed in systems with an immobilized antigen (or antibody) and antibody (or antigen) present in solution. Data in Table 1 reveal that for suspensions of P(S/ PGL)-HSA the size of the microspheres depends on the concentration of the anti-HSA serum proteins. One has to note also that aggregation of P(S/PGL)-HSA microspheres did not occur when the serum free from antiHSA has been added to the suspension of microspheres. The above observation proves that the aggregation of microspheres is due only to specific HSA-anti-HSA interactions. An electric charge of P(S/PGL)-HSA microspheres and microsphere aggregates could be evaluated from measurements of ζ-potentials and diameters of these objects. The average charge on particles and/or particle aggregates (Q) is related to ζ-potential and the average diameter of these objects (D h n) according to eq 2 Q ) 2πζD hn
(2)
The ζ-potential was determined parallely to measurements of electrophoretic mobility (u) of microspheres (the relation between u and ζ-potential is described by eq 1). The average diameter of microspheres and microsphere aggregates was measured by photon correlation spectroscopy (PCS).
Dependence of electrostatic charge of particles (Q) on concentration of anti-HSA serum proteins is shown in Figure 4. As it is shown in Figure 4, the average charge of microspheres and/or of microsphere aggregates becomes rapidly less negative when the concentration of anti-HSA in solution is increased. Then, when the concentration of antiHSA exceeded 50 µg/mL, Q began to decrease. Apparently, attachment of anti-HSA onto P(S/PGL)-HSA microspheres (through the antigen-antibody interactions) results in a partial charge compensation. However, at higher concentrations of anti-HSA, particle aggregates are efficiently formed. The total charge of an aggregate is higher (more negative) then the charge of a single microsphere. Similar behavior was noticed when interactions of CRP (C-reactive protein) with polystyrene particles bearing IgG or F(ab′)2 against CRP were monitored nephelometrically.14 Photon correlation spectroscopy (PCS) could be used not only for the determination of the average diameter of microspheres and microsphere aggregates but also for the determination of the fraction of not aggregated particles (data treatment assuming multimodal distribution of diameters of particles). Figure 5 shows three-dimensional plots illustrating relations between the concentration of the serum (without and with anti-HSA component), the electrophoretic mobility of P(S/PGL)-HSA microspheres and their aggregates, and the fraction of not aggregated particles. According to this figure, the addition of serum proteins without anti-HSA leads to some changes of electrophoretic mobility of P(S/PGL)HSA microspheres, but not to particle aggregation (primary P(S/PGL)-HSA microspheres constitute ca. 100% of all particles in suspension). On the other hand, the addition of anti-HSA serum results in aggregation and in the changes of electrophoretic mobility of P(S/PGL)-HSA microspheres. Both of these effects are combined in a product (denoted as Π) of the electrophoretic mobility of P(S/PGL)-HSA microspheres and/or their aggregates (u) and the fraction of aggregated P(S/PGL)-HSA particles in suspension (1-f). Figure 6 shows the dependence of Π on the concentration
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spheres for detection of antigens and/or antibodies other than anti-HSA. Typical latex tests are based exclusively on aggregation phenomena. By electrophoretic mobility measurements, we include an additional factor increasing sensitivity of determinations. In the case of the discussed microspheres-HSA/ anti-HSA system, the maximal changes of the electrophoretic mobility of microspheres (∆u) were ca. 1.5 µm cm/Vs (cf Figure 2), and the changes that still could be reliably detected (∆minu) were ca. 0.2 µm cm/Vs (standard error in determination is ca. 0.1 µm cm/Vs). Thus, the measurements of electrophoretic mobility of microspheres could increase the sensitivity of the test by a factor close to ∆u/∆minu ≈ 7. It is obvious that this effect may vary for detection of various proteins and should be determined for each particular test separately. Conclusions Figure 5. Relation between the concentration of serum proteins (with or without anti-HSA), average size of aggregates and electrophoretic mobility of P(S/PGL)-HSA. Surface concentration of HSA Γcov ) 3.69 × 10-4 g/m2, concentrated of particles, c ) 1.5 × 10-4 g/mL, experiment performed in phosphate buffer, PB, pH ) 7.2, I ) 2 × 10-3 M.
It has been shown that for a model system [P(S/PGL) microspheres with immobilized antigen (HSA) and antibodies against HSA] the combination of electrophoretic mobility measurements and measurements of the fraction of not aggregated particles allows determination of antibodies in concentration range from 0.1 to 150 µg/mL. Despite the fact that the principle of this test has been verified only in the case of the HSA-anti-HSA system, one could expect that it could be useful also for design of other tests based on antigen-antibody interactions. References and Notes
Figure 6. Dependence of the product of electrophoretic mobility of P(S/PGL)-HSA microspheres and their aggregates (Π) and the fraction of aggregated P(S/PGL)-HSA particles in suspension (Π ) u(1 - f)) as a function of serum concentration.
of anti-HSA serum. For comparison there is shown also a similar plot obtained by using serum without anti-HSA. It is evident that Π is very sensitive to variations in concentration of anti-HSA and could be used for the quantitative determination of anti-HSA. In the case of the analyzed model system, the proposed test based on the combined electrophoretic mobility and particle aggregation measurements could be used for determination of anti-HSA in the range from 0.1 to 150 µg/mL. Remembering that blood serum contains ca. 6-8 wt % of proteins40 and that for aggregation and ζ-potential measurements there is needed ca. 0.5 mL of serum, the abovementioned range means that the lowest amount of anti-HSA serum needed for analysis would be ca. 1 µL. Obviously, further studies would be needed to demonstrate applicability of the tests based on the electrophoretic mobility of micro-
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