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Effects of the Ionic Environment, Charge, and Particle Surface Chemistry for Enhancing a Latex Homogeneous Immunoassay of C-Reactive Protein Soledad Perez-Amodio,‡ Peter Holownia,*,† Carol L. Davey,§ and Christopher P. Price†
St. Bartholomew’s and Royal London Hospital’s School of Medicine and Dentistry, Department of Clinical Biochemistry, Turner Street, London E1 2AD, U.K.
The role of the solution environment for a light-scattering, latex-particle-enhanced, homogeneous immunoassay of C-reactive protein (CRP) has been investigated in order to assess and optimize the immunoagglutination response. Latex particles of 50-170-nm sizes were covalently coupled with an IgG polyclonal antibody and subjected to an extensive optimization regime. This consisted of conditions responsible, in different degrees, for the principal attractive/repulsive forces affecting both colloidal stability and the antibody/antigen interaction: particle size, antibody concentration, ionic strength and species, pH, and amino acid chemistry of the particle surface. Careful control of these parameters was found to be necessary to achieve the desired effects of balancing high colloidal stability in the absence of antigen but promoting a rapid, sensitive, and dose-dependent agglutination with pathological serum samples. In addition, the estimation of fundamental properties governing intermolecular interaction (i.e. the “Hamaker” constant and critical coagulation concentration) was attempted to order to investigate a simple, practical means of defining a colloidal/immunoassay system under “real conditions” as well as “real time”. It is concluded that because each antibody system is unique, a similar optimization should be performed in diagnostic immunoassays of this type to maximize their clinical utility. There is a growing need to enhance the performance of immunoassay in order to achieve greater sensitivity and more rapid reaction rates for both automated and miniaturized clinical analysis.1 A key format is the homogeneous immunoassay, which provides a simple, single step and precise procedure by virtue of a signal from a labeled species being directly modified, in quantitative fashion, by the event of an antibody/antigen interaction.2 It is found that the latex-enhanced assays of this type are * Corresponding author. Fax: 00(44)-20-7377-1544. E-mail: p.g.holownia@ mds.qmw.ac.uk. † St. Bartholomew’s and Royal London Hospital’s School of Medicine and Dentistry. ‡ Present address: Department of Periodontology, Academic Centre for Dentistry, Amsterdam (ACTA), and Department of Cell Biology and Histology, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, Netherlands. § Present address: Department of Biochemistry, Oxford University, South Parks Road, Oxon, OX1 3QU, U.K. (1) Price, C. P. Clin. Chem. Lab. Med. 1998, 36, 341-347. 10.1021/ac001529h CCC: $20.00 Published on Web 06/15/2001
© 2001 American Chemical Society
the most simple and versatile, that is, particle-enhanced turbidimetric immunoassay (PETIA) and its immunoinhibitory counterpart, particle-enhanced turbidimetric inhibition immunoassay (PETINIA)3 as opposed to, for example, the cloned enzyme donor immunoassay (CEDIA), which although an elegant procedure, is a complex, multistep system and is more vulnerable to sample specific variation in background. In addition, latex assays, unlike the other main homogeneous assays of fluorescence polarization (FPIA) or enzyme-multiplied assays (EMIT), have no restriction on antigen size and can also be readily adapted onto automated instruments that use spectrophotometric detection,4 this being the case for the majority of clinical analyzers and near-patient testing devices, thus avoiding the need for dedicated detection systems. The aim of this study was to optimize the environment in a typical PETIA so that control of (a) the specific antibody/antigen interaction5,6 and (b) macromolecular and particle colloidal stability6,7 is achieved to extend working ranges to lower limits. The clinical utility of assays may thus be enhanced as well making possible the measurements of those other analytes present in the circulation below the lower limits of detection using existing latex particle-enhanced technologies3. This procedure is complementary to the antibody engineering techniques of generating high specificity/affinity antibodies.8 The immunoagglutination response was measured by an automated analyzer for the analyte CRP, an important clinical marker of the human acute phase response to infection and disease.9 In latex particle assays, the interactions can be between particle and particle, particle and protein, or protein and protein, which will show varying sensitivities to the environment.10 The surface chemistry of the latex particle reagent should be considered in (2) Bangs, L. B. Pure Appl. Chem. 1996, 68, 1873-1879. (3) Newman, D. J.; Thakker, H.; Price, C. P. Immunodiagnostics, 1st ed.; Oxford University Press: Oxford; 1999, Chapter. (4) Gorman, E. G.; Arentzen, R.; Bedzyk, W.; Cassidy, L. A. Principles and Practice of Immunoassay, 2nd ed.; Macmillan Reference Ltd.: U.K.; 1997; Chapter 13. (5) Absolom, D. R.; VanOss, C. J.; CRC Crit. Rev. Immunol. 1986, 6, 1-46. (6) Ortega-Vinuesa, J. L.; Hidalgo-Alvarez, R.; de-las-Nieves, F. J.; Davey, C. L.; Newman, D. J.; Price, C. P. J. Colloid Interface Sci. 1998, 204, 300-311. (7) Molina-Bolivar, J. A.; Galisto-Gonzalez, F.; Ortega-Vinuesa, J. L.; Schmitt, A.; Hidalgo-Alvarez, R. J. Biomater. 1999, 10, 1093-1105. (8) George, A. J. T. Principles and Practice of Immunoassay, 2nd ed.; Macmillan Reference Ltd.: U.K.; 1997; Chapter 4. (9) Fleck, A. Proc. Nutr. Soc. 1989, 48, 347-354. (10) Price, C. P.; Newman, D. J. Principles and Practice of Immunoassay, 2nd ed.; Macmillan Reference Ltd.: U.K.; 1997; Chapter 18.
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its entirety, of which the antibody in a PETIA format typically occupies a “footprint” area of around 10-20% in many developed assays. The residual area will consist of suitably capped reactive groups of the shell polymer, for example, chloromethyl groups blocked with glycine. According to the Deryagin-LandauVerwey-Overbeek (DLVO) theory,11 the stability of colloidal dispersions, including both particles and macromolecules, is maintained by the sum of the electrostatic force (long-range and usually repulsive) and the attractive London-van der Waals force (short range). In addition, structural forces consisting of the steric force, arising from entropic repulsion and the osmotic effect, are also present. The antibody/antigen interaction12 relies on overcoming the long-range electrostatic repulsion between species by a complementary array of charged and hydrophobic groups together with the nonspecific hydrophobic effect due to the cohesive properties of water. At closer ranges, the attractive London-van der Waals forces, salt bridging, and secondary attraction cement the binding, and the process is also facilitated by conformational changes. The unique nature of any particular interaction also creates a varied sensitivity to the presence or absence of solvent water molecules both within the paratope/ epitope region and the overall molecular surfaces, where enhancement or a decrease can occur by the various mechanisms described in the discussion. Thus, the nature of the reaction medium will balance the repulsive forces maintaining colloidal stability, together with the opposing attractive forces governing the specific antigen/antibody binding event. For a given antibody (monoclonal or a polyclonal population), the interaction will be dependent on the relative magnitudes of these forces and the types of mechanism by which an immunoreaction occurs. The principal variables10 in this study consisted of the ionic concentration and species type, buffer pH, and latex surface blocking agents (e.g., amino acids such as glycine), with appropriate reference to the lyotrophic series and stability ratios (W).13,14 Other factors, such particle sizes, monitoring wavelengths, and antibody loading, were also investigated. By careful optimization it should be possible to (a) minimize nonspecific binding (NSB), (b) increase the reaction rate, and (c) enhance the signal (i.e., sensitivity). The aforementioned stability ratio is a frequently used kinetic criterion of particle suspensions or macromolecular solutions. It is defined as the ratio of the maximum agglutination rate (rapid agglutination regime where every collision between particles is effective) to the observed agglutination rate (slow agglutination regime where not every collision results in agglutination). From this and the critical coagulation concentration, CCC, an approximation for the Hamaker constant can also be derived to characterize the strength of the total intermolecular interaction.14,15 We suggest that this could be used to provide an alternative means, with supporting evidence of charge measurement, of accurately classifying antibody performance in a particle immu(11) Everett, D. H. Basic Principles of Colloid Science, 1st ed.; Royal Society of Chemistry: U.K., 1988; Chapter 3. (12) VanOss, C. J. Mol. Immunol. 1995, 32, 199-211. (13) Everett, D. H. Basic Principles of Colloid Science, 1st ed.; Royal Society of Chemistry: U.K., 1988; Chapter 9. (14) Hunter, R. J. Foundations of Colloid Science, 1st ed.; Oxford University Press: Oxford; 1987; Vol. 1, Chapter 7. (15) Ortega-Vinuesa, J. L.; Martin-Rodriguez, A.; Hidalgo-Alvarez, R. J. Colloid Interface Sci. 1996, 184, 259-267.
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noassay under real conditions as well as real time in any given affinity of antibody, which other techniques such as surface plasmon resonance (SPR) can only approximate as a result of, for example, exclusive focus on just the antibody/antigen interaction16 or the inevitable restrictions imposed by immobilization to solid surfaces, as opposed to a colloidal dispersion. Other known factors of control, such as polyelectrolytes and detergents, are the subject of further work which continues on the aims of this paper for defining and improving the performance of this type of immunoassay. EXPERIMENTAL SECTION Synthesis of Antibody/Particle Reagent and Sample Preparation. Latex particles were obtained in three sizes from Bangs Laboratories, Inc. (9025 Technology Drive, Fishers, IN 460382886), as 10% (w/v) suspensions of 50-, 100-, and 170-nm diameters consisting of a polyvinylnaphthalene core and a chemically reactive shell of chloromethylstyrene (CMST). The antibody was an IgG fraction, sheep antihuman C-reactive protein polyclonal bought from The Binding Site Ltd. (PO Box 4073, Birmingham, B29 6AT, U.K.) cat. no. PNO 44.X, lot 162308). Throughout the development work, a 1% w/v concentration of these particles was used for the preparation of the antibody/particle reagent. Prior to use, the antibody was dialyzed in 15 mmol/L sodium phosphate buffer, pH 7.4, over 24 h at 4 °C. The synthesis and characteristics are detailed elsewhere,17 but in essence consist of a gentle, overnight mixing of antibody with latex, where coupling of the amino groups to the CMST shell of the latex at 37 °C occurs. This is followed by four wash steps of ultracentrifugation/resuspension of the latex pellet at room temperature. An immunologically active antibody particle reagent is produced that has a proven stability18 at 4 °C in excess of a year and calibration stability of several months. Indeed, this type of coupling chemistry is standard in many turbidimetric tests developed commercially by some of the major multinational diagnostic companies, for example, Dade Behring.17 The loadings of antibody onto each particle size were 1.5 g/L, 2.0 g/L, and 2.5 g/L with coupling efficiencies at ∼90% ((5%). These were determined from the absorbance at 280 nm of the free uncoupled protein present in the primary supernatant after coupling, assuming a 1.43 extinction coefficient at 1% w/v concentration normally used for protein at this wavelength. The amount of protein coupled onto the particle is expressed as a percentage of the initial antibody concentration. The percentage of particle surface area occupied by the antibody was estimated at 10% in the case of the 2.5 mg/L loading and 50-nm-diameter latex, assuming an IgG antibody footprint area of 15 nm2. The ζ-potential of the latex particles and particle reagent was measured on a Malvern Zetasizer 3 instrument (Malvern Instruments, U.K.), only in conditions without Gafac surfactant because of the unavoidable interference from microbubbles, were of sufficient magnitude to be considered colloidally stable (-35mV and -25mV, respectively), and consisted of a mean derived from five replicate readings with CVs ranging from 4 to 8%. The pI (isoelectric point) of the polyclonal antibody ranged between pH (16) Karlsson, R.; Roos, H. Principles and Practice of Immunoassay, 2nd ed.; Macmillan Reference Ltd.: U.K.; 1997; Chapter 5. (17) Holownia, P.; Newman, D. J.; Thakker, H.; Bedzyk, W. D.; Crane, H.; Olabiran, Y.; Davey, C. L.; Price, C. P. Clin. Chem. 1998, 44, 1316-1324. (18) Thakkar, H.; Davey, C. L.; Medcalf, E. A.; Skingle, L.; Craig, A. R.; Newman, D. J.; Price, C. P. Clin. Chem. 1991, 37, 1248-1251.
4.5 and 7.5 and was obtained from isoelectric focusing using the Pharmacia IPGphor strip system (Amersham Pharmacia Biotech U.K., Ltd). The final working dilution particle reagent for immunoassay was adjusted with diluent buffer consisting of 5 mmol/L glycine pH 7.4, 0.005% Gafac (nonylphenoxypolyethyleneoxy phosphate ester, RE16) obtained from Gafac Inc, Manchester, U.K., and 0.001% sodium azide such that the absorbance at the active measuring wavelength of 340 nm was 1000 ( 25 milliabsorbance units (mAU) before the addition of the sample. Samples containing CRP consisted of five patient serum pools with values previously assigned by the Dade Behring Nephelometer Array analyzer (BNA, supplied by Dade Behring Ltd; Walton Manor, Walton, Milton Keynes, MK7 7AJ, U.K.) using the standard CRP kit (Behring OSCD 15) of
