Anal. Chem. 2001, 73, 3426-3431
Effect of Poly(ethylene glycol), Tetramethylammonium Hydroxide, and Other Surfactants on Enhancing Performance in a Latex Particle Immunoassay of C-Reactive Protein Peter Holownia,* Soledad Perez-Amodio,† and Christopher P. Price
Department of Clinical Biochemistry, St. Bartholomews and Royal London Hospital’s School of Medicine and Dentistry, Turner Street, London E1 2AD, U.K.
The influence of a variety and combination of both ionic surfactants and different chain lengths of the polyelectrolyte poly(ethylene glycol) (PEG) on the performance characteristics (with particular reference to signal response) of a homogeneous, latex agglutination immunoassay was investigated. The test analyte was human serum C-reactive protein (CRP), and the antibody reagent consisted of a sheep polyclonal anti-CRP IgG fraction covalently coupled to 50-nm-sized latex including a glycinecapped chloromethylstyrene shell. The amount and rate of immunoagglutination was monitored turbidimetrically after sample addition. It was found that 2.5 mmol/L concentrations of the small cationic surfactant tetramethylammonium hydroxide (TMH), when present alone, substantially increased both reaction rates and sensitivity in the lower clinical ranges of CRP concentration when compared to normally used assay conditions containing PEG and the anionic detergent Gafac. The nonspecific binding (NSB) was also found to be unchanged. Evidence is presented that the TMH enhances the actual antibodyantigen interaction as opposed to the known effects of other surfactants in immunocomplex dissociation or in maintenance of colloidal stability. We suggest that the enhancement seen with TMH could be an alternative to PEG and may provide a new means of further extending detection limits. The utility of this type of immunoassay technology could therefore be increased whenever clinically required. In previous work,1 methods of jointly controlling the physicochemical processes of immunological recognition, aggregation, and colloidal stability were examined by optimization of the reaction medium in a particle-based immunoassay of C-reactive protein (CRP). The balance in maintaining the stability of aggregates against the opposing immunospecific forces of attraction * Corresponding author: (e-mail)
[email protected]; (fax) 00(44)20-7377-1544. † Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA) and Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, P. O. Box 22700, 1100 DE Amsterdam, The Netherlands. (1) Perez-Amodio, S., Holownia, P., Price, C. P. 2001, 73, 3417-3425.
3426 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
responsible for antigen-antibody binding were shown to define the conditions for minimal nonspecific binding but maximum rate and sensitivity for this particular type of immunoassay system. The aim of the present study was to continue this optimization in order to show whether these characteristics can be further improved to extend assay ranges and to examine the possibility of applying this technology to the measurement of other analytes, which are not normally detectable by these means. The effects of surfactants (detergents) and polyelectrolytes (nonionic polymers) are known to play important roles in control and enhancement of immunoassay performance2,3 and form the main subject of this study. Surfactants have been shown4 to remove both nonspecific and specific interactions as well as to promote antibody particle reagent stability. In optimized concentrations, a bilayer is coated on to the particle surface which results in steric repulsion5 as well as the formation of a charged layer surrounding both the particles and the epitope/paratope regions, thus enhancing the electrostatic repulsion6,7 under appropriate conditions of pH. In contrast, polyelectrolytes, such as poly(ethylene glycol) (PEG), are commonly used in agglutination immunoassays as rate accelerators or as precipitants for separation in heterogeneous types. The mechanism is considered to be one of steric exclusion where large macromolecules such as the antibody and the immune complex are excluded from the volume occupied by PEG and are thereby present in a greater concentration in the bulk phase compared to smaller ones such as solvent, hapten, and to a lesser extent free antigen.3,8 Equivalence points, signal, and rate of precipitation are thereby all increased as PEG counteracts the solubilization of the immune complex. In the present work, both cationic and anionic surfactants of varying chain length, i.e., nonylphenoxypolyethoxylated phosphate ester (Gafac), caprylic acid, tetramethylammonium hydroxide (TMH), and dodecyltrimethylammonium bromide (DTB) as well (2) Price, C. P., Newman, D. J., Eds. Principles and Practice of Immunoassay, 2nd ed.; Macmillan Reference Ltd.: London, 1997; Chapter 18. (3) Hellsing, K. In Automated Immunoanalysis, 1st ed.; Ritchie, R. F., Ed.; Marcel Dekker Inc.: New York, 1978; Part 1, Chapter 3. (4) Absolom, D. R.; Van-Oss, C. J. CRC Crit. Rev. Immunol. 1986, 6, 1-46. (5) Peula, J. M.; Hidalgo-Alvarez, R.; Nieves, F. J. J. Colloid Interface Sci. 1998, 201, 139-145. (6) Brown, W.; Zhao, J. Macromolecules 1993, 26, 2711-2715. (7) Stutzenberger, F. J. Anal. Biochem. 1992, 207, 249-254 (8) Lizana, J.; Hellsing, K. Clin. Chem. 1974, 20, 415-420. 10.1021/ac001530g CCC: $20.00
© 2001 American Chemical Society Published on Web 06/15/2001
as the polyelectrolyte PEG were investigated as potential controls of immunoassay performance. This was performed by automated turbidimetric monitoring of immunoagglutination and colloidal stability, size and charge measurement by photon correlation spectroscopy,9 and determination of the total intermolecular interaction (Haamaker constant) from stability ratios and the critical coagulation concentration10 (CCC) in the presence and absence of antigen. It was found that low concentrations (0.52.5 mmol/L) of TMH significantly improved performance in the low range of CRP concentration, (1 and 10 mg/L in others, e.g., infection.12 Once a rigorous clinical validation is performed,11 with particular reference to rheumatoid factor, paraprotein, and other more common and sample specific interferences,13 it is suggested that this strategy could be applied to other clinical analytes present at lower serum concentrations: for example, R-fetal protein (0.02-0.1 mg/L), ferritin (0.007-0.6 mg/L), and placental lactogen (0.3-11 mg/ L).14 The practical range of latex particle technology could thus be extended as well as increasing the speed of the actual test. EXPERIMENTAL SECTION Instrumentation. A Cobas Bio centrifugal analyzer (Roche Diagnostic Systems) was used to perform automated turbidimetric measurement of all aggregation phenomena as described previously.1 Photon correlation spectroscopy9 was used to determine ζ potential (being the effective potential difference at the shear plane between static layers on a molecule/particle and the mobile bulk phase) and hydrodynamic diameter. This was done by the introduction of a 2-mL aliquot of test suspension into the capillary cell of a Malvern Zetasizer 3 instrument (Malvern Instruments). All results presented were the mean of five replicate measurements, with CVs of 4-8% for both types of measurement. Materials. Anionic detergents with different chain lengths, Gafac (C33) and caprylic acid (C8), were used in the synthesis of particle reagent in order to obtain a final suspension containing 0.001, 0.005, and 0.01% (v/v) of each. Cationic surfactants TMH (C4) and DMB (C15) were used to obtain a final reagent suspension of 0.5, 1, 2.5, 5, 10, and 20 mmol/L and 0.5, 1, and 2.5 mmol/L, respectively. Gafac was obtained from the Gafac Corp. (Manchester U.K.), while the others were from Sigma-Aldrich Co. Ltd. (Poole, U.K.). Different sized polymers of PEG, likewise purchased (9) Bloomfield, V. A. Dynamic Light Scattering, 1st ed.; Plenum Press: New York, 1985; Chapter 12. (10) Ortega-Vinuesa, J. L.; Martin-Rodriguez, A.; Hidalgo-Alvarez, R. J. Colloid Interface Sci. 1996, 184, 259-267. (11) 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. (12) Fleck, A. Proc. Nutr. Soc. 1989, 48, 347-354. (13) Selby, C. Ann. Clin. Biochem. 1999, 36, 704-721. (14) Burtis, C. A., Ashwood, E. R., Eds. Tietz Textbook of Clinical Chemistry, 3rd ed.; W. B. Saunders Co.: Philadelphia, PA, 1999; Chapter 50.
from Sigma-Aldrich, i.e., 4000, 6000, 8000, and 20000 were used, each at concentrations of 0.1, 1.4, and 4% (w/v), in addition to the standard conditions defined in the next section. Procedure for Immunoassay. The synthesis of antibody/ particle reagent, incubation procedures, medium conditions, sample preparation, and measurement of response have been previously described.1 Serum samples were representative of the
