monoclonal CRP immunoassay, opening the door to a less expensive automated method with similar sensitivity and a wide measuring range. For practical use, it is essential to obtain microparticle reagents colloidally stable under reaction and storage conditions, with the stability often depending on protein coverage. Colloidal particles coated with polyclonal IgG usually are not very stable, mainly because of the high antibody coverage needed to obtain good sensitivities (3.6 mg/m 2 in our case). Posttreatment with additives (BSA, surfactants) has been proposed (13, 14), although it re- duces IgG charge and reactivity. We suggest an alterna- tive approach that uses a small amount of immunopuri- fied antibody and stabilizes the reagent by saturation of free surface with BSA in a two-step sequential covalent procedure. In this way, the inactive IgG molecules are replaced with molecules of BSA, remarkably increasing the latex stability by electrostatic repulsion. Moreover, both IgG and BSA could be added in only one step if the right conditions are found (14 ). In conclusion, a low coverage (0.3 mg/m 2 ) of immuno- purified IgG provides a reagent with immunoreactivity similar to or better than that of microparticles totally covered (at saturation) by a nonimmunopurified IgG antibody (3.6 mg/m 2 ), but with higher colloidal stability (the suspension remained stable for more than 3 months when it was stored at 4 °C). This approach to antibody immunopurification could be extended to obtain reagents useful for the measurement of several other proteins at low concentrations (15 ). This research was supported in part by the Comision Interministerial de Ciencia y Tecnologı ´a (CICYT), Projects MAT99-0662-C03-02 and -03. References 1. Thompson D, Milford-Ward A, Whicher JT. The value of acute phase protein measurements in clinical practice. Ann Clin Biochem 1992;29:123–31. 2. Haverkate F, Thompson SG, Pyke SDM, Gallimore JR, Pepys MB. 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A sensitive, particle-enhanced assay for ferritin on IMMAGE TM immunochemistry system [Abstract]. Clin Chem 1999;45(Suppl 6):A45. 13. Peula JM, Hidalgo-Alvarez R, de las Nieves FJ. Coadsorption of IgG and BSA onto sulfonated polystyrene latex. II. Colloidal stability and immunoreactiv- ity. J Biomater Sci Polym Ed 1995;3:241–51. 14. Peula JM, Puig J, Serra J, de las Nieves FJ, Hidalgo-Alvarez R. Electrokinetic characterization and colloidal stability of polystyrene latex particles partially covered by IgG/a-CRP and m-BSA proteins. Colloids Surf A Physicochem Eng Aspects 1994;92:127–36. 15. Borque L, Rus A, Bellod L, Seco ML. Development of an automated immunoturbidimetric ferritin assay. Clin Chem Lab Med 1999;37:899 –905. Flexibility of Melting Temperature Assay for Rapid Detection of Insertions, Deletions, and Single-Point Mutations of the AGXT Gene Responsible for Type 1 Primary Hyperoxaluria, Doroti Pirulli, 1,2 Michele Boniotto, 1 Daniela Puzzer, 1 Andrea Spano `, 1 Antonio Amoroso, 1,2 and Sergio Crovella 1,2* ( 1 Servizio di Genetica Medica, IRCCS Burlo Garofolo, 34137 Trieste, Italy; 2 Sezione di Genetica Medica, Dipartimento Scienze della Riproduzione e dello Sviluppo, Universita ` di Trieste, 34137 Trieste, Italy; * ad- dress correspondence to this author at: Servizio di Ge- netica, IRCCS Burlo-Garofolo, Via dell’Istria 65/1, 34137 Trieste, Italy; fax 39-040-3785210, e-mail crovella@burlo. trieste.it) Primary hyperoxaluria type 1 (PH1; OMIM 259900) is a rare autosomal recessive disorder characterized by im- paired hepatic detoxification of glyoxylate. PH1 is caused by a deficiency of alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which catalyzes the transamination of glyoxylate to glycine. This defect leads to endogenous overproduction of oxalate and glycolate, producing oxalic and glycolic hyperacidurias, which are the hallmarks of the disease (1). The AGT enzyme is encoded by a single-copy gene (AGXT), which consists of 11 exons ranging from 65 to 407 bp and spanning a 10-kb DNA segment in the 2q37.3 human region. AGT is a 392-amino acid protein with a molecular mass of 43 kDa (2). Several technical approaches have been used to identify 7 polymorphisms and 26 mutations in the AGXT gene (3–6). Here we describe a rapid, flexible, and inexpensive method for detection of the different types of mutations (insertions, deletions, point mutations) of the AGXT gene. Our method is based on the ability to distinguish between PCR amplification products by their melting tempera- tures (T m ) (7–9 ). Nine PH1 patients, whose mutations had first been analyzed by the single-strand conformation polymor- phism (SSCP) technique and then by sequencing of ab- normal mobility bands of four AGXT exons (5), were studied comparatively by the melting temperature assay (MTA). Heterozygous relatives of three patients were also included in this study. Five healthy Italian subjects served 1842 Technical Briefs