Such assays are suitable for the measurement of proteins at concentrations >5– 10 mg/L. .. Development of an automated immunoturbidimetric ferritin assay. May 3, Different laboratory methods are used to quantify ferritin . assays and automated equipment developments, and in routine laboratory assays and Seco M. Development of an automated immunoturbidimetric ferritin assay. J. Colloid. Interface Sci. (2): – 3. Borque, L., Rus, A., Bellod, L., and Seco, M.L. Development of an automated immunoturbidimetric ferritin assay.
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C-Reactive protein CRP is a marker of acute phase response to inflammation that increases rapidly within hours of disease onset. CRP measurements have been used for many years in the management of a variety immunoturbidiemtric clinical situations, such as bacterial infections, ischemic necrosis of tissue, and active inflammatory conditions 1.
Recent studies of CRP concentrations within the conventional reference interval have suggested new clinical applications. CRP is a prognostic risk factor for coronary heart disease in both patients with angina stable or not and in apparently healthy subjects 2and distinguishes rapidly destructive vs slowly progressive osteoarthritis 3.
Recent reports show median values in healthy controls of 0. Nevertheless, these results can be influenced by the sensitivity of the methodology used for CRP quantification. Among the many commercially available assays for CRP, the most common use fluid-phase or particle-enhanced turbidimetric or nephelometric procedures 7.
New methods of measuring CRP have lower limits of detection one-tenth those of earlier methods 8 9. Some use expensive monoclonal antibodies, and others use higher sample volumes and antibody concentrations in the reaction mixtures, which can increase nonspecific reactions We describe a microparticle reagent that we use in an optimized turbidimetric procedure with immunopurified polyclonal antibodies against CRP.
It allows lower antibody coverage on the microparticle reagent and provides highly sensitive and robust particle-enhanced turbidimetric immunoassay for serum CRP. In addition, we present a new procedure of sequential covalent coupling of IgG and bovine serum albumin BSA that improves the reagent colloidal stability and could eliminate most of the drawbacks of the light-scattering immunoassays, such as nonspecific agglutination.
The microparticle reagents were prepared according to a previously published method by the carbodiimide [1-ethyl 3-dimethylamino-propyl carbodiimide chloride] procedure For the immunopurified anti-CRP antibodies, we used a modified procedure consisting of two sequential covalent coupling steps.
Coverage was calculated by measuring the uncoupled IgG present in the supernatants of the first centrifugation, after filtering with a Nucleopore polycarbonate filter pore diameter, 0. After elution, the released protein was measured using the BCA procedure. The covalently coupled isotherms of IgG anti-CRP on carboxyl-modified polystyrene particles nm diameter are shown in Fig.
The maximum adsorption plateau is achieved immunofurbidimetric a polyclonal antibody concentrations of 3. The immunopurified antibody showed a similar trend, having a covalently coupled adsorption isotherm according to that of the nonimmunopurified antibody in the range studied 0. The microparticle reagent response of the different antibodies to increasing CRP concentrations can be seen in Fig. The most striking feature in Fig. We found that at any given working dilution of microparticles studied, the initial absorbance decreases with increasing wavelength and increases with microparticle diameter, according to the light-scattering theory.
The effect of varying the particle size on a reagent with a constant charge of 0. Thus, a compromise between sensitivity and measurement range is demanded.
A microparticle size of nm and a wavelength of nm were selected as the best choice because of the high slope and wide dynamic range showed. We chose this microparticle reagent, with a coverage of 0. Polyethyleneglycol PEG is commonly used to increase the immunoturbidimetriv kinetics. We monitored the reaction kinetics at pH values of 6.
We then selected 0. The turbidimetric method affords a measuring range of 0.
Samples with CRP values above the upper limit of the calibration curve can be immhnoturbidimetric after automated or manual sample dilution. The detection limits for CRP, defined as the mean plus 3 SD of the zero signal saline solutionwere 0. Parameter values abuatomated coefficients rand confidence regions were as follows: 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.
Development of an automated immunoturbidimetric ferritin assay.
Posttreatment with additives BSA, surfactants has been proposed 13 14although it reduces IgG charge and reactivity. We suggest an alternative approach that uses a small amount of immunopurified 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.
In conclusion, a low coverage 0. This approach to antibody immunopurification could be extended to obtain reagents useful for the measurement of several other proteins at low concentrations Covalent isotherm A and dose—response curves as a function of antibody type and coverage B and particle size C.
Jadsamount of antibody adsorbed; Jinamount of antibody added. Ceffect of particle size on absorbance at nm. Skip to main content. Research Article Technical Brief. View inline View popup. Conditions for CRP determination. Download figure Open in new tab. The value of acute phase protein measurements in clinical practice. Ann Clin Biochem ; Production of C-reactive protein and risk of coronary events in stable and unstable angina. Lancet ; Increased serum C-reactive protein levels by immunonephelometry in patients with rapidly destructive hip osteoarthritis.
Rev Rheum Engl Ed ; C-reactive protein and bacterial infection in preterm infants. Eur J Pediatr ; Variability in the measurement of C-reactive protein in healthy subjects: Clin Chem ; A rapid and sensitive automated light scattering immunoassay for serum C-reactive protein and the definition of a reference range in healthy blood donors. Clin Chem Lab Med ; Development and validation of a particle enhanced turbidimetric immunoassay for C-reactive protein.
J Immunol Methods ; Evaluation of an improved immunonephelometric assay for C-reactive protein [Abstract].
Clin Chem or 42 Suppl 6: Evaluation of four automated high-sensitivity C-reactive protein methods: A new method of measuring C-reactive protein, with a low limit of detection, suitable for risk assessment of coronary heart disease. J Clin Immunoassay ; Clin Chem ; 45 Suppl 6: Colloidal stability and immunoreactivity. J Biomater Sci Polym Ed ; 3: Development of automafed automated immunoturbidimetric ferritin assay. Table of Contents Index by author. Clinical Chemistry Nov46 11.
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