Peptide assays

Several pathological self-polymerizing systems have been biophysi-cally characterized sufficiently to permit identification of protein or peptide species that could serve as molecular targets in a structure-activity relationship. These include transthyretin (TTR) [73-76], serum amyloid A protein (SAA) [77], microtubule-associated protein tau [78-80], amylin or islet amyloid polypeptide (IAPP) [81,82], IgG light chain amyloidosis (AL) [83-85], polyglutamine diseases [9,86], a-synuclein [47,48] and the Alzheimer s (3 peptide [87-96]. A variety of A(3 peptide assay systems have been established at Parke-Davis to search for inhibitors of fibril formation that could be therapeutically useful [97]. 

Enzyme activities are based on rates of casein hydrolysis under defined conditions. The products of casein hydrolysis, as defined in this protocol, are those peptides soluble in 5% TCA that can be detected by the bicinchoninic acid (BCA) protein assay (unitbi.i). The amount of TCA-soluble peptide generated during the course of the reaction can actually be quantified by any one of several protein/peptide assays. The color yield in these assays is assumed to be proportional to the amount of peptide in solution. The amount of product/peptide in the reaction mixture is often reported as bovine serum albumin (BSA) equivalents—since standard curves based on this protein may be used to calibrate the assay. Thus, activity units can be expressed as the amount of BSA equivalents generated per unit time. 

Many methods are available for measuring TCA-soluble peptides. Possibly the easiest is to measure the absorbance of the solution at 280 nm, as the absorbance at this wavelength is a function of the aromatic amino acid content of the solution. This approach requires a UV spectrophotometer, and the sensitivity of the assay is likely to be lower than that of some of the colorimetric assays. There are also several colorimetric peptide assays that can be applied to this type of peptidase assay, such as the Biuret, Lowry, and Bradford dye-binding methods (for comparison see Piyachomkwan and Penner, 1995). All of these methods measure a relative value rather than an absolute amount of peptide in solution. The results should thus be reported in terms of equivalents, such as BSA equivalents when using a calibration curve prepared using a BSA standard solution. 

Many tests substances change the secretion of hypothalamic hormones, either by direct action or more generally by a feedback effects at the hypothalamic level. Under these conditions, the tissue concentration in hypothalamic specimens of treated rats is of interest, especially because several hypothalamic peptide assays are available and can be measured in the same specimen. In contrast to the tissue concentration, for many hypothalamic hormones it is difficult to measure the circulating concentrations, due to analytical problems of low concentration and rapid inactivation by enzymes. Circulating concentrations would also change immediately due to the interference of anesthesia and of the autopsy procedure. It is therefore recommended to take samples of hypothalamic tissue, and control samples of cerebral cortical tissue. 

The most definitive laboratory test to distinguish type 1 from type 2 diabetes is the C-peptide assay, which is a measure of endogenous insulin production. With type 2 diabetes, proinsulin can be split into insulin and C-peptide lack of C-peptide indicates type 1 diabetes. The presence of anti-islet antibodies (to glutamic acid decarboxylase, insulinoma associated peptide-2 or insulin) or absence of insulin resistance (determined by a glucose tolerance test) is also suggestive of type 1. 

High-Pressure Liquid Chromatography (HPLC) Peptide Assay... 

Measurement of C-peptide has a number of advantages over insulin measurement. Because hepatic metabolism is negligible, C-peptide concentrations are better indicators of P-ceU ffinction than is peripheral insulin concentration. Furthermore, C-peptide assays do not measure exogenous insulin and do not cross-react with insulin antibodies, which interfere with the insulin immunoassay. 

Wu ABH, Tate J, et al. Quality specifications for B-type natriuretic peptide assays. Clin Chem 2005 51 486-93. 

Kazanegra R, Clopton P, Maisel A. Utility of a rapid B-natriuretic peptide assay in differentiating congestive heart failure from lung disease in patients presenting with dyspnea. J Am Coll Cardiol 2002 39 202-9. 

Wieczorek SJ, Wu AH, Christenson R, Krishnaswamy P, Gottlieb S, Rosano T, et al. A rapid natriuretic peptide assay accurately diagnoses left ventricular dysfunction and heart failure a multicenter evaluation. Am Heart J 2002 144 834-9. 

Deftos LJ. Bone protein and peptide assays in the diagnosis and management of skeletal disease. Clin Chem 1991 37 1143-8. 

Logeart D, Thabut G, Jourdain P, et al. Predischarge B-type natriuretic peptide assay for identifying patients at high risk of readmission after decompensated heart failure. J Am Coll Cardiol 2004 43 635-641. 

The measurement of C-peptide provides a fully validated means of quantifying endogenous insulin secretion, preventing influence of exogenous insulin or insulin antibody. However, most C-peptide assay kits cannot differentiate C-peptide from proinsulin and proinsulin conversion products. The influence of proinsulin may be significant in cases where serum proinsulin is elevated, as in Type 2 diabetes, familial hyperproinsulinemia and in patients with proinsulin antibody. 

All evaluations of this C-peptide assay performance were performed by LUMIPULSE FORTE. The method used on LUMIPULSE FORTE was as follows 20 pL of serum or standard was mixed with 250 pL of antibody-coated ferrite particle suspension and the mixture was incubated for 37 °C, 10 min. After B/F separation, 250 pL of Enzyme-labeled antibody was added to the ferrite particle suspension and the mixture was incubated for 37 °C, 10 min. After the second B/F separation, 200 pL of AMPPD solution was added. During the washing steps, the ferrite particles were magnetically separated from the bulk solution on the wall of the cartridge. After the enzyme reaction had proceeded for 5 min, the chemiluminescent light emission was measured for 2 s. 

Figure 1. Precision profile for C-peptide assay Broken lines indicate functional sensitivity (CV=10%) of the assay...

We showed reactivity to C-peptide, proinsulin and insulin of the C-peptide assay (Fig. 2). Cross-reactivity to proinsulin and insulin was <1.7% (proinsulin) and <0.03% (insulin) of C-peptide on a molar basis. 

The use of a monoclonal antibody that recognizes the N-terminal of the C-peptide molecule made it possible to realize a low cross-reactivity to proinsulin. Our results indicate that the C-peptide assay on the LUMIPULSE system shows good specificity, sensitivity, precision and linearity. Accordingly, the C-peptide assay enables for the accurate measurement of C-peptide at low concentrations. Especially, it is useful for the presumption of residual P-cell function of diabetic patients who are nearly depleted of insulin secretion. 

FKBP60 and FKBP65 are ER-resident proteins with four FKBP domains and a Ca2+-binding motif. They are active in the peptide assay as well as in the catalysis of folding of type III collagen (Zeng et al., 1998 Shadidy et al., 1999). 

FIGURE 25.2 Effects of NIPP-1 and NIPP-2 on procollagen synthesis in LX-2 cells. Ten percent FBS was used for the tests to eliminate the interference of serum with the produced protein. Procollagen type IC-peptide assay was done after 24 h (A) and 48 h (B) of treatment. Each value was expressed as the mean SD of triplicate experiments. P< 0.05, P< 0.01, and p< 0.001 as compared with TGF-pi-treated cells. P< 0.001 as compared with non-TGF-pi-treated cells. 

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