Development and Evaluation of Novel ELISA for Determination of Urinary Pentosidine

J Nutr Sci Vitaminol, 65, 526–533, 2019 Development and Evaluation of Novel ELISA for Determination of Urinary Pentosidine Shoji KASHIWABARA1, Hiroaki HOSOE1, Rei-ichi OHNO2, Ryoji NAGAI2 and Masataka SHIRAKI3 1 Research & Development, SB Bioscience Co., Ltd., 33–94 Enoki-cho, Suita, Osaka 564–0053, Japan 2 Laboratory of Food and Regulation Biology, Graduate School of Agriculture, Tokai University, Toroku 9–1–1, Higashi-ku, Kumamoto, Kumamoto 862–8652, Japan 3 Research Institute and Practice for Involutional Diseases, Nagano 399–8101, Japan (Received June 12, 2019) Summary Pentosidine is the most well-characterized advanced glycation end product (AGE). It has been measured by HPLC, although this approach cannot be adapted to analyze many clinical samples and is also time-consuming. Furthermore, the detection of pentosidine using a reported ELISA kit and HPLC system requires pretreatment by heating, which generates artificial pentosidine leading to overestimation. We developed a novel pentosidine ELISA system that don’t require sample pretreatment for analyzing urine samples. We then analyzed the accuracy, precision, and reliability of this system. Urinary samples for analysis were obtained from healthy volunteers and stored urinary samples from the participants of the Nagano cohort study were also used. The LoB and LoD were 4.25 and 6.24 pmol/mL, respectively. Intra- and inter-assay coefficients of variation were less than 5%. The spiking and dilution recoveries were 101.4% and 100.5%, respectively. Analysis of the cross-reac- tivities against seven compounds representative of AGEs and structurally similar to pentosidine showed no significant cross-reactivity. The correlation coefficient between the concentrations of pentosidine obtained from HPLC and ELISA for the same urine samples was r50.815. The urinary excretion of pentosidine upon overnight fasting was lower than that after a meal, suggesting the presence of diurnal variation in urinary pentosidine. In con- trast, day-to-day variation was not observed. These results indicate that the ELISA system has sufficient reliability, accuracy, and precision for measuring urinary pentosidine. Sam- pling of fasting urine is suitable for minimizing variation. In conclusion, this ELISA system is promising to evaluate the effect of AGE on lifestyle-related diseases. Key Words advanced glycation end-product (AGE), cross-reactivity, spiking and dilution recoveries, diurnal variation, least significant change (LSC) Cardiovascular disease (CVD) and fracture are seri- tosidine is important to evaluate metabolic disorders. ous health problems in the elderly. These morbidities are However, this has not spread widely in a clinical context associated with a reduction in the activities of daily life; because the main approach for assaying pentosidine therefore, there is an urgent need to establish a system has been HPLC, which is time consuming. Furthermore, for their early detection. Although the exact mecha- the detection of pentosidine using a reported ELISA kit nisms behind these morbidities are complex, tissue oxi- and HPLC system requires pretreatment (pronase and dation and glycation are recognized as important causal acid hydrolysis, respectively) by heating, which gener- processes. To prevent CVD or fracture, biomarkers to ates artificial pentosidine leading to overestimation. evaluate tissue glyco-oxidation are urgently required in The application of heat treatment to protein solutions a clinical context. Among several biomarkers to detect containing glucose lead to the generation of (artificial) tissue glyco-oxidation, pentosidine is the most well- AGEs (6). Therefore, an assay method with the ability to characterized advanced glycation end product (AGE). analyze large numbers of samples with sufficient speci- This compound is formed in collagen fibers in bone and ficity and accuracy without heating is required. Here, vessels non-enzymatically and accumulates with age. we describe the development of a new ELISA system to In fact, the urinary excretion of pentosidine has been measure the urinary excretion of pentosidine and its reported to increase with aging (1) and can be predictive application to clinical samples. of future fracture in men (2) and women (3) and dia- MATERIALS AND METHODS betic patients (4). In addition, the association of serum or urinary pentosidine with CVD has been reported (5). Subjects. A test was performed to analyze the cor- These reports indicated that the measurement of pen- relation between the results of HPLC and ELISA for human urine samples (n5105) from the Nagano cohort E-mail: [email protected] study, an ongoing study of outpatients at a primary care 526 Development ELISA for Urinary Pentosidine 527 Fig. 1. Method of sample collection for the determination of LSC. Samples were collected three times a day on three arbitrary days during 1 wk: (m) second voiding urine in the morning, (a) afternoon (after lunch), and (e) evening (after dinner). institute in Nagano Prefecture, Japan (7, 8). To deter- plates were washed with PBS-T and then supplemented mine least significant change (LSC) and day-to-day with secondary antibody [HRP-labeled goat anti-rabbit and diurnal variations, urine samples from six healthy IgG (Scantibodies Laboratory, Santee, CA, USA)] diluted male volunteers were obtained after obtaining written 1 : 1,000 in blocking buffer. After incubation for 1 h at informed consent. room temperature, plates were washed with PBS-T and Chemicals for cross-reactivity evaluation. Pentosidine supplemented with TMB substrate (Agilent Technolo- {aS-amino-2-[(4S-amino-4-carboxybutyl) amino]-4H- gies, Santa Clara, CA, USA) followed by stop solution imidazo(4,5-b)pyridine-4-hexanoic acid, molecular after 30 min. Absorbance (OD450 and OD630 for refer- weight: 378.4} was purchased from Cayman Chemical ence) was measured using an ELx808 microplate reader (Ann Arbor, MI, USA). CML [Ne-(carboxymethyl) lysine] (BioTek Instruments, Winooski, VT, USA) and data were and CMA [N-(carboxymethyl) arginine] were from analyzed using Gen5 data analysis software. Nippi, Inc. (Tokyo, Japan). 3-Deoxyglucosone (3-deoxy- Evaluation of the ELISA. The use of the ELISA for glucosone) was from Dojindo Laboratories (Kumamoto, analyzing human urinary pentosidine was examined Japan). L(1)Arginine, L-histidine, and D(2)ribose were using the limits of blank and detection (LoB, LoD), intra- from FUJIFILM Wako Pure Chemical Corporation and inter-assay variation, recovery, dilution, and cross- (Osaka, Japan). L(1)Lysine was from Tokyo Chemical reactivity test. Industry Co., Ltd. (Tokyo, Japan). The limit of blank (LoB) and limit of detection (LoD) Development of competitive ELISA. Female New Zea- were determined using methods similar to those out- land White SPF rabbits that were 6 wk old were first lined in the classical approach of Clinical Laboratory immunized with pentosidine conjugated with keyhole Standards Institute (CLSI, Wayne, PA, USA) guideline limpet hemocyanin (KLH) in complete Freund’s adju- EP17-A2 (9). Briefly, this involved measurements being vant via subcutaneous injection. Six booster immuni- performed on both a set of blank samples and a set of zations were given at 2-wk intervals via subcutaneous low pentosidine level samples containing a measurand injection with incomplete Freund’s adjuvant. The rab- targeted around the assumed LoD. Depending upon the bits were exsanguinated on day 91, which was 7 d after distributions of the blank and low-level sample results, a the final immunization. Affinity chromatography was nonparametric data analysis option was selected to cal- performed on a Protein A-coupled column (GE Health- culate the LoD and LoD estimates. Specifically, two lots care, Buckinghamshire, England, UK) for purification of of ELISA kits and five urine samples from healthy volun- the polyclonal IgG. teers were used. The healthy samples were used as low- ELISA 96-well Nunc Maxisorp plates (Thermo Fisher level samples. Blank samples were made by performing Scientific, Waltham, MA, USA) were coated with 0.3 mg immunoadsorption for each specimen to remove endog- per well of recombinant streptavidin (Thermo Fisher enous pentosidine. Then, five blank samples for LoB and Scientific) in PBS overnight at 4˚C, were washed with five low-level samples for LoD were measured in quadru- PBS containing 0.05% Tween 20 (PBS-T), and then plicate on three test days. were blocked with Block Ace (Megmilk Snow Brand, To analyze intra-assay variation, eight replicates of Tokyo, Japan) for 3 h at room temperature. Biotin-con- three different samples were run in one assay. For inter- jugated pentosidine in PBS was added to the plate and assay variation, three different samples were run in five incubated at room temperature for 2 h. After the plates independent assays. The intra- and inter-assay variation had been washed with PBS-T, a pentosidine-coated plate was calculated as the coefficient of variation (CV%). was obtained as immobilized pentosidine was left in the The spike recovery test was used to determine whether plates. Then, 200 mL of PBS and 10 mL of test sample analyte detection was affected by a difference between or calibrator (0, 10, 30, 60, 120, or 180 pmol/mL) were the diluent used to prepare the standard curve and added to a microplate for dilution. Moreover, 30 mL of the urine sample matrix. It was performed using three the diluted sample and 70 mL of the polyclonal anti- samples: two with known pentosidine concentrations pentosidine

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