Increased concentration of two different advanced glycation end-products detected by enzyme immunoassays with new monoclonal antibodies in sera of patients with rheumatoid arthritis

Antigens were prepared according to the following three procedures.

Procedure A: reaction of protein carriers (BSA, bovine crystallin, porcine thyroglobulin, ovalbumin) with ribose. Proteins (50 mg/ml) were dissolved in 0.25 M phosphate buffer (pH 7.8) containing 0.5 M ribose. After addition of 2 drops of toluene, the samples were incubated for 6 weeks at 37°C. All samples were dialyzed extensively against phosphate-buffered saline (PBS) after 6 weeks to remove free sugar, then incubated again at 37°C for another 2 weeks to allow formed glycation products to ripen.

Procedure B: reaction of protein carriers with glyoxylic acid in the presence of sodium cyanoborohydride [18]. This reaction generates mainly CML.

Procedure C: reaction of protein carriers with pyruvate in the presence of sodium cyanoborohydride [19]. This reaction generates mainly CEL.

Splenocytes of Balb/c mice immunized with particular antigens were electrofused [20] with cells from the myeloma line Sp2/0. Hybridoma supernatants were screened over several subsequent rounds to gradually focus the selection. 1) Screening against the immunizing hapten, bound to a different protein carrier. 2) Screening against all non-modified protein carriers (BSA, crystallin, thyroglobulin, ovalbumin) and these same protein carriers modified by glycation ("ribosylation," change of lysine to CML or CEL), coated to a solid phase. 3) Selected pairs of glycated carriers were tested for the ability to mutually inhibit binding of corresponding antibodies to coated antigens. In this step, we compared the affinity of antibodies for antigens presented either in solid or liquid phase. The requirement of an antibody with similar affinity for antigen presented in solid or liquid phases is necessitated by its intended use in an inhibition enzyme-linked immuno-sorbent assay (ELISA) depicted in the flow chart shown in Figure 1. The interaction of an antigen with an antibody that occurs in an inhibition mixture established on a low-binding plate (left side of the flow chart) takes place in liquid phase; the interaction of the antigen with free antibody, that occurs after transferring the inhibition mixture onto a high-binding plate coated with the antigen (right side of the flow chart), takes place in solid phase. It is important that antigen-antibody complexes that formed in the liquid phase should remain stable during this latter step. 4) Concentrations of antigens used for coating plates and dilutions of culture media containing particular antibodies were optimized by titration. Selected conditions were used to test the inhibition of mAb binding to corresponding antigens coated to plates by diluted human sera. This test directly addressed the presence of measurable hapten levels in human serum (i.e., use of the corresponding mAb for analysis of human sera by inhibition ELISA).

Two mAbs were selected for further work: 103-E3 (unidentified antigen formed in the reaction of proteins with ribose), and 8-C1 (CEL). Both these mAbs are IgG₁, kappa. These mAbs were isolated from culture media on Protein G, labeled with biotin, and used to develop inhibition ELISAs.

We have developed an indirect inhibition ELISA for use with our mAbs. The steps for this procedure are shown in Figure 1.

We have used an AGE-modified high-molecular-weight protein as a temporal working standard, expressing results as its weight equivalents. Standards were either BSA glycated with ribose (Rib-BSA) for mAb 103-E3, or CEL-crystallin for mAb 8-C1. Either the same (Rib-BSA for mAb 103-E3) or the next from the set of proteins modified with corresponding AGE (CEL-ovalbumin for mAb 8-C1) was used as an antigen for coating high-binding plates. Maximum amplification of the signal was achieved by using a combination of biotin-labeled mAb and ultra-sensitive streptavidin-peroxidase polymer for detection.

It was necessary to use extreme dilution of antibodies in the inhibition mixture to achieve the required sensitivity in both assays, as the antigen concentrations in the samples (sera) are very low. Therefore, we blocked not only the coated MaxiSorp plates used for ELISA, but also the low-binding plates used for incubation of the inhibition mixture. We used synthetic polymer polyvinylpyrrolidone (PVP) [21] for blocking the low-binding plates, and we also added it to all solutions used for dilution of antibodies and standards. Results can be affected, as any protein added to the inhibition mixture can hypothetically bind the antibody due to its natural content of AGE. Coated MaxiSorp plates were blocked with BSA; at this stage, the results can no longer be affected by any possible natural content of AGE in albumin.

Both ELISAs are very sensitive; titration curves of sera diluted 20×, 40×, and 80× in the inhibition mixture were practically parallel with the 4-parameter calibration curve that was obtained by titration of standards. Representative examples of both calibration curves are shown in Figure 2. Intra-assay variability of both ELISAs was approximately 5%. Inter-assay variability was higher (10-20%), possibly due to an operator change during the course of the study. Sera sets that should be directly compared were measured, if possible, on one plate, to avoid possible bias from high inter-assay variability. For larger sets that had to be accommodated on several plates, coded sera samples of all diagnoses were grouped, and then the order of all samples was randomized. Results measured on different plates were always normalized to the mean of the octuplicate control (pooled) serum that was included on every plate. Results were taken from one of at least two independent measurements, performed at least one week apart.

Labeled mAbs stored at 4°C, and standards divided into small aliquots and stored at -20°C are stable for a minimum of several months. Serum samples are stable for one week if stored unfrozen at 4°C; frozen serum samples (-70°C) are stable for a minimum of 10 freeze/thaw cycles. However, levels measured in the same sample differ depending on the storage method. Following the first freeze/thaw cycle, levels measured in frozen sera were approximately 10% higher than samples that were never frozen. This change of approximately 10% remained unaltered during further freeze/thaw cycles. This phenomenon can be explained, in our opinion, by partial denaturation of serum proteins, leading to increased epitope accessibility for the antibody.

Since pentosidine prevails among species generated during in vitro glycation of proteins with ribose [18], we measured the level of 103-E3 antigen in a collection of sera with a known concentration of pentosidine determined by HPLC [8]. However, we found no correlation between the concentration of 103-E3 antigen measured with ELISA and the concentration of pentosidine (Braun and Vilim, unpublished results). This observation indicates that ribose-derived 103-E3 antigen is different from pentosidine. Since pentosidine is a product of glycoxidation, this conclusion is consistent with our addition of a drop of toluene to the ribosylation mixture to avoid bacterial contamination. Thus, conditions were created that preferred simple glycation over glycoxidation by blocking oxygen access to the glycation mixture.

This study included 45 RA patients, 40 patients with SLE, 48 patients with OA of the knee, and 51 healthy volunteers (HC). Informed consent for the study was given by all subjects, and the study was approved by the local ethics committee. Basic demographic, clinical and laboratory data corresponding to date of sera collection are summarized for all cohorts in Table 1. Clinical data include Disease Activity Score 28 (DAS28) [22] for RA patients, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [23] for SLE patients, and grading according to Kellgren and Lawrence (K/L grade) [24] for OA patients. All RA patients were treated with anti-TNF-α therapy (infliximab) in combination with methotrexate (MTX). Infliximab was administered by intravenous infusion at a dosage of 3 mg/kg every 8 weeks; MTX was administered at a constant dosage of 10-20 mg/week. In addition, 40/45 RA patients (89%) received a constant dosage of glucocorticoids (either methylprednisolone 4-10 mg/day or prednisolone 2.5-10 mg/day or every other day). Sera were collected during the first year of treatment with infliximab; prior to serum collection, each RA patient received at least 4 infliximab infusions. This RA patient cohort was chosen for the treatment with infliximab due to extremely high disease activity, and because they failed to respond to previous treatment. The cohort also included a high proportion of non-respondents to the infliximab therapy. 87% of RA patients were RF positive, and 69% were anti-CCP positive.

Sera were stored at -70°C until used. Levels of the following markers were determined using commercially-available kits, according to the manufacturers' protocol: high-sensitive CRP (CRP Latex, Olympus-Diagnostic, Olympus Czech Group, Praha, Czech Republic), anti-cyclic citrullinated peptide antibodies (Anti-CCP ELISA, Euro-Diagnostica AB, Malmö, Sweden), and rheumatoid factor (Particle Agglutination Test Serodia-RA, Fujirebio Inc., Tokyo, Japan).

Sera levels of AGE of analyzed cohorts were compared using nonparametric tests; two cohorts were compared by the Mann-Whitney test, and three cohorts were compared by the Kruskal-Wallis test, followed by mutual comparison of all groups by Dunn's post test. The value 1.0 was assigned to samples with the antigen level below the limit of ELISA detection, to allow their inclusion in statistical analyses with nonparametric tests. The relationship between two variables was estimated by Spearman's nonparametric correlation. The proportion of males in all four cohorts was evaluated by chi-square test. P values lower then 0.05 were considered to be significant. Statistical analyses were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA).

We observed accumulation of both measured glycation products (CEL and antigen binding to mAb 103-E3) in sera of RA patients (Figure 3). The difference was highly significant (P < 0.0001) when all three cohorts shown in Figure 3 (RA, SLE, HC) were simultaneously compared. The significant increase of AGE levels in RA sera versus SLE sera, as well as versus HC sera, was confirmed (P < 0.001) by Dunn's post-test. Levels of both antigens in SLE and HC sera did not differ. Means ± standard deviation (SD) determined for all three cohorts and both antigens are shown in Table 2. OA joined the list of diagnoses with significantly lower serum levels of antigenic AGE when compared with RA. OA sera were analyzed together with repeated analysis of RA sera, enabling direct comparison of both diagnoses (Figure 4); the difference was again highly significant (P < 0.0001). Means ± SD appear in Table 2.

Neither the levels of CEL nor the levels of 103-E3 antigen in RA sera correlated with RA activity measured by DAS28 score (Table 3). Levels of both antigens in RA sera did not correlate with age, gender, treatment with corticosteroids, levels of CRP, anti-CCP antibodies, or RF in sera (Table 3). The only significant correlation we observed (P = 0.0317) was the negative correlation between disease duration and concentration of 103-E3 antigen (Table 3). This correlation should not be considered significant, as we report the results of testing 7 independent null hypotheses for each ELISA; applying the Bonferroni correction for multiple hypothesis testing, a P-value accepted as significant at the 5% level should be less then 0.0073. However, also the P-value of the negative correlation between disease duration and CEL concentration was the lowest P-value calculated, although it did not reach statistical significance. Decrease of apparent AGE levels in RA sera with disease duration would make sense if, for example, autoantibodies reacting to AGE and interfering in assays gradually accumulated in RA sera during the course of the disease. Our preliminary data (Vilim and Vytášek, unpublished data) suggest that this hypothesis may be correct.