|Molecular Vision 1999;
Received 3 May 1999 | Accepted 11 July 1999 | Published 14 July 1999
Development of a Polyclonal Antibody with Broad Epitope Specificity for Advanced Glycation Endproducts and Localization of these Epitopes in Bruch's Membrane of the Aging Eye
Behnom Farboud,1 Amy Aotaki-Keen,1 Toshio
Miyata,2 Leonard M.
Hjelmeland,1,3 James T.
1Departments of Ophthalmology and 3Molecular and Cellular Biology, UC Davis, Davis, California; 2Institute of Medical Sciences and Department of Medicine, Tokai University School of Medicine, Japan
Correspondence to: James T. Handa, M.D., Vitreoretinal Research Laboratory, School of Medicine, University of California, One Shields Avenue, Davis, CA, 95616-8794; Phone: (530) 754-6229; FAX: (530) 752-2270; email: email@example.com
Purpose: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes.
Methods: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis.
Results: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe.
Conclusions: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.
Long-lived proteins are modified by the nonenzymatic Maillard reactions between the protein's primary amino groups and carbohydrate-derived aldehyde groups to produce Schiff bases and Amadori products in vivo . Further reactions such as dehydration, condensation, and rearrangements yield the so-called advanced glycation endproducts (AGEs). The formation of AGEs can be enhanced by oxidation, a process that has been termed glycoxidation . AGEs have been implicated in diabetes and age-associated diseases such as cataract, Alzheimer's disease, and atherosclerosis [3-6]. However, the most dramatic rise in AGEs has been observed in uremia, regardless of the presence of hyperglycemia or diabetes . It is believed that high oxidative stress with the utilization of substrates other than sugars accelerates AGE formation in renal failure. While a wide variety of AGE structures are possible, only a few have been characterized. Two of these compounds are carboxymethyllysine (CML), a highly prevalent AGE, and pentosidine, a lysine-arginine crosslink [8,9].
Our laboratory has been studying the hypothesis that the age-dependent formation of AGEs in Bruch's membrane alters the phenotype of the retinal pigment epithelium, and that this altered phenotype could contribute to the development of age-related macular degeneration. Previously, we showed an age-related increase in pentosidine and localized pentosidine and CML in aged Bruch's membranes and choroid . This age-associated increase in pentosidine was documented biochemically by HPLC analysis in 23 eyes aged 28 to 71 years old. The distribution of AGEs in Bruch's membrane including basal laminar deposits and drusen, choroidal extracellular matrix, and vasculature was determined immunohistochemically in a smaller set of eyes using polyclonal anti-pentosidine and monoclonal anti-CML antibodies . Ishibashi et al. also provided immunohistochemical evidence of CML in drusen, basal laminar and basal linear deposits, and RPE cells from globes and surgically excised choroidal neovascular membrane samples . To date, polyclonal anti-AGE antibodies have been found to recognize CML as the major epitope [12-16]. The purpose of this work was to develop an anti-AGE antibody that recognizes both pentosidine and CML as well as other unidentified AGE epitopes for use in determining if a diversity of AGE products form in Bruch's membrane and choroid with age.
In vitro incubation experiments
Essentially fatty acid-free grade bovine serum albumin (BSA; Sigma, St. Louis, MO) was incubated with glucose (0.5 M), ascorbate (0.5 M), or glycolaldehyde (0.0078-0.0313M) in 1.0 ml of Phosphate Buffered Saline (PBS, pH 7.4) under air at 37 °C for 3 and 7 days. Unbound material was removed by extensive dialysis against PBS.
Pentosidine measurement by high performance liquid chromatography (hplc) assay
For quantification of pentosidine, samples (50 µl) were mixed with equal volume of 10% trichloroacetic acid and centrifuged at 5000 x g for 5 min. The supernatant was discarded and the pellet was washed with 300 µl of 5% trichloroacetic acid. The pellet was dried under a vacuum and hydrolyzed by 100 µl of 6 N HCl for 16 h at 110 °C under nitrogen, followed by neutralization with 100 µl of 5 N NaOH and 200 µl of 0.5 M PBS (pH 7.4), then filtered through a 0.5 µm-pore filter, and diluted in PBS. Pentosidine in glycated BSA was analyzed by reverse-phase HPLC according to a method previously described by one of us [17,18]. Briefly, a 50 µl solution of acid hydrolysate was injected into an HPLC system and separated on a C18 reverse-phase column (Waters, Tokyo, Japan). The effluent was monitored using a fluorescence detector (RF-10A, Shimadzu, Columbia, MD) and an excitation-emission wavelength of 335/385 nm. Synthetic pentosidine was used to obtain a standard curve .
AGE modified keyhole limpet hemocyanin (KLH, Calbiochem, Inc., La Jolla, CA) and AGE modified BSA were prepared by incubating KLH (20 mg/ml) and BSA (50 mg/ml) in 0.5 M glycolaldehyde (Sigma) in PBS, pH 7.4 under sterile conditions at 37 °C for 7 days, as previously described . After incubation, unbound material was removed by extensive dialysis against PBS and lyophilized. Fluorescence of AGEs was confirmed using a Hitachi F-2000 fluorescence spectrometer with an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The AGE-KLH (0.5 mg) was dispersed in 5 ml PBS and emulsified in 5 ml complete Freund's adjuvant containing 5 mg of heat-killed Tubercle bacillus (Difco, Detroit, MI). Immunization of rabbits followed a previously published protocol .
Affinity purification and immunoabsorption
Crude antibody was affinity purified as previously described . Affinity resin was prepared by coupling AGE-BSA to AffiGel 10 (Bio-Rad Laboratories, Richmond, CA) according to the manufacturer's recommendations. Fractions of purified antibody were dialyzed and titered by immunoblot analysis. The IgG content of the pooled fractions was determined by immunoblot analysis comparing serial dilutions of the pooled fractions to serial dilutions of purified rabbit IgG standards.
The specificity of the affinity purified antibody was determined by immunoblot analysis using the Bio-Dot microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA) according to our previously described protocol . The antigen of interest (AGE-BSA, pentosidine-BSA , CML-BSA [8,20], Amadori product 6-(N-fructosyl) aminocaproic acid (ALT988; Alteon Inc., Ramsey, NJ), KLH, BSA, human collagen IV (Collaborative Biomedical Products, Becton Dickinson Labware, Bedford, MA), and mouse laminin (Collaborative Biomedical Products)) was prepared in TBS containing 10 µg/ml BSA and applied to a nitrocellulose membrane (Schleicher and Schuell Inc., Keene, NH). The membrane was then blocked with 10% non-fat dry milk dissolved in TBS with 0.05% Tween 20 (Blotto) for 1 h at room temperature, incubated with 1 µg/ml anti-AGE antibody diluted in 3% Blotto overnight at 4 °C. The membrane was incubated with 0.2 µg/ml alkaline phosphatase conjugated goat anti-rabbit IgG (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD) in 3% Blotto for 1 h at room temperature, followed by color development with BCIP/NBT (Kirkegaard and Perry Laboratories, Inc.). For competition experiments, anti-AGE antibody was preincubated for 3 h at room temperature with increasing amounts of the antigen of interest prior to incubating the membrane.
Human globes were obtained from a 20 month old child with Sickle disease who died from sepsis (Centralized Pathology Unit for Sickle Cell Disease, Birmingham, AL), an 82 year old nondiabetic donor who died from pneumonia (National Disease Research Interchange, Philadelphia, PA), and an age-matched 82 year old diabetic who died from endstage cardiomyopathy (Medical Eye Bank of Maryland, Baltimore, MD) within 24 h of death for immunohistochemical evaluation. None of the eyes were known to have AMD. Eyes were fixed in 2% paraformaldehyde and cryoprotected using graded sucrose infiltration.
RPE-Bruch's membrane-choroidal cryostat sections (10 µm) were rinsed in PBS, and incubated in 0.3% H2O2-100% methanol for 30 minutes to block endogenous peroxidase. The tissue was blocked with 3% goat serum and 3% blotto for 30 minutes at room temperature, and anti-AGE antibody (1 µg/ml) was incubated overnight at 4 °C in a humidified chamber. Detection was performed using an ABC staining kit (Vector laboratories, Burlingame, CA). Sections were counterstained with Nuclear Fast Red (Vector Laboratories). Control sections were incubated with non-immune rabbit IgG (Vector Laboratories). Competition experiments were performed by preincubating the anti-AGE antibody for 3 h with a 1:200 excess of AGE-BSA, CML-BSA, or pentosidine-BSA.
Quantification of pentosidine in glycated BSA
We were interested in developing an antibody that detects a broad spectrum of epitopes. We chose glycolaldehyde to prepare our AGE immunogen because it significantly accelerates the formation of AGEs compared to glucose [21,22]. While known to be an efficient precursor for CML, glycolaldehyde's ability to form other less prevalent AGEs such as pentosidine, is unknown [23,24]. Therefore, we performed experiments to determine if glycolaldehyde modification leads to pentosidine formation, reasoning that if pentosidine is formed, AGEs other than CML could also be formed. BSA was glycated with glycolaldehyde (0.0078-0.0313M), ascorbate (0.5 M), and glucose (0.5 M) for 3 or 7 days in air at 37 °C. HPLC analysis showed that significant pentosidine, up to 4497 pmol/ml, was generated in a time and concentration dependent manner with glycolaldehyde (Table 1). The formation of pentosidine with glycolaldehyde was immediate, faster and in greater quantity than when ascorbate or glucose were used as substrate.
KLH was glycated with glycolaldehyde for 7 days. The glycated KLH was assessed for fluorogens with excitation/emission maxima at 370/440 nm as an indicator of AGE formation. Glycated KLH showed a 740% increase in fluorescence compared to control samples. Anti-AGE antibodies were made by injecting AGE-KLH into rabbits, and the antiserum was affinity purified. Immunoblot analysis shows that the affinity purified anti-AGE antibody reacts with AGE-BSA, CML-BSA, and pentosidine-BSA, but not KLH, BSA, or the major basement membrane proteins collagen IV or laminin (Figure 1A). For comparison, a different polyclonal anti-AGE antibody is shown that recognizes CML, but not pentosidine  (Figure 1B).
Carboxymethyllysine and pentosidine are only two of many possible AGE structures. Experiments were conducted to determine if other AGE epitopes were recognized by our antibody. Our antibody was preincubated with CML-BSA and pentosidine-BSA, and the preabsorbed antibody was subjected to dot blot analysis using AGE-BSA as the antigen. Figure 2A shows that while preincubation with CML-BSA and pentosidine-BSA prevents the recognition of these antigens, a marked reaction persists against AGE-BSA. The antibody was then preincubated with an Amadori product, and subjected to dot blot analysis. These experiments showed no reduction in staining, suggesting that our antibody recognizes little or no early glycation products (data not shown). Finally, preincubation of our antibody with pentosidine-BSA, CML-BSA, and Amadori product, despite competing out pentosidine and CML, shows that significant AGE epitopes remain (Figure 2B).
Comparative immunostaining of human Bruch's membrane with antibodies against CML, pentosidine, and complex mixtures of AGEs
We previously documented the distribution of CML and pentosidine in aged human Bruch's membrane and choroid using anti-CML and anti-pentosidine antibodies . Experiments were conducted with our anti-AGE antibody to compare the distribution of AGE structures with that of CML and pentosidine. In an 82 year old nondiabetic eye without a known history of AMD, Bruch's membrane, the extracellular matrix of the choroid, and choroidal vasculature showed immunohistochemical evidence of AGEs (Figure 3). In this eye, basal laminar deposits (BLDs) and drusen were identified that showed prominent staining with the anti-AGE antibody (Figure 3A and Figure 3B). As with the nondiabetic globe, an 82 year old diabetic eye showed similar staining for AGEs in Bruch's membrane, the choroidal extracellular matrix, and vasculature (Figure 3C). In contrast, the RPE-Bruch's membrane-choroid from a 20 month old nondiabetic donor showed no immunohistochemical evidence of AGEs (Figure 3D). The distribution of AGEs in aged, but not young Bruch's membrane and choroid with our anti-AGE antibody was similar to anti-CML and anti-pentosidine staining. Significant staining remained in the 82 year old nondiabetic globe after the antibody was preincubated with excess CML-BSA and pentosidine-BSA, suggesting that other AGE epitopes were present (Figure 3E). The same concentrations of CML-BSA and pentosidine-BSA blocked immunohistochemical reaction with anti-CML and anti-pentosidine antibodies, respectively (data not shown). Minimal immunoreaction was observed in the old or diabetic eyes incubated with nonimmune rabbit IgG (Figure 3F). No reaction was seen in the 82 year old nondiabetic globe with the anti-AGE antibody preincubated with excess AGE modified BSA (Figure 3G).
The first aim of this study was to develop an anti-AGE antibody that would recognize AGE epitopes other than CML in order to survey aged Bruch's membrane and choroid for a broad spectrum of AGEs. Previous studies have reported CML as the predominant epitope recognized by polyclonal anti-AGE antibodies [12-16]. While protein modification by glycolaldehyde had not previously been known to form pentosidine (a low abundance AGE [21,22]), our HPLC analysis not only confirmed that glycolaldehyde is a precursor of pentosidine, but that glycolaldehyde is efficiently converted to pentosidine. These results suggested that a glycolaldehyde modified immunogen would be a suitable antigen to produce an antibody that recognizes a spectrum of AGE epitopes. Our antibody appears to be unique with respect to other antibodies in that it recognizes pentosidine in addition to CML. The other polyclonal anti-AGE antibody used in this study, which recognizes predominantly CML, was prepared by incubating BSA in glucose for 60 days . We found that the staining pattern of our antibody was similar to what we found in aged Bruch's membrane, basal laminar deposits, drusen, and choroid from our previous work using anti-pentosidine and anti-CML antibodies .
The competition experiments imply that our antibody recognizes other AGE structures in addition to CML and pentosidine. It is possible that some of the uncharacterized epitopes represent early glycation products. The competition experiments with 6-(N-fructosyl) aminocaproic acid, an Amadori product, suggest otherwise. Since this compound is small, it probably does not attach to the membrane used in the immunoblot analysis. However, it has been successfully used in competition binding studies such as those utilized in our experiments (Personal communication, H.W. Founds, Ph.D., Alteon Inc., 1998). It is unlikely that our antibody recognizes proteins of major abundance in Bruch's membrane since it did not recognize collagen IV or laminin. Our dot blot experiments provide indirect evidence that our anti-AGE antibody recognizes other AGE epitopes. The use of an in vitro AGE-immunogen produces a number of AGEs, not all of which have been structurally defined [12-16]. However, we hypothesize that other AGE structures such as pyrraline  or crossline  could be present. Furthermore, we postulate that these less abundant AGEs were formed during our prolonged glycolaldehyde incubation of KLH, and served as epitopes that were recognized during the synthesis of our antibody. Application of these in vitro results with an interpretation of our immunohistochemical results must be made with caution. Significant staining with our anti-AGE antibody did remain after preincubation with CML-BSA and pentosidine-BSA. It is possible that reactivity of our antibody to CML and pentosidine could remain after blocking with CML-BSA and pentosidine-BSA. However, the same concentrations of CML-BSA and pentosidine-BSA blocked immunohistochemical reaction with anti-CML and anti-pentosidine antibodies. This information provides indirect evidence that AGEs other than CML and pentosidine are being detected in Bruch's membrane. AGEs are a heterogenous group of structures. It is possible that specific AGEs could induce unique biological effects. This is supported by the recent work of Horie et al where formation formation of pentosidine and CML, but not pyrraline, was correlated with diabetic nephropathy . With more extensive characterization of specific AGEs that develop in Bruch's membrane, it may be possible to determine the key AGE structures that are critical to aging of the RPE.
The utility of the antibody we developed is based on its ability to detect AGEs other than pentosidine and CML. Our antibody might find its best use in surveys of the age-dependent accumulation of a large number of epitopes that might not be revealed by antibodies with a narrower epitope reactivity such as to pentosidine and CML. Ishibashi et al documented CML in drusen, basal laminar and basal linear deposits, and RPE cells from aged eyes and surgically excised choroidal neovascular membrane samples . Our previous work extended their observations. For example, we quantitated by biochemical analysis an age-dependent increase of pentosidine in 23 eyes . The immunohistochemical analysis was more qualitative, and showed not only CML, but also pentosidine accumulation in Bruch's membrane including basal laminar deposits and drusen . It is important to note that the previous immunohistochemical data results from antibodies specific for pentosidine and CML. Questions concerning the diversity of AGE epitopes and their age dependent formation in Bruch's membrane in vivo will be a reasonable next study utilizing the antibody we report here. Ultimately, we hope that our studies will support our hypothesis that aging of Bruch's membrane through the formation of AGEs changes the phenotype of RPE cells in contact with these structures, and that this new phenotype could contribute to the development of age-related macular degeneration.
The authors thank Gerard Lutty, Ph.D. for providing tissue from the Centralized Pathology Unit for Sickle Cell Disease, Birmingham, AL; National Disease Research Interchange, Philadelphia, PA; Medical Eye Bank of Maryland, Baltimore, MD. for the tissue. Support for this work was from the National Eye Institute EY00344 (JTH), EY06473 (LMH), Research to Prevent Blindness (RPB) "Manpower Award" (JTH), RPB Senior Scientist Award (LMH), and an unrestricted RPB award.
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