Natural antibodies are those antibodies which are present in individuals who do not have a known history of sensitization against the relevant antigen (Calne, 1970). The natural antibodies characterized most extensively in recent years are xenoreactive antibodies, typically defined as those antibodies which bind to the cells of a foreign species (Platt et al., 1990a). Xenoreactive natural antibodies initiate the 'hyperacute rejection" of pig-to-primate xenografts. Hyperacute rejection almost invariably destroys organ xenografts within hours of reperfusion unless steps are taken to control the natural antibody-initiated response (Cooper et al., 1988; Platt et al., 1991; Lin et al., 1997). Xenoreactive antibodies are also thought to play a key role in the 'acute vascular rejection" of xenografts (Cotterell et al., 1995; Parker et al., 1996c; Platt, 1998), a pathologically distinct form of rejection which typically destroys xenografts over a period of days to weeks (Leventhal et al., 1993; Magee et al., 1995; Kobayashi et al., 1997).
Greater than 80% of human xenoreactive natural antibodies are specific for Gal[alpha]1-3Gal (Good et al., 1992; Sandrin et al., 1993; Collins et al., 1994; Parker et al., 1996b), which is expressed by all mammals except apes, humans, and Old World monkeys (Galili et al., 1987b). Natural anti-Gal[alpha]1-3Gal antibodies are members of a family of natural antibodies which includes isohemagglutinins (Parker et al., 1996b). Natural anti-Gal[alpha]1-3Gal antibodies are found in the serum of all immunocompetent individuals and consist of IgM, IgA in at least some individuals, and, depending on blood type, IgG (Sandrin et al., 1993; Rieben et al., 1995; Yu et al., 1996). However, it is natural anti-Gal[alpha]1-3Gal IgM, rather than other isotypes, which activate complement on the surface of endothelium (Parker et al., 1994) and which is apparently responsible for the rejection of xenografts in nonhuman primates (Cotterell et al., 1995; Parker et al., 1996c; Platt, 1998).
Some antibodies which recognize a terminal [alpha]-galactose may not be xenoreactive. Naturally occurring antibodies which bind to Gal[alpha]1-2Gal (Wieslander et al., 1990) and antibodies which bind Gal[alpha]1-4Gal (Wieslander et al., 1990) do not bind to Gal[alpha]1-3Gal, the [alpha]-galactosyl structure known to be expressed by lower mammals, suggesting that these anti-[alpha]-galactosyl antibodies are not xenoreactive. In the present study, natural antibodies are identified in human serum which bind to Gal[alpha]1-6Glc (melibiose; the structure first used to characterize anti-[alpha]-galactosyl antibodies (Bird and Roy, 1980; Galili et al., 1987a), but not Gal[alpha]1-3Gal, suggesting that these antibodies are not xenoreactive. This finding, together with prior studies (Galili et al., 1987a; Wieslander et al., 1990; Parker et al., 1996a), demonstrates that all Gal[alpha]1-xHexosepyrranosides (Gal[alpha]1-2, Gal[alpha]1-3, Gal[alpha]1-4, and Gal[alpha]1-6) are recognized by natural antibodies in human serum but that all anti-Gal[alpha]1-xHexosepyrranosides do not bind to Gal[alpha]1-3Gal. Antibodies which recognize Gal[alpha]1-2Gal were found in the serum of a number of species, including a number of lower mammals, suggesting that at least some anti-Gal[alpha]1-2Gal antibodies might not be xenoreactive. Further, a significant fraction of natural anti-[alpha]-galactosyl antibodies which bind to Gal[alpha]1-3Gal under optimum conditions in vitro did not bind to a porcine xenograft and were not detected by ELISA using cultured porcine aortic endothelial cells as a target. Thus, naturally occurring anti-[alpha]-galactosyl antibodies are more diverse and more widely distributed in phylogeny than previously known.
Overlap in specificity between anti-Gal[alpha]1-3Gal and anti-Gal[alpha]1-6Glc antibodies
Anti-[alpha]-galactosyl antibodies were initially identified (Bird and Roy, 1980), isolated, and quantitated (Galili et al., 1987a) using Gal[alpha]1-6Glc as a solid phase ligand. However, some xenoreactive anti-Gal[alpha]1-3Gal IgM were later shown not to recognize Gal[alpha]1-6Glc (Parker et al., 1996a). To determine whether some anti-Gal[alpha]1-6Glc IgM might not bind Gal[alpha]1-3Gal and would thus be non-xenoreactive, we evaluated the binding of anti-Gal[alpha]1-6Glc IgM to Gal[alpha]1-3Gal under various conditions. We focused on IgM rather than IgG because IgM but not IgG are deposited on the endothelium of hyperacutely rejecting xenografts (Platt et al., 1991), and natural IgM but not natural IgG fix complement on porcine cell surfaces (Parker et al., 1994; Platt et al., 1990b). Moreover, natural anti-Gal[alpha]1-3Gal IgG have not been detected in the serum of most baboons and some humans regardless of the assay used (Yu et al., 1996; McCurry et al., 1997). As shown in Figure
Figure 1. The extent to which anti-Gal[alpha]1-3Gal antibodies and anti-Gal[alpha]1-6Glc antibodies are the same antibodies. (A) The binding of 125I labeled anti-Gal[alpha]1-6Glc IgM to Gal[alpha]1-6Glc-agarose (melibiose-agarose) and Gal[alpha]1-3Gal-agarose (galactobiose-agarose) determined by radioimmunoassay. The matrix was used at two different concentrations (6.25% and 12.5% volume) to determine whether binding sites were in excess compared to antibody. The binding of 125I labeled anti-Gal[alpha]1-6Glc IgM in the presence of 300 mM Gal[alpha]1-6Glc, which was about 10% of the total binding, was taken to be 'background" binding, and was subtracted from the total binding. Thirty percent to 40% less anti-Gal[alpha]1-6Glc IgM bound to Gal[alpha]1-3Gal-agarose compared to Gal[alpha]1-6Glc-agarose, suggesting that a significant fraction of anti-Gal[alpha]1-6Glc IgM do not recognize Gal[alpha]1-3Gal. (B) Inhibition of binding of 125I labeled anti-Gal[alpha]1-6Glc IgM to Gal[alpha]1-6Glc-agarose by Gal[alpha]1-6Glc and by Gal[alpha]1-3Gal determined by radioimmunoassay. At high concentrations of saccharide (>500 mM), Gal[alpha]1-6Glc inhibited the binding of anti-Gal[alpha]1-6Glc IgM to Gal[alpha]1-6Glc-agarose about 40% more than did Gal[alpha]1-3Gal at the same concentration. The experiment was repeated and similar results were obtained. This result further suggests that a significant fraction of anti-Gal[alpha]1-6Glc IgM does not recognize Gal[alpha]1-3Gal. (C) Inhibition of binding of anti-Gal[alpha]1-3Gal IgM to porcine cells by Gal[alpha]1-6Glc and by Gal[alpha]1-3Gal determined by ELISA. Gal[alpha]1-6Glc inhibited about 70% of the binding of anti-Gal[alpha]1-3Gal antibodies to porcine cell surfaces, indicating that ~30% of the anti-Gal[alpha]1-3Gal antibodies did not bind Gal[alpha]1-6Glc. Phylogenetic distribution of anti-Gal[alpha]1-2Gal antibodies
While the phylogenetic distribution of anti-Gal[alpha]1-3Gal antibodies is one factor which had suggested that these antibodies might be xenoreactive, the phylogenetic distribution of other anti-[alpha]-galactosyl antibodies is unknown. To determine the distribution of other anti-[alpha]-galactosyl antibodies in phylogeny, the presence of anti-Gal[alpha]1-2Gal antibodies in the serum of a variety of different species was evaluated by ELISA. Anti-Gal[alpha]1-2Gal antibodies which bound to immobilized Gal[alpha]1-2Gal and which were inhibited by soluble Gal[alpha]1-2Gal but not by soluble Gal[alpha]1-3Gal or Gal[beta]1-4Glc were found by ELISA in the serum of humans and baboons, which produce anti-Gal[alpha]1-3Gal antibodies, and also found in the serum of squirrel monkeys, horses, cows, goats, dogs, cats, and rats, which do not produce anti-Gal[alpha]1-3Gal antibodies. These results show that at least some anti-[alpha]-galactosyl antibodies are widely distributed in phylogeny, suggesting these antibodies may not be xenoreactive in many instances. Reactivity of xenoreactive anti-Gal[alpha]1-3Gal IgM and other anti-Gal[alpha]1-3Gal IgM with Gal[alpha]1-3Gal[beta]1-4GlcNAc-albumin at 4°C
Previous studies have shown that all anti-[alpha]-galactosyl antibodies which bind to porcine cell surfaces react with the Gal[alpha]1-3Gal, and do not require a trisaccharide or larger sugar for binding (Parker et al., 1996a). All xenoreactive anti-[alpha]-galactosyl antibodies also react with the trisaccharide Gal[alpha]1-3Gal[beta]1-4GlcNAc, which is present on porcine tissues (Oriol et al., 1993). Thus, either synthetic Gal[alpha]1-3Gal or synthetic Gal[alpha]1-3Gal[beta]1-4GlcNAc might be suitable for use in assays for xenoreactive anti-Gal[alpha]1-3Gal antibodies. Accordingly, Gal[alpha]1-3Gal[beta]1-4GlcNAc has been conjugated with albumin and the conjugate used as a target in solid phase assays (Galili et al., 1997a). We therefore tested whether some antibodies specific for Gala1-3Gal might in fact not bind to a xenograft. Ascertaining whether antibodies which bind to Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA might fail to bind to a porcine xenograft is precluded if the porcine organ transplant becomes saturated with antibody, leaving anti-Gal[alpha]1-3Gal antibodies in the circulation which could bind to the organ if antigen were available. To overcome this obstacle, we used a system which ensured that porcine organs did not become saturated with antibodies. Thus, transplants were carried out in baboons which were immunodepleted of anti-Gal[alpha]1-3Gal antibodies as shown in Figure
Figure 2. Experimental model for analysis of xenoreactive antibodies in vivo. Baboons were depleted of anti-Gal[alpha]1-3Gal Ig by treatment with three to four immunoadsorption sessions (indicated by long arrows) performed at intervals of 2-3 days. At the time of the first immunoadsorption session, baboons were placed on immunosuppression as described in Materials and methods and a splenectomy was performed. The final immunoadsorption session was performed 2-6 h prior to the xenotransplant procedure(s). Blood taken from baboons following xenotransplantation was then analyzed for anti-Gal[alpha]1-3Gal antibodies.
Figure 3. Binding of xenoreactive IgM and other anti-Gal[alpha]1-3Gal IgM to immobilized Gal[alpha]1-3Gal[beta]1-4GlcNAc at various temperatures as determined by ELISA. Serum was isolated from blood taken from baboons before any treatment (pre), immediately prior to the second immunoadsorption, (see Figure 2), immediately post absorption (post column) using a column bearing Gal[alpha]1-3Gal, and at various times after the transplantation of a porcine heart. Some samples were incubated with immobilized Gal[alpha]1-3Gal[beta]1-4GlcNAc in the presence of 20 mM Gal[alpha]1-3Gal (+ GB). The data shown are representative of all cases tested, including pre- and posttransplant samples from 10 heart and 8 kidney xenotransplants which have been analyzed at 4°C and at 37°C.
Several lines of evidence suggested that the anti-Gal[alpha]1-3Gal IgM in the blood of baboons with functioning porcine xenotransplants either did not bind or bound only weakly to the porcine organ transplants because the antibodies did not recognize antigen on the endothelium of the xenograft. First, the level of anti-Gal[alpha]1-3Gal IgM in the serum of the xenograft recipients did not depend on the length of time the organ was in place (Figure
Figure 4. Binding to porcine cardiac xenografts of xenoreactive IgM in normal baboons and in baboons depleted of anti-Gal[alpha]1-3Gal antibodies. Porcine hearts were transplanted heterotopically into normal baboons (A) or into baboons depleted of anti-Gal[alpha]1-3Gal antibodies (B). Biopsies were performed 1 h after the transplant. Biopsy sections were snap-frozen, sectioned, and stained for IgM. IgM binding to porcine organs in baboons depleted of anti-Gal[alpha]1-3Gal antibodies was substantially less than that in normal baboons. This result indicates that there is unbound antigen in the porcine organ transplants in immunodepleted baboons.
We next tested the possibility that although some anti-Gal[alpha]1-3Gal IgM did not bind to a porcine xenograft, it might bind to porcine cells under some conditions. To this end, the binding of anti-Gal[alpha]1-3Gal IgM (those detected by ELISA using Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA at 4°C as a target) to cultured porcine cells was evaluated at 4°C, as it has been previously shown that the binding of anti-Gal[alpha]1-3Gal IgM is temperature dependent, maximum binding occurring at temperatures approaching 0°C (Parker et al., 1994). As shown in Figure
Figure 5. The extent to which anti-Gal[alpha]1-3Gal antibodies bound by cultured porcine cells and antibodies bound by immobilized Gal[alpha]1-3Gal[beta]1-4GlcNAc are the same antibodies. (A) The binding of anti-Gal[alpha]1-3Gal IgM in normal human serum or serum absorbed with cultured porcine endothelial cells was assayed by ELISA using cultured porcine aortic endothelial cells as a target. The absorption on porcine cells and the ELISA were carried out at 4°C. The ELISA was performed in duplicate and the standard error is shown. (B) The binding of anti-Gal[alpha]1-3Gal IgM in normal serum or serum adsorbed with immobilized Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA was assayed by ELISA using Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA as a target. The adsorption on immobilized Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA and the ELISA were carried out at 4°C. The ELISA was performed in duplicate, and the standard error is shown.
Nor did there exist a fraction of anti-Gal[alpha]1-3Gal antibodies which would bind to porcine cells, but not to Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA. To determine this, we evaluated the fraction of anti-Gal[alpha]1-3Gal IgM bound to porcine cells that was adsorbed by Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA immobilized on a solid surface. As shown in Figure Temperature dependence of reactivity of anti-Gal[alpha]1-3Gal IgM with Gal[alpha]1-3Gal[beta]1-4GlcNAc-albumin
Since xenoreactive antibodies bind less avidly at higher temperatures (Collins et al., 1994), the 'stringency" of an assay for anti-Gal[alpha]1-3Gal antibodies might be varied by varying the temperature at which it is performed. As shown in Figure Reactivity of anti-Gal[alpha]1-3Gal IgM with porcine cells
As shown in Figure
Figure 6. Binding of xenoreactive anti-Gal[alpha]1-3Gal IgM to cultured porcine cells as determined by ELISA. Serum was isolated from blood taken from baboons before any treatment (pre), immediately prior to the second immunoadsorption, (see Figure 2), immediately following immunoadsorption (post column), and at various times post transplant(post Tx). The binding to cultured porcine cells before and after treatmentof the cells with [alpha]-galactosidase is shown. The ELISA was performed in duplicate, and the standard error is shown. Quantitation of anti-Gal[alpha]1-3Gal IgG by ELISA using Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA and cultured porcine cells as targets
The amount of anti-Gal[alpha]1-3Gal IgG, which might play a role in the elicited immune response to xenotransplantation (Galili et al., 1997b), was evaluated using various methods. The amount of human anti-Gal[alpha]1-3Gal IgG in 14 samples of serum from different individuals as determined by direct ELISA using Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA as a target (ELISA at room temperature) was about 5-fold greater than the amount determined by direct ELISA using cultured porcine cells (ELISA at 4°C; Table I). On the other hand, the amount of human anti-Gal[alpha]1-3Gal IgM in 13 serum samples from different individuals as determined by ELISA using Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA at room temperature as a target was similar to amounts detected in the same samples by ELISA using cultured porcine cells at 4°C (Table I). These results suggest that most anti-Gal[alpha]1-3Gal IgG detected by ELISA using Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA are not detected by ELISA using cultured porcine cells. The difference between IgG binding to Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA and cultured porcine cells probably reflects the lower avidity of anti-Gal[alpha]1-3Gal IgG compared to anti-Gal[alpha]1-3Gal IgM (Yu et al., 1996).
Early studies on natural antibodies suggested that a significant fraction of human antibodies are specific for [alpha]-galactose (Bird and Roy, 1980; Galili et al., 1984; Lalezari and Jiang, 1984; Lalezari et al., 1984). Subsequent studies focused on IgG specific for Gal[alpha]1-3Gal (Galili et al., 1987a,b, 1986). Later it was found that anti-[alpha]-galactosyl antibodies constituted the primary humoral barrier to xenotransplantation (Good et al., 1992). However, it was not clear which anti-[alpha]-galactosyl antibodies actually bind to an organ and initiate rejection. Subsequent studies showed that xenoreactive anti-[alpha]-galactosyl antibodies comprise only a fraction of naturally occurring anti-[alpha]-galactose antibodies (Wieslander et al., 1990), and the present studies suggest that that fraction is even less than might have been previously thought. Here we show that a significant fraction of anti-[alpha]-galactosyl antibodies recognize Gal[alpha]1-6Glc but not Gal[alpha]1-3Gal. Human serum also contains anti-Gal[alpha]1-2Gal and anti-Gal[alpha]1-4Gal antibodies, which do not recognize Gal[alpha]1-3Gal (Wieslander et al., 1990). Taken together, our work and the work Wieslander (1990) indicate that most anti-[alpha]-galactosyl antibodies are not specific for Gal[alpha]1-3Gal. Perhaps more important from a medical perspective is our finding that ~20% of anti-Gal[alpha]1-3Gal IgM do not bind to an organ xenograft.
The phylogenetic distribution of natural antibodies has important implications. The original studies on anti-Gal[alpha]1-3Gal antibodies concluded that these antibodies are restricted to higher primates (Galili et al., 1987b; Galili, 1993). These studies, in conjunction with studies on the phylogenetic distribution of Gal[alpha]1-3Gal expression, clearly showed that anti-Gal[alpha]1-3Gal antibodies might potentially be xenoreactive (Galili et al., 1986, 1987a,b). Although some might assume that all natural anti-[alpha]-galactosyl antibodies are produced only by higher primates, studies on the phylogenetic distribution of natural anti-[alpha]-galactosyl antibodies other than anti-Gal[alpha]1-3Gal antibodies are lacking. Here we show antibodies against [alpha]-galactosyl sugars are broadly expressed in phylogeny, providing insight into the possible xenoreactive nature of the antibodies. In addition, the phylogenetic distribution of these antibodies, which is the broadest thus far identified of any natural anti-carbohydrate antibody, suggests a greater importance of these antibodies than might otherwise be thought.
The results reported here suggest that natural anti-Gal[alpha]1-6Glc antibodies are different from other anti-[alpha]-galactosyl antibodies. Previous studies identified natural anti-Gal[alpha]1-6Glc which bound to the antigen passively adsorbed to human erythrocytes (Bird and Roy, 1980; Lalezari and Jiang, 1984). However, anti-Gal[alpha]1-6Glc antibodies which recognize Gal[alpha]1-6Glc coupled by the reducing terminus to proteins have not been observed (Wieslander et al., 1990). Our own unpublished results confirm that anti-Gal[alpha]1-6Glc antibodies do not bind to Gal[alpha]1-6Glc covalently bound to protein by the reducing terminus, although the saccharide in this form is recognized by lectins specific for [alpha]-galactose. Nevertheless, the present studies show that anti-Gal[alpha]1-6Glc antibodies bind to Gal[alpha]1-6Glc when immobilized to agarose through hydroxyl groups. These observations suggest that anti-Gal[alpha]1-6Glc antibodies recognize carbohydrate in a manner different from other anti-[alpha]-galactosyl antibodies, as the latter recognize their respective disaccharides when the saccharides are coupled through the reducing terminus to a protein (Wieslander et al., 1990).
Table I.
The results reported here, together with prior results, indicate that natural anti-[alpha]-galactosyl antibodies are comprised of a diverse group of antibodies, some of which may recognize only a single Gal[alpha]1-xHexosepyrranoside while others recognize more than one Gal[alpha]1-xHexosepyrranoside. Although speculative, it may be that there are other natural anti-carbohydrate antibodies that exhibit similar properties. For example, anti-blood group A antibodies (specific for GalNAc[alpha]1-3(Fuc[alpha]1-2)Gal) may be members of a broader group of anti-[alpha]-N-acetylgalactosaminyl and/or anti-[alpha]-fucosyl antibodies as some, but not all, anti-blood group A antibodies (in the serum of blood group O individuals) also react with blood group B (Gal[alpha]1-3(Fuc[alpha]1-2)Gal) (Kabat, 1956).
The present studies have important implications regarding the assay of the anti-Gal[alpha]1-3Gal antibodies important in xenotransplantation. First, the detection of xenoreactive anti-Gal[alpha]1-3Gal antibodies depends absolutely on the use of Gal[alpha]1-3Gal rather than other [alpha]-galactosyl saccharides as a target, since the use of other [alpha]-galactosyl saccharides will fail to detect some xenoreactive anti-Gal[alpha]1-3Gal antibodies and will detect some anti-[alpha]-galactosyl antibodies which are not xenoreactive. Second, factors other than the disaccharide target influence the detection of xenoreactive anti-Gal[alpha]1-3Gal antibodies. For example, some assays utilizing Gal[alpha]1-3Gal as a target detect substantial amounts of anti-Gal[alpha]1-3Gal antibodies which do not bind to xenogeneic organs. The recognition of anti-Gal[alpha]1-3Gal antibodies which do not bind to xenogeneic organs can be avoided by adjusting the 'stringency" of a given assay. The importance of stringency suggests that the anti-Gal[alpha]1-3Gal antibodies which do not bind to xenogeneic organs bind less avidly to Gal[alpha]1-3Gal than anti-Gal[alpha]1-3Gal antibodies which do bind to the organs, perhaps due to differences in fine specificity, which are typical of anti-carbohydrate antibodies (Chen and Kabat, 1985; Gooi et al., 1985; Neethling et al., 1996). While we have used temperature to adjust stringency, other factors such as pH and detergents may also prove useful. For example, the use of Tween 20 as a blocking reagent in ELISA's employing Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA as the antigen dramatically decreases 'nonspecific" binding (not shown).
There are other considerations regarding assays for xenoreactive anti-Gal[alpha]1-3Gal antibodies which are not addressed by the present studies. First, since we have used saccharides immobilized on solid supports or cultured cells as targets, we cannot be certain that we will detect all antibodies which might bind to a given saccharide in solution or all antibodies which might bind to a transplanted organ. A second consideration stems from the fact that some alkaline phosphatase used as an enzyme conjugate contains Gal[alpha]1-3Gal modifications. The use of such conjugates may result in a relatively greater signal generated by anti-Gal[alpha]1-3Gal IgM than by other IgM since anti-Gal[alpha]1-3Gal IgM may bind directly to the enzyme. Further, unless a particular alkaline phosphatase conjugate is adsorbed against human serum, the conjugate will react with anti-Gal[alpha]1-3Gal IgM. For example, purified anti-Gal[alpha]1-3Gal IgM react directly with goat anti-human IgG conjugated to alkaline phosphatase (by binding to Gal[alpha]1-3Gal on the alkaline phosphatase). A third consideration is that treatment of cells with [alpha]-galactosidase may generate or expose new epitopes. Generation of new epitopes in this manner is seen only rarely (Parker et al., 1994) on porcine cells cultured in a standard fashion, but frequently on porcine cells which have been cultured in the presence of human serum for extended periods of time (Parker et al., 1998). A fourth consideration is that treatment of cells with most fixatives is known to alter properties of the cell surface (Luft, 1976; Nermut, 1989) and may therefore alter the binding of antibody to the cell surface. For example, the rate of binding and the amount of binding at equilibrium of xenoreactive antibody to porcine cells fixed with 0.2% glutaraldehyde is severalfold greater than the rate and amount of binding to live cells. Materials
Filter paper (grade 415) was from VWR Scientific Products (West Chester, PA) and rayon/polyester gauze (Nu Gauze) was from Johnson and Johnson Medical Inc. (Arlington, TX). Plates for tissue culture (24-well, Primaria type) were obtained from Becton Dickinson Labware (Lincoln Park, NJ). Alkaline phosphatase-conjugated, affinity-isolated goat antibodies specific for human µ-chain; alkaline phosphatase-conjugated, affinity-isolated goat antibodies specific for human [gamma]-chain; alkaline phosphatase-conjugated, affinity-isolated goat antibodies specific for rabbit Ig; affinity-isolated goat antibodies specific for human µ-chain; affinity-isolated goat antibodies specific for human [gamma]-chain; purified human IgM; purified human IgG; affinity isolated rabbit antibodies against bovine IgG, goat IgG, rat IgG, horse IgG, and dog IgG; dithiothreitol; [beta]-mercaptoethanol; polyoxyethylenesorbitant monolaurate (Tween 20); Gal[alpha]1-6Glc (melibiose); Gal[alpha]1-6Glc attached to 4% beaded agarose (divinyl sulfone activation, 5 atom spacer); and bovine serum albumin (BSA) were from Sigma Chemical Co. (St. Louis, MO). Nunc Maxisorb ELISA plates were from Gibco Scientific Inc. (Coon Rapids, MN). Human alpha thrombin was from Enzyme Research Labs (South Bend, IN); nitro blue tetrazolium (NBT) and bromochloroindolyl phosphate (BCIP) were from Promega Corp. (Madison, WI). The enzyme [alpha]-galactosidase (from green coffee beans) was obtained from Boehringer Mannheim (Indianapolis, IN). Lectin I, isolectin B4 from Griffonia simplicifolia (GS-IB4; specific for [alpha]-d-galactose) (Hayes and Goldstein, 1974; Wood et al., 1979) and biotinylated GS-IB4 were from Vector Laboratories, Inc. (Burlingame, CA). Gal[alpha]1-3Gal[beta]1-4GlcNAc attached to BSA was from V-Labs Inc. (Covington, LA). The disaccharide 3-O-[alpha]-d-galactopyranosyl-d-galactose (galactobiose; Gal[alpha]1-3Gal) was obtained from Toronto Research Chemicals Inc. (Downsview, Canada). Gal[alpha]1-3Gal[beta]1-O(CH2)3NH- linked to a glass matrix (pore diameter 2000 Å, particle size 0.1-0.3 mm) by a poly[N-(2-hydroxyethyl)acryl-amide] spacer-arm, Gal[alpha]1-3Gal[beta]1-O(CH2)3NH- linked to agarose by a poly[N-2-hydroxyethyl)acrylamide] spacer-arm, and Gal[alpha]1-2Gal[beta]1-O(CH2)3NH- linked to biotin by a poly[N-(2-hydroxy-ethyl)acrylamide] spacer-arm were purchased from GlycoTech (Rockville, MD). Iodogen was from Pierce (Rockford, IL), and 125I was from Dupont NEN (Boston, MA). Serum
Serum was used as a source of natural antibodies. Some serum was obtained from healthy volunteers. Other human serum was generated from fresh frozen plasma obtained from the American Red Cross. For this purpose, 1 ml of 10% CaCl2 and 100 units of human alpha thrombin were added to each unit of plasma and the fibrin allowed to precipitate overnight at 4°C. The resulting mixture was centrifuged for 30 min at 12,300 × g and 4°C and the supernatant was then filtered sequentially through rayon/polyester gauze, VWR Grade 415 filter paper, and finally through Whatmann 3 mm filter paper. The serum was then stored at -80°C until needed. Serum was also obtained from baboons which received porcine cardiac xenografts as described below. All serum was heat inactivated by incubation for 30 min at 56°C. Sera used for analysis of anti-[alpha]-galactosyl IgG were incubated with 10 mM dithiothreitol at 37°C for 15 min to depolymerize IgM. Xenotransplantation
Transplantation of porcine hearts into baboons was carried out as described previously (Table I) (Lin et al., 1997). Immunosuppression and depletion of anti-Gal[alpha]1-3Gal antibodies were begun 5-7 days before transplantation as shown in Figure
Anti-Gal[alpha]1-3Gal antibodies were depleted from baboons by plasmapheresis combined with immunoabsorption using columns containing matrix conjugated with Gal[alpha]1-3Gal as previously described (Lin et al., 1997). Five to seven days before transplan-tation and prior to the first immunoabsorption session, with baboons under general anesthesia, a splenectomy was performed and a catheter inserted in the internal jugular vein. The blood was separated into plasma and cellular components and the plasma filtered through Sepharose columns bearing Gal[alpha]1-3Gal[beta]1-4GlcNAc. The absorbed plasma was recombined with the blood cells and returned to the baboon through the catheter. For each treatment, three to five plasma volumes were passed through the columns. Prior to receiving a xenograft, baboons received two to four immunoabsorption sessions each separated by 2-3 days (Figure Purification of human anti-Gal[alpha]1-6Glc IgM
Anti-Gal[alpha]1-6Glc IgM were isolated from human serum by affinity chromatography using Gal[alpha]1-6Glc agarose followed by euglobulin precipitation. For this procedure, 400 ml of human serum was passed through a column packed with 20 ml of Gal[alpha]1-6Glc-agarose. The column was washed sequentially with 60 ml of PBS, 60 ml of 300 mM lactose, and 60 ml of PBS, and eluted with 300 mM Gal[alpha]1-6Glc. The eluate was then dialyzed overnight against deionized water. The precipitate was collected by centrifugation for 20 min at 15,000 × g and dissolved in 10 mM phosphate, 140 mM NaCl, 50% (v/v) glycerol, pH 7.4. Samples were flash frozen in liquid nitrogen and stored at -80°C until use. IgG and albumin were the only contaminants detected, constituting ~30% of the preparations as judged by densiometric scanning of silver and Coomassie stained samples resolved on SDS polyacrylamide gels. Greater than 90% of the IgM in the preparations bound to Gal[alpha]1-6Glc as judged by binding to Gal[alpha]1-6Glc-agarose.
Purified human anti-Gal[alpha]1-6Glc IgM were labeled asfollows: two-hundred micrograms of purified anti-Gal[alpha]1-6Glc IgM were incubated with 0.3 mCi [125I]NaI and 5 µg Iodogen for 30 min at 21°C. The reaction was stopped by addition of KI to a final concentration of 0.1 M. The 125I labeled anti-Gal[alpha]1-3Gal IgM were separated from free 125I using a PD-10 column. Purification of human anti-Gal[alpha]1-3Gal IgM
Anti-Gal[alpha]1-3Gal IgM were isolated from human serum by affinity chromatography using Gal[alpha]1-3Gal-agarose followed by euglobulin precipitation. For this procedure, 600 ml of human serum were passed through a column packed with 20 ml of Gal[alpha]1-3Gal-agarose. The column was washed with 60 ml of PBS and eluted with 0.2 M glycine, pH 2.8. The eluate was immediately neutralized with about 70 µl of 0.9 M potassium phosphate, pH 8.0 per ml of eluate. Fractions containing greater than 0.4 absorbance units at 280 nm were pooled and dialyzed against three changes of deionized water. The precipitate was collected by centrifugation, dissolved, and flash frozen as described above. IgG and albumin were the only contaminants detected, constituting ~30% of the preparations as judged by densiometric scanning of silver and Coomassie stained samples resolved on SDS polyacrylamide gels. Sixty percent to 70% of the IgM in the preparations bound to Gal[alpha]1-3Gal as judged by precipitation with Gal[alpha]1-3Gal conjugated polyacrylamide immobilized on glass beads. Precipitation with blood group A conjugated polyacrylamide immobilized on glass beads was measured as a control. Quantitation of anti-Gal[alpha]1-3Gal IgM by direct ELISA using cultured porcine aortic endothelial cells as a target
Cultured porcine aortic endothelial cells were used as a target to model antibody-antigen interactions which might occur in a transplanted organ. The binding of human or baboon IgM to porcine endothelial cells was evaluated by ELISA as previously described (Platt et al., 1990a). Endothelial cells cultured in 96-well plates were blocked for 1 h with 1-3% BSA in PBS and then incubated with a source of human or baboon antibodies. After incubation for 3 h at 4°C the endothelial cells were washed three times with cold (4°C) PBS, and then alkaline phosphatase-conjugated goat antibodies specific for human µ-chain and known to be cross-reactive with baboon µ-chain were added and allowed to incubate for 1 h at 4°C. The endothelial cells were then washed four times with PBS, and incubated at room temperature with a developing solution consisting of 1.0 mg/ml p-nitrophenyl phosphate in 100 mM diethanolamine, 0.5 mM MgCl2 and 0.2 % (w/v) NaN3, pH 9.5. Absorbance at 405 nm (A405) was determined using an EL 340 Bio Kinetics Reader (Bio-Tek Instruments; Winooski, VT). The binding of anti-Gal[alpha]1-3Gal IgM to the porcine cells was taken to be the binding which was eliminated by (1) the removal of [alpha]-galactosyl residues from the surface of the porcine cells with [alpha]-galactosidase (enzymatic digestion described below), or (2) the presence of maximal inhibitory concentrations of Gal[alpha]1-3Gal (= 3.5 × 106 mol of Gal[alpha]1-3Gal per mole of xenoreactive antibody) as described previously (Parker et al., 1996a). Typically, 50-90%, depending on the cells used, of the antibody bound to porcine cells was bound to Gal[alpha]1-3Gal. The concentration of anti-Gal[alpha]1-3Gal antibodies in serum was determined based on comparison with a well characterized serum, as previously described (Parker et al., 1994). The concentrations obtained by this method agreed within 10% with concentrations determined using purified anti-Gal[alpha]1-3Gal IgM (purification described above) as a standard. Treatment of porcine aortic endothelial cells with [alpha]-galactosidase
Porcine aortic endothelial cells cultured in 96-well plates were fixed with glutaraldehyde, blocked with 1% BSA in PBS and washed as described above. Fifty microliters of solution containing 20 mU of [alpha]-galactosidase in 100 mM NaCl, 50 mM sodium acetate, pH 5.0 was added to each well, and the plates were incubated at 37°C for 1 h. Buffer without enzyme was used as a control. The reaction of the enzyme was judged to be complete when: (1) further digestion resulted in no further decrease in the binding of xenoreactive natural antibodies or GS-IB4, a lectin which is specific for [alpha]-galactosyl residues, and (2) soluble [alpha]-galactosyl saccharides did not inhibit the binding of xenoreactive natural antibodies or GS-IB4 to the treated cells. [alpha]-galactosidase was previously shown to specifically remove [alpha]-galactosyl residues from the porcine cell surface (Collins et al., 1994). Quantitation of anti-Gal[alpha]1-3Gal IgG using porcine aortic endothelial cells as a target
Anti-Gal[alpha]1-3Gal IgG were quantitated using a 'direct" ELISA similar to that used to quantitate anti-Gal[alpha]1-3Gal IgM. Cultured porcine aortic endothelial cells were fixed, blocked and washed as described above. The cells were then incubated with human serum as a source of anti-Gal[alpha]1-3Gal IgG. After washing, the cells were incubated with alkaline phosphatase-conjugated goat antibodies specific for human [gamma]-chain and then washed and developed as described above. The binding of anti-Gal[alpha]1-3Gal IgG to the porcine cells was taken to be the binding which was eliminated by (1) the removal of [alpha]-galactosyl residues from the surface of the porcine cells with [alpha]-galactosidase or (2) the presence of maximal inhibitory concentrations of Gal[alpha]1-3Gal as described above. To determine approximate concentrations of antibody, dilutions at known concentration of purified human IgG immobilized on the plate with goat anti-human [gamma]-chain were used as 'standards."
To verify the concentrations in serum of anti-Gal[alpha]1-3Gal IgG determined by the above method, anti-Gal[alpha]1-3Gal IgG from some (71%) of the samples tested by the above method were affinity-isolated using porcine cells as ligands and quantitated by sandwich ELISA. Dilutions of serum were incubated with cultured endothelial cells at 25.0 ± 1.0°C for 6 h. Cells were then washed eight times with PBS and incubated with a 10 mM solution of lactose or Gal[alpha]1-3Gal in PBS at the same temperature; Gal[alpha]1-3Gal at 10 mM was sufficient to elute all anti-Gal[alpha]1-3Gal antibodies. After 6 h, the supernatant containing the eluant was removed, and the eluted IgG were quantitated by sandwich ELISA. IgG eluted by lactose were taken to be bound 'nonspecifically" and were subtracted from the total. Concentrations determined by this method agreed very well (within 40%) with concentrations determined by direct ELISA on porcine cells (described above). Quantitation of anti-Gal[alpha]1-3Gal IgM and IgG by ELISA using Gala1-3Gal[beta]1-4GlcNAc-BSA as a target
The binding of anti-Gal[alpha]1-3Gal IgM and IgG to Gal[alpha]1-3Gal[beta]1-4GlcNAc was assayed by ELISA as follows. Maxisorp 96 well plates were coated with 5 µg/ml of Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA overnight at 4°C. Plates were washed and then blocked with 1% BSA and 0.05% Tween 20 in PBS, pH 7.4. Serial dilutions of heat inactivated human or baboon serum in PBS were then added and allowed to incubate for 3 h. Wells were then washed and incubated for 1 h with alkaline phosphatase conjugated goat anti-human µ chain, or alkaline phosphatase conjugated goat anti-human [gamma] chain in 1% BSA and 0.05% Tween 20 in PBS, pH 7.4. The incubations with enzyme conjugate were performed at the same temperature as the incubation with serum for all experiments. Plates were washed, developed, and the absorbance at 405 nm determined as described above. The binding of IgM or IgG to unconjugated BSA (generally < 3% of the total binding in normal sera) was taken to be 'background" and was subtracted from the total binding. To provide estimations of the amount of anti-Gal[alpha]1-3Gal antibodies, dilutions at known concentration of purified human IgM or IgG immobilized on the plate with goat-anti-human µ-chain or goat anti-human [gamma]-chain were used as standards. Evaluating the presence of anti-Gal[alpha]1-2Gal Ig by ELISA using immobilized Gala1-2Gal as a target
The binding of anti-Gal[alpha]1-2Gal Ig to immobilized Gal[alpha]1-2Gal was assayed by ELISA as follows. Maxisorp 96-well plates were coated with 5 µg/ml of avidin overnight at 4°C. Plates were washed, incubated for 1 h with 3.5 µg/ml Gal[alpha]1-2Gal[beta]1-O(CH2)3NH- linked to biotin by a poly[N-( 2-hydroxyethyl)acryl-amide] spacer-arm, and then blocked with 1% BSA and 0.05% Tween 20 in PBS, pH 7.4. Some wells were incubated with no conjugate or with Glc[beta]-O(CH2)3NH- linked to biotin by a poly[N-( 2-hydroxyethyl)acrylamide] spacer-arm. Serial dilutions of heat inactivated serum in PBS were then added and allowed to incubate for three hours. Wells were then washed and incubated for 1 h with alkaline phosphatase conjugated polyclonal anti-Ig in 1% BSA and 0.05% Tween 20 in PBS, pH 7.4. Alternatively, wells were washed following incubation with heat inactivated serum, incubated for 1 h with unconjugated rabbit anti-Ig, washed, and then incubated with alkaline phosphatase-conjugated anti-rabbit Ig in 1% BSA and 0.05% Tween 20 in PBS, pH 7.4. The presence of antibodies from each species was evaluated using anti-Ig reagents developed against that particular species, except feline IgY were detected using antibodies against canine Ig, and baboon and squirrel monkey Ig, which were detected using antibodies against human Ig. Plates were washed, developed, and the absorbance at 405 nm determined as described above. Anti-Gal[alpha]1-2Gal antibodies were considered to be present in a given serum if the absorbance in wells coated with immobilized Gal[alpha]1-2Gal was greater than in wells not coated with saccharide and in wells coated with glucose, and if soluble Gal[alpha]1-2Gal but not soluble Gal[beta]1-4Glc inhibited the binding of antibody.
We thank Patricia Rowe and Patricia Hart for help with preparation of the manuscript. This work was supported by grants from the NIH (HL50985 and HL52297) and from Nextran.
Discussion
Antibodies
No. of samples tested
Target
Porcine cells
Gal[alpha]1-3Gal[beta]1-4GlcNAc-BSA
Anti-Gal[alpha]1-3Gal IgM
13
20.8 ± 4.8 µg/ml
21.9 ± 3.5 µg/ml
Anti-Gala1-3Gal IgG
14
1.9 ± 0.6 µg/ml
9.7 ± 2.0 µg/ml
Materials and methods
Acknowledgments
References
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 25 Aug 1999
Copyright©Oxford University Press, 1999.