Division of Clinical Immunology, F79, IMP1, Karolinska Institutet, Huddinge University Hospital AB, S-141 86 Stockholm, Sweden
Received on August 6, 2001; revised on October 25, 2001; accepted on November 2, 2001.
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Abstract |
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Key words: ABO incompatible/alpha1,2-fucosyltransferase/antibody absorption/glycoconjugate/mucin
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Introduction |
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Glycans carrying the ABH determinants are found on glycoproteins, on glycolipids, or as free oligosaccharides. ABH antigens can be found on N- or O-linked glycans. The most common core structures described so far on O-linked glycans are core 1 (Galß3GalNAc), core 2 (Galß3(GlcNAcß6)GalNAc), core 3 (GlcNAcß3GalNAc), and core 4 (GlcNAcß3(GlcNAcß6)GalNAc). These have been shown to carry type 1 (Galß3GlcNAc), type 2 (Galß4GlcNAc), and type 3 (Galß3GalNAc) structures (Takasaki et al., 1978
; Donald, 1981
; Clausen and Hakomori, 1989
; Holgersson et al., 1992
; Hakomori, 1999
; Lowe, 1999
). Type 1 structures are mainly found as extensions of the core 3 and 4 structures, whereas type 2 chains (polylactosamine) are seen as extensions on the GlcNAcß1,6 branch of core 2 structures (Clausen and Hakomori, 1989
; Lowe, 1999
).
There are numerous reports on the results of either transfection of different cells with the different 1,2 FTs (Ernst et al., 1989
; Rajan et al., 1989
; Prieto et al., 1995
; Liu et al., 1998
, 1999; Zerfaoui et al., 2000
), or on the specificity in vitro of the
1,2 FTs with synthetic substrates (Beyer et al., 1980
; Kumazaki and Yoshida, 1984
; Betteridge and Watkins, 1985
; Ernst et al., 1989
; Rajan et al., 1989
; Kyprianou et al., 1990
; Larsen et al., 1990
; Sarnesto et al., 1990
, 1992). However, less is known about the synthesis of the various ABH antigens by these enzymes in different cell types on a defined carrier protein. There is one study that was undertaken investigating the substrate specificity of the H type
1,2 FT on various glycoprotein acceptors (Prieto et al., 1997
). As far as we know, there has not been any equivalent study on the Se gene encoded
1,2 FT or a comparison of the action of the two
1,2 FTs on one single glycoprotein produced in different cell lines.
Transplantation (Tx) across the ABO barrier is usually avoided in organ Tx because the risk of antibody-mediated rejection (AMR) due to preformed antibodies is often high (Porter, 1963; Gugenheim et al., 1990
; Sanchez-Urdazpal et al., 1993
; Farges et al., 1995
). This may also hold true in bone marrow transplantation (Benjamin and Antin, 1999
; Benjamin et al., 1999
), though it has long been the belief that blood group ABH incompatibility does not affect the outcome (Gale et al., 1977
). However, in some cases it would still be desirable to transplant across the ABO barrier, one of the reasons being that it widens the pool of available donors for a particular recipient, even if it does not increase the total number of donors (Gugenheim et al., 1990
; Farges et al., 1995
; Gibbons et al., 2000
).
Removal of anti-A or anti-B antibodies by extracorporeal immunoabsorption (EIA) or plasmapheresis (PP) has been shown to improve graft survival following ABO-incompatible organ Tx (Eid et al., 1998; Tamaki et al., 1998
; Alkhunaizi et al., 1999
). Another method used for the prevention of AMR in both ABO-incompatible Tx and xeno-Tx is infusion of free oligosaccharides (Cooper et al., 1993
; Simon et al., 1998
), but low affinity of antibodies for free saccharides (Galili and Matta, 1996
) and the short half-life of low-molecular-weight oligosaccharides in the circulation (Ye et al., 1994
; Simon et al., 1998
) may prevent a wider use. Synthetic immunoabsorbents for extracorporeal absorption of antiblood group antibodies are available (Rieben et al., 1995
) and have been used both in animal models and clinically (Bensinger, 1981
; Bensinger et al., 1981a
,b, 1982).
Here, we describe the production in various host cells of a recombinant P-selectin glycoprotein-1/mouse IgG2b (PSGL-1/mIgG2b) chimera substituted with blood group H and A determinants. The repertoire of core saccharide structures carrying H epitopes was investigated by western blotting using core-specific anti-H monoclonal antibodies. The A epitope density was correlated to the type of host cell used for transfection and to the 1,2 FT gene used to obtain H precursor chains. Furthermore, the ability of blood group A substituted PSGL-1/mIgG2b chimeras to absorb antiblood group A antibodies from human blood group O serum was evaluated.
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Results |
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Both FUT1 and FUT2 could with the 1,3 GalNAcT support the biosynthesis of blood group A chains on the PSGL-1/mIgG2b (B). The combination of FUT2 and the
1,3 GalNAcT seemed to create more A epitopes on PSGL-1/mIgG2b than the combination of FUT1 and the
1,3 GalNAcT (cf. these lanes in B). On the other hand, FUT1 alone supported expression of abundant H type 2 structures (D), whereas FUT2 gave rise to few H type 2 epitopes on the mucin/Ig (D). There were no detectable H type 1 structures on PSGL-1/mIgG2b (C). The H epitopes created by FUT2 were based almost exclusively on type 3, that is, Fuc
2Galß3GalNAc
-R, because no H type 1 structures and few H type 2 structures were made on PSGL-1/mIgG2b by this enzyme (E). Interestingly, cotransfection of both the H or Se gene with the A gene gave rise to abundant epitopes on the PSGL-1/mIgG2b reactive with the anti-H type 3 antibody (E). Blood group A, H type 2 and 3 epitopes were mainly O-linked, because peptide:N-glycosidase F (PNGaseF) treatment of PSGL-1/mIgG2b did not decrease antibody staining using antibodies specific for these epitopes (Figure 2BD). Efficient N-glycan deglycosylation was indicated by a complete mobility shift of the PSGL-1/mIgG2b (Figure 2AD).
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Chinese hamster ovary (CHO) cells.
Figure 4AE, shows the staining of PSGL-1/mIgG2b made in CHO cells. PSGL-1/mIgG2b carried blood group A epitopes following coexpression of FUT1 or FUT2 cDNAs with the 1,3 GalNAcT (B). Neither H type 1 (C) nor H type 2 (D) structures could be detected on the PSGL-1/mIgG2b chimera made in CHO cells following cotransfection with either FUT1 or FUT2, suggesting that H type 3 structures are the sole precursors available for the
1,3 GalNAcT. Staining with the anti-H-type 3 antibody was seen with both FUT1 and FUT2 (E), supporting the theory that the A epitopes are based solely on core 1 structures in CHO cells. Further support to this was obtained after PNGaseF treatment, which did not affect the antibody staining intensity, indicating an O-glycan restricted A epitope expression (data not shown). The number of A epitopes and H type 3 epitopes on the PSGL-1/mIgG2b chimera were clearly higher with FUT2 as compared to FUT1 suggesting that the Se gene product is superior to the H gene product in terms of
1,2-fucosylation of core 1 (Galß3GalNAc
-Ser/Thr) structures.
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The relative blood group A epitope density on PSGL-1/mIgG2b produced in different host cells
To semi-quantify the relative number of A epitopes on the PSGL-1/mIgG2b chimera made in various host cells expressing the A gene with either of the 1,2 FTs, western blotting with anti-A antibodies and anti-mIgG antibodies followed by chemiluminescence detection in a Fluor-S®Max MultImager was used. The ratios of the blood group A and the mIgG reactivities for each PSGL-1/mIgG2b are shown in Figure 5 (one of three representative experiments shown). As seen, the A epitope density was highest on the PSGL-1/mIgG2b made in CHO cells expressing the A gene together with the Se gene encoded
1,2 FT. This PSGL-1/mIgG2b carried approximately three times more A epitopes than the PSGL-1/mIgG2b made in 293T cells transfected with FUT2 and the A gene; the PSGL-1/mIgG2b having the second highest A epitope density (Figure 5). In each cell line, the FUT2 gave higher A epitope density together with the A gene than did FUT1 together with the A gene.
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Discussion |
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For elucidation of the mechanisms behind precursor protein selectivity, a model glycoprotein (such as our recombinant PSGL-1/mIgG2b) can be very useful. Our experiments show that the glycosylation of recombinant PSGL-1/mIgG2b does indeed correlate with in vitro studies. FUT1 has a marked preference for type 2 precursor chains, whereas FUT2 clearly prefers type 3/core 1 structures. However, as seen in Figure 1D, FUT2 is able (though rather poorly) to use type 2 chains as precursors for fucosylation of glycoproteins, as has been suggested by studies on the substrate specificity of FUT1 and FUT2 (Beyer et al., 1980; Kumazaki and Yoshida, 1984
; Le Pendu et al., 1985
; Betteridge and Watkins, 1985
; Kyprianou et al., 1990
; Larsen et al., 1990
; Sarnesto, 1990
, 1992; Kelly et al., 1995
; Rouquier et al., 1995
) and, as seen in Figure 4E, FUT1 can make H type 3 structures on PSGL-1/mIgG2b. Because we could not detect any H type 1 or 2 structures, but only H type 3 structures on PSGL-1/mIgG2b made in CHO cells following transfection with the H gene alone or in combination with the A gene (Figure 3CE), and that these cells have been reported not to extend core 1 structures (Yeh et al., 2001
), we conclude that the A and H epitopes are substitutions on core 1 O-linked glycans. This indicates that FUT1, as previously suggested in transfections of COS cells, can use core 1 precursors on glycoproteins (Liu et al., 1999
).
Our results from western blot analysis further show that the blood group A antigen density on recombinant PSGL-1/mIgG2b when made in CHO cells by a combined H and A gene expression is low. When the enzymes were expressed in 293T cells in the same way, the A epitope density on the PSGL-1/mIgG2b was higher than in CHO cells (Figure 5), reflecting that there are likely to be more lactosamine sequences available for the FUT1 enzyme in the former cell type. In spite of the presence of H type 2 precursor chains in COS cells (Figure 3D), the A epitope density on the PSGL-1/mIgG2b made in COS cells with FUT1 and the 1,3 GalNAcT, was lower than on PSGL-1/mIgG2b made in CHO cells with the same enzymes (Figure 5). A possible explanation for this may be that more H type 3 precursor chains are available in CHO cells transfected with the H gene.
With regard to the number of H type 3 chain epitopes expressed (Figures 1 and 3E), PSGL-1/mIgG2b produced in 293T cells showed a different staining pattern compared to PSGL-1/mIgG2b produced in COS cells. The low expression of H type 3 chain structures on PSGL-1/mIgG2b made in COS cells indicate low levels of the core 1 precursor chain, or that this chain has been blocked by the action of another enzyme, for example, a sialyltransferase. Only a structural characterization of the glycans carried by PSGL-1/mIgG2b can resolve this issue.
Organ Tx across the ABO barrier is characterized by AMR mediated by preformed or induced antibodies against the donor organ blood group (Porter, 1963; Sanchez-Urdazpal et al., 1993
; Farges et al., 1995
; Tanabe et al., 1998
; Alkhunaizi et al., 1999
). There are several methods described for the purpose of removing anti-ABO antibodies, such as cryofiltration (Tamaki et al., 1998
), plasma exchange (Bensinger et al., 1987
), double filtration PP (Ishikawa et al., 1998
), and EIA (Rieben et al., 1995
). We propose to use a recombinant PSGL-1/mIgG2b carrying A determinants as the absorber in an EIA setting. Our results show that approximately 20 pmoles of recombinant PSGL-1/mIgG2b (calculated on a molecular weight of 300 kDa), corresponding to 2 nmoles of A determinants (based on 100 A determinants per mole of PSGL-1/mIgG2b) (Wilkins et al., 1996
; Aeed et al., 1998
, 2001) made in CHO-K1 with FUT2 and
1,3 GalNAcT could absorb 60% of the A-PAA-reactive antibodies. A-PAA-MPG corresponding to approximately 164,000 pmol of A trisaccharides (calculated on 2 µmol/g) was needed to absorb the same amount of anti-A antibodies. The amount of nonspecific protein absorption for both compounds was also assessed, and the nonspecific absorption was almost fourfold higher with the PAA-MPG-based compounds (data not shown). We believe the relatively higher absorption efficacy of mucin-based absorbers may depend on (1) multivalent carbohydrate substitution, (2) close spacing of carbohydrate epitopes, and (3) a structural versatility of the core saccharide chains that carry the immunodominant determinant. However, only a detailed structural characterization can verify these explanations. Furthermore, changing the protein backbone, for example, by making a synthetic mucin-type protein constructed by optimized mucin tandem repeats, may improve the absorber (Silverman et al., 2001
). Also, cells engineered to express different combinations of GalNAc:polypeptide transferases may improve absorption efficacy by optimising the O-glycan substitution density.
In conclusion, we have investigated the repertoire of blood group A and H epitopes carried on recombinant PSGL-1/mIgG2b produced in various host cells expressing FUT1 or FUT2 together with the A gene 1,3 GalNAcT. In accordance with previous studies FUT1 gave rise to mostly H type 2 structures, whereas FUT2 in the absence of type 1 chain precursors (-Galß3GlcNAc-), clearly preferred O-linked core 1 precursors and thus mainly made H type 3 structures. The A epitope density on the PSGL-1/mIgG2b was highest in the CHO made protein, which on a carbohydrate epitope molar basis was at least 80 times better than A-PAA-MPG in absorbing anti-A antibodies.
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Materials and methods |
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Purification of HH14 antibodies
Supernatant was collected from cultured HH14 cells. Two liters of supernatant was affinity purified on a goat anti-mouse IgM (Sigma) column, using a Bio-Rad LP chromatograph (Bio-Rad, Hercules, CA). The bound proteins were eluted by 0.1 M glycine-Cl, pH 2.5, and the eluate was immediately neutralized using 1 M TrisCl, pH 7.5. The eluate was dialyzed against 1% phosphate-buffered saline (PBS) and lyophilized. Lyophilized proteins were dissolved in distilled H2O to a final concentration of 3 µg/µl, as measured by the BCA assay.
Construction of expression vectors
The human blood group A gene was polymerase chain reaction (PCR) amplified off cDNA made from total RNA isolated from the MKN-45 cell line, using 5'-cgc ggg aag ctt gcc gag acc aga cgc gga-3' as forward primer and 5'-cgc ggg cgg ccg ctc acg ggt tcc gga ccg c-3' as reverse primer. The amplified cDNA (A gene) was subcloned into the polylinker of CDM8 (Seed, 1987) using Hind III and Not I. The blood group H gene was PCR amplified in two pieces using a human tonsil stroma library (Holgersson and Seed, unpublished data) as template. An internal Sse I site was created by PCR using internal overlapping primers changing nucleotide 775 (GenBank accession no. M35531) to a C, creating a Sse I site of the Pst I site. The cDNA encoding the carboxy terminal of FUT1 was amplified using 5'-ggg gac tac ctg cag gtt atg cct cag cgc-3' as forward primer and 5'-cgc ggg gcg gcc gct tca agg ctt agc caa tgt-3' as reverse primer, cleaved by Sse I and Not I, and subcloned into the CDM8 expression vector digested with Pst I and Not I. The cDNA encoding the amino terminal of FUT1 was amplified using 5'-cgc ggg aag ctt acc atg tgg ctc cgg agc cat-3' as forward primer and 5'-cca gcg ctg agg cat aac ctg cag gta gtc-3' as reverse primer, cleaved by Hind III and Sse I, and subcloned into CDM8 carrying the carboxy terminal following Hind III and Sse I cleavage.
The Se gene was similarly PCR amplified from cDNA reversely transcribed from total RNA isolated from peripheral blood mononuclear cells donated by a blood group A2Le(a-b+)Se individual, using 5'-cgc ggg aag ctt acc atg ctg gtc gtt cag atg-3' as forward primer and 5'-cgc ggg cgg ccg ctt agt gct tga gta agg g-3' as reverse primer. The Se gene cDNA was subcloned into CDM8 using Hind III and Not I. Glycosyltransferase cDNAs were sequenced and the enzymatic activity they encoded checked by flow cytometric analysis of transiently transfected cells using blood group H and Aspecific monoclonal antibodies. The PSGL-1/mIgG2b chimera was constructed as described before (Liu et al., 1997).
Transfections and production of secreted PSGL-1/mIgG2b chimeras
The transfection cocktail was prepared by mixing 39 µl of 20% glucose, 39 µg of plasmid DNA, 127 µl dH2O, and 15.2 µl 0.1M polyethylenimine (25 kDa; Aldrich, Milwaukee, WI) in 5-ml polystyrene tubes. In all transfection mixtures, 13 µg of the PSGL-1/mIgG2b plasmid was used. Thirteen micrograms of the plasmid for the different glycosyltransferases were added, and, when necessary, the CDM8 plasmid was added to reach a total of 39 µg of plasmid DNA. The mixtures were left in room temperature for 10 min before being added in 10 ml of culture medium to the cells, at approximately 70% confluency. After 7 days, cell supernatants were collected, debris spun down (1400 x g, 15 min) and NaN3 was added to a final concentration of 0.02% (w/v).
Purification of secreted PSGL-1/mIgG2b for SDSPAGE and western blot analysis
PSGL-1/mIgG2b fusion proteins were purified from collected supernatants on 50 µl goat anti-mIgG agarose beads (100 µl slurry; Sigma) by rolling head over tail overnight at 4°C. The beads with fusion proteins were washed three times in PBS and used for subsequent analysis. Typically, the sample was dissolved in 50 µl of 2x reducing sample buffer and 10 µl of sample was loaded in each well.
PNGaseF treatment of affinity-purified PSGL-1/mIgG2b
A PNGaseF kit (Roche Diagnostics, Indianapolis, IN) was used for N-glycan deglycosylation. A slight modification of the protocol provided by the manufacturer was used. In 1.5-ml Eppendorf tubes, 20 µl of reaction buffer was mixed with purified PSGL-1/mIgG2b on agarose beads and boiled for 3 min. The mixture was spun down, and 10 µl of the supernatant was transferred to a new Eppendorf tube. Ten microliters of PNGaseF or, as a negative control, 10 µl of reaction buffer were added. The tubes were incubated for 1.5 h at 37°C. After incubation, 20 µl of 2x reducing sample buffer and 10 µl of H2O was added, and the samples were boiled for 3 min.
ELISA for determination of PSGL-1/mIgG2b concentration in supernatants
Ninety-six-well ELISA plates (Costar 3590, Corning, NY) were coated with 0.5 µg/well of affinity-purified goat anti-mIgG specific antibodies (Sigma) in 50 µl of 50 mM carbonate buffer, pH 9.6, for tw2o h in room temperature. After blocking o/n at 4°C with 300 µl 3% bovine serum albumin (BSA) in PBS with 0.05% Tween (PBS-T) and subsequent washing, 50 µl sample supernatant was added, serially diluted in culture medium. Following washing, the plates were incubated for 2 h with 50 µl of goat anti-mIgM-HRP (Sigma), diluted 1:10,000 in blocking buffer. For the development solution, one tablet of 3, 3', 5, 5'-tetramethylbenzidine (Sigma) was dissolved in 11 ml of 0.05 M citrate/phosphate buffer with 3 µl 30 % (w/v) H2O2. One hundred microliters of development solution was added. The reaction was stopped with 25 µl 2 M H2SO4. The plates were read at 450 and 540 nm in an automated microplate reader (Bio-Tek Instruments, Winooski, VT). As a standard, a dilution series of purified mIgG Fc fragments (Sigma) in culture medium was used in triplicate.
SDSPAGE and western blotting
SDSPAGE was run by the method of Laemmli (1970) with a 5% stacking gel and an 8% resolving gel, and separated proteins were electrophoretically blotted onto HybondTM-C extra membranes as described before (Liu et al., 1997
). Following blocking overnight in Tris-buffered saline with 0.05% Tween-20 (TBS-T) with 3% BSA, the membranes were washed three times with TBS-T. They were then incubated for 1 h in room temperature with mouse anti-human blood group A all types (mIgM, Dako, Carpinteria, CA) or anti-human H type 1 (mIgG3, Signet; Dedham, MA), H type 2 (mIgM, Dako) or H type 3 (mIgM, hybridoma HH14, ATCC HB9299). All antibodies were diluted 1:200 in 3% BSA in TBS-T, except for the H type 3 antibody, which was diluted to a concentration of 1 µg/ml in 3% BSA in TBS-T. The membranes were washed three times with TBS-T before incubation for 1 h at room temperature with secondary horseradish peroxidase (HRP)conjugated antibodies, goat anti-mIgM (Cappel, Durham, NC) or goat anti-mIgG3 (Serotec, Oxford, England) diluted 1:2000 in 3% BSA in TBS-T. Bound secondary antibodies were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the instructions of the manufacturer. For detection of the PSGL-1/mIgG2b itself, HRP-labeled goat anti-mIgG (Sigma) was used at a dilution of 1:10,000 in 3% BSA in TBS-T as described, but without incubation with a secondary antibody.
Determination of the relative blood group A epitope density on PSGL-1/mIgG2b
Western blots were run as described. The membranes were visualized in a Fluor-S Max MultImager carrying a CCD camera operating at 35°C (BioRad). Using the volume tool in the analysis window of the Quantity One software (BioRad), the volume (sum of the intensities of the pixels within the volume boundary x pixel area) for the blood group A reactivity was divided by the volume for the mIgG reactivity for the PSGL-1/mIgG2b made in COS, CHO, and 293T cells. To compare the A epitope/mouse IgG ratios between PSGL-1/mIgG2b made in the different host cells, the ratios were normalized to the ratio obtained from the A substituted PSGL-1/mIgG2b made in COS cells transfected with the H and A genes.
Absorption of serum
Six hundred microliters slurry of goat anti-mIgG agarose beads (Sigma) was transferred into 1.5-ml Eppendorf microcentrifuge tubes. The beads were spun down by a quick spin at 400 x g. The supernatant was then removed, and the beads were washed once with 1 ml PBS, spun down again, and transferred to 180 ml of supernatant from CHO cells transfected with cDNAs encoding PSGL-1/mIgG2b, the Se and the A gene. Supernatants containing agarose beads were incubated head over tail o/n at 4°C. For collection, the beads were spun down at 400 x g, 15 min, at room temperature, and transferred to 1.5-ml Eppendorf microcentrifuge tubes. Washing was done three times with PBS. These beads are referred to as A mucin-beads. Six hundred microliters of anti-mIgG agarose beads were also prepared in the same manner, but they were used for dilution of the A mucin-beads to obtain a dilution series of the A mucin-beads as absorbents. These beads are referred to as goat anti-mIgG beads. The beads were aliquoted into 4-ml Ellerman tubes according to Table I. A-PAA-MPG (Syntesome, Münich, Germany) and B-PAA-MPG (Syntesome) were weighed and aliquoted into Ellerman tubes according to Table I, and thereafter washed once with PBS.
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Five hundred microliters of serum were added to each tube and mixed with the beads for 4 h on a rolling table at 4°C. Following absorption, the beads were spun down and the absorbed serum transferred to new Ellerman tubes. The absorbed serum was stored at 20°C until further analysis.
To determine the amount of PSGL-1/mIgG2b on the A mucin-beads, a goat anti-mIgG Fc ELISA (see previous procedure) was run on the supernatant before and after incubation with agarose beads.
ELISA for quantification of anti-A antibodies
Ninety-six-well ELISA plates (Costar 3590, Corning) were coated for 2 h at room temperature with 0.05 µg of A-PAA-biotin (Syntesome) in 50 µl per well of 50 mM carbonate buffer, pH 9.6. Following blocking o/n at 4°C with 300 µl 3% BSA in PBS-T and subsequent washing, 50 µl of serum serially diluted in PBS were added and the plate was incubated at room temperature for 2 h. After washing, the incubation was done with 50 µl of mouse-anti-human IgA, G and M-HRP (Jackson, PA) diluted 1:10,000 in blocking buffer. Development and reading of the plates was done as described previously.
Determination of total protein concentration in serum
The total protein concentration in serum was determined before and after absorption of anti-A antibodies. The microtiter plate protocol of the BCA (bicinchoninic acid) Protein Assay Reagent (Pierce, Rockford, IL) was used according to the manufacturers instructions, and samples were run in duplicates or triplicates.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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