Optimization of phage-immobilized ELISA for autoantibody profiling in human sera
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- Woo, M., Heo, C., Hwang, H. et al. Biotechnol Lett (2011) 33: 655. doi:10.1007/s10529-010-0483-6
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Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.
KeywordsBlocking bufferCoating bufferHuman serumPhage ELISAPre-adsorption
Multiplex antigen arrays have been used to identify signature autoantibody profiles in human diseases. Although these arrays are usually constructed using known antigenic proteins, in autoimmunity and cancer research, identification of novel self-antigens recognized by autoimmune sera has been long considered as an important research goal, often leading to new diagnostic and therapeutic paradigms (Di Niro et al. 2009). Therefore, profiling autoantibody repertoires by screening phage display cDNA libraries prior to the identification of individual antigens, has been suggested to be a powerful tool for analysis of autoimmune diseases (Somers et al. 2009; Jadali et al. 2005) and cancers (Wang et al. 2005; Zhong et al. 2008).This is possible because antibody-antigen binding can be induced by only a small antigenic structure derived from whole antigens. In this aspect, phage display random peptide libraries (Cortese et al. 1995; Cwirla et al. 1990; Felici et al. 1991) are also useful for autoantibody profiling.
In our previous studies, an anti-fatty acid synthase (FASN) autoantibody was identified in a hepatocellular carcinoma (HCC) model mouse (Heo et al. 2010). In addition, the mimotope phage displaying the specific epitope for anti-FASN autoantibody was selected from a phage display cyclic random hepta-peptide peptide library and was suggested to be a binder of anti-FASN autoantibody in human sera. The reaction of human sera to autoantigenic mimotope phages usually has been measured by indirect ELISAs, but nonspecific background signals caused by the human sera are a major obstacle hindering specific detection. Although there have been many studies on autoantibody profiling using epitope-display phages, optimized conditions of phage ELISAs for human sera have not been proposed. Therefore, in this study, various conditions within the ELISA procedure, including coating, blocking, and pre-adsorption of human sera, were tested to optimize phage ELISAs for the analysis of human sera.
Materials and methods
Phage K10P6 and K39P15
The phage K10P6 and K39P15 are epitope display phages which were selected from the Ph.D.-C7C Phage Display Peptide Library (New England BioLabs, Ipswich, MA) through specific binding to K10 and K39 monoclonal autoantibodies (mAb) derived from an H-ras12V transgenic HCC model mouse (Heo et al. 2010). For biopanning, 300 ng purified antibody K10 or K39 diluted in 200 μl TBST (50 mM Tris/HCl, 150 mM NaCl and 0.1% Tween 20, pH 7.4) was mixed with 10 μl of the phage library (2 × 1011 phages) and incubated for 20 min at room temperature (RT). Protein L agarose (Pierce, Rockford, IL) was washed with TBST and blocked with 5% (w/v) bovine serum albumin in 0.1 M Na2CO3/NaHCO3 buffer, pH 8.6, for 1 h at 4°C. After washing with TBST four times, 50 μl Protein L agarose (50% v/v aqueous suspension) was mixed with the antibody/phage mixture and incubated for 15 min at RT. After the beads were washed with TBST ten times, bound phages were eluted by the addition of 1 ml 0.2 M glycine/HCl buffer, pH 2.2, and immediately neutralized with 150 μl 1 M Tris/HCl buffer, pH 9.1. Small aliquots of eluted phages were used for phage titration and the remaining phages were amplified for subsequent panning. Three rounds of panning were performed and the concentration of Tween 20 in the washing buffer was increased stepwise (0.1, 0.3, 0.5%). For phage amplification, E. coli strain ER2738 (New England BioLabs) was used as host cell. Pre-cultured ER2738 was inoculated into 20 ml of LB broth and incubated for 1 h at 37°C. Eluted phages were added to the ER2738 culture and further incubated for 4.5 h at 37°C. After centrifugation at 18,700×g for 10 min at 4°C, the supernatant was transferred to a new tube and re-centrifuged again. Supernatant (15 ml) was combined with 3 ml 20% (w/v) polyethylene glycol 8000 containing 2.5 M NaCl (PEG/NaCl), and incubated overnight at 4°C. After centrifugation at 18,700×g for 15 min at 4°C, phage pellets were resuspended in 1 ml TBS, combined with 200 μl PEG/NaCl, and incubated for 1 h at 4°C. The mixture of phage and PEG/NaCl solution was centrifuged for 15 min at 16,000×g at 4°C, and the pellet was resuspended in 150 μl TBS containing 0.02% (w/v) NaN3 and stored at 4°C. After three rounds of panning, 20 phage clones were selected from the titration plate and amplified. Their reactivity against target antibody was measured by ELISA described below and the sequence of peptide insert was determined following the manufacturer’s manual (New England BioLabs). K10P6 or K39P15 is the most reactive phage antigen against K10 or K39 mAb respectively.
Phage ELISA for monoclonal antibody
96-well ELISA plates were coated with 100 μl of 1011 phages/ml of 0.1 M Na2CO3/NaHCO3 buffer, pH 8.6 and incubated overnight at 4°C. Plates were washed three times with TBST and blocked with 5% (w/v) skim milk in TBST for 2 h at RT. After discarding the blocking buffer, 1 μg K10 or K39 mAb diluted in 100 μl blocking buffer was added to each well and incubated for 1 h at RT. Plates were washed six times with TBST before the addition of 100 μl horseradish peroxidase (HRP)-conjugated anti-mouse polyvalent immunoglobulin solution (Sigma), 1:2000 diluted in TBST, and incubated for 1 h at RT. After six washes with TBST, 50 μl 1-Step UltraTMB ELISA substrate solution (Pierce, Rockford, IL) was added and allowed to react for 5 min at RT. Reactions were stopped by the addition of 50 μl 2 M H2SO4. Absorbance values were measured at 450 nm using a microplate reader. All ELISAs were performed in duplicate and assays were repeated to establish reproducibility of results. Bars in results show the mean ELISA signal of duplicate wells and (±) errors bars represent standard deviation.
Hepatocellular carcinoma and normal serum
Sera from HCC patients and normal subjects were obtained from Seoul Catholic Hospital and numbered arbiturarily.
Selection of coating buffer for phage ELISA
The first trial of the phage ELISA for human sera was performed by following conditions described above with human sera as primary antibody and HRP-conjugated anti-human polyvalent immunoglobulin antibody (Abcam, Cambridge, MA) as the secondary antibody, but it resulted in high background signals in all wells. Therefore, reaction conditions for each step in ELISA were optimized.
As phage coating buffer, three buffers of different pHs, TBS (50 mM Tris/HCl buffer containing 150 mM NaCl, pH 7.4), 0.1 M Na2CO3/NaHCO3 buffer (pH 8.6), and 0.1 M NH4HCO3/(NH4)2CO3 buffer (pH 9.6) were tested following the procedures described above. To confirm the coating efficiency of phage on the plates, an HRP-conjugated anti-M13 antibody (Abcam; 1 μg/ml diluted in each blocking buffer) was used as a probe.
Optimization of phage number for coating
To determine a suitable number of phages for coating, K10P6 was serially diluted from 2 × 1012 phages/ml to 1.6 × 1011 phages/ml in each coating buffer. 5% (w/v) skim milk in TBST was used as the blocking buffer and 1 μg K10 mAb diluted in 100 μl of blocking buffer was used as the primary antibody. The remaining procedures were performed as described above.
Optimization of blocking buffer
To select appropriate blocking buffer for human sera, following blocking buffers were tested: 5% (w/v) skim milk (Difco, Detroit, MI) in TBS, 5% (w/v) bovine serum albumin in TBS, 10% (v/v) fetal bovine serum in TBS, and protein-free blocking buffer (PFBB; Pierce). Without coating phage antigens, 96-well plates were blocked with each blocking buffer and incubated for 2 h at RT. After discarding blocking buffer, normal serum or from human hepatocellular carcinoma (1:100 diluted in each blocking buffer) was added to each well and incubated for 1 h at RT. After six washes with TBST, HRP-conjugated anti-human polyvalent immunoglobulin secondary antibody solution (Abcam; 1:2000 diluted in blocking buffer) was added, and plates were incubated for 1 h at RT. Plates were washed and developed with TMB as described above.
Pre-adsorption conditions for human sera
Human sera were pre-adsorbed with ER2738 phage host cell extracts before used in phage ELISA. ER2738 host cells were cultured by inoculating 2 ml pre-cultured ER2738 in 200 ml LB broth, followed by shaking incubation at 37°C for 5 h. Cultured ER2738 cells were harvested by centrifugation at 18,700×g for 15 min, and the cell pellet was resuspended in 10 ml TBS and sonicated for cell disruption at 4°C. Sonicated cell extracts were centrifuged at 18,700×g for 15 min and supernatants were used for adsorption of human sera. HCC serum was added to serially-diluted ER2738 extracts (diluted from 1 mg/ml to 16 μg/ml in PFBB) at a 1:500 ratio, and was incubated for 1 h at RT. To confirm pre-adsorption efficiencies, 96-well plates were coated with 100 μl of 2.5 × 1011 empty phages/ml of TBS (pH 7.4) overnight at 4°C and, after blocking with PFBB, 100 μl pre-adsorbed human sera was added. Empty phages mean phages displaying no peptide insert. To determine suitable temperatures for pre-adsorption, the pre-incubation of human sera with ER2738 cell extracts was performed at RT or 37°C. The remaining ELISA procedures were performed as described above.
Results and discussion
The optimal coating buffer for ELISA can vary depending on the type of surface and can be specific for each protein or antibody to be adsorbed onto the surface. For some proteins or antibodies, coating buffers with a neutral pH of 7.4 is optimal, however, other proteins may require coating buffers with more alkaline pHs around 9.6 to obtain enhanced immobilization. To examine the different variables that affect phage coating, two phages, K10P6 and K39P15, were utilized, which display epitopes with different structural characteristics. K10P6 and K39P15 phages were selected from the Ph.D.-C7C Phage Display Peptide Library displaying disulfide-constrained cyclic hepta-peptides that are useful for mimicking structural epitopes. To determine whether these phages bound to their target antibodies (K10 and K39, respectively) in a structure-dependent manner, ELISA was performed after the disulfide constraints of phage antigens were loosen by dithiothreitol and iodoacetamide treatment and compared with ELISA using original phage antigens displaying cyclic peptides (data not shown). The binding efficiencies of non-reduced K10P6 and K39P15 for their respective antibodies were high (OD450nm of 3.5 and 2.8, respectively). However, binding of K10 mAb to reduced K10P6 was nearly eliminated (OD450nm of 0.05), and binding of K39 mAb to reduced K39P15 was only approximately 15% (OD450nm of 0.4) of what it was to non-reduced phage. These results indicate that binding of K10 mAb to K10P6 is structure-dependent, whereas the binding of K39 to K39P15 is less structure-dependent.
To determine the optimal phage titer for coating, plates were coated with serially-diluted K10P6 in TBS and treated with the K10 antibody (Fig. 1b). The wells that were coated with the greatest phage titer (2 × 1011 phages/well) resulted in the highest OD value (3.8). Wells coated with 2.5 × 1010 phages/well gave an OD450nm of 1.9. Based on these results, the appropriate phage titer for coating was 2.5 × 1010 phages/well.
In indirect ELISA using human serum as primary antibody, non-specific background signals are usually very high compared with ELISA using monoclonal antibodies, likely due to multiple components in human sera. When the detection of low-titer autoantibodies in human sera with phage antigens screened out in our studies was performed following the procedure of phage ELISA for monoclonal antibody, the background signals were too high to discriminate the specific signals. Therefore, background signals must be abolished or at least greatly decreased to detect specific autoantibodies in human sera.
Pre-adsorption of human sera
To determine the optimal temperature for pre-adsorption, human sera were diluted 1:500 in 1 mg/ml ER2738 cell extract and incubated at RT or 37°C for 1 h (Fig. 3b). The average background OD450 value for pre-adsorbed human sera was 0.36 when incubated at RT, but decreased to 0.16 when incubated at 37°C. Therefore, pre-adsorption efficiency was enhanced when it was performed at 37°C. Background signals could also be induced by non-specific interactions of human sera components with coated phages. To examine whether pre-adsorption with empty phage (phage without peptide inserts) decreases background signals, human sera was combined with ER2738 cell extract (1 mg/ml) at a 1:500 ratio with or without 2.5 × 1011 of empty phages (data not shown). However, pre-adsorption with empty phages did not reduce background signals anymore.
Optimization of phage ELISA
This research was supported by grants from the KRIBB Research Initiative Program (KGM2251012) and the Korea Science and Engineering Foundation (KOSEF) (OGM2000912).