Journal of Immunological Methods 406 (2014) 34–42

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Research paper

Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA) J.J. Flanagan a,⁎, A. Arjomandi a, M.L. Delanoy a, E. Du Paty b, P. Galea b, D. Laune b, F. Rieunier b, R.P. Walker a, S.R. Binder a a b

Clinical Diagnostics Group, Bio-Rad Laboratories, Inc., Benicia, CA, USA SysDiag CNRS/Bio-Rad, UMR3145, Montpellier, France

a r t i c l e

i n f o

Article history: Received 17 January 2014 Accepted 18 February 2014 Available online 25 February 2014 Keywords: Pre-haptoglobin-2 Zonulin Monoclonal antibodies Enzyme-linked immunosorbent assay Intestinal permeability Autoimmune

a b s t r a c t Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1 ng/mL to 200 ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2 ng/mL (95% CI: 106.5–335.9 ng/mL) and a median value of 23.9 ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Haptoglobins (HPs) are members of the alpha 2-globulin family of proteins whose main function is to bind free hemoglobin, thereby preventing oxidative damage (Gutteridge, 1987). In addition to their role as hemoglobin scavengers, HPs

⁎ Corresponding author at: Bio-Rad Laboratories, Inc., 5500 E Second St, Benicia, CA 94510. Tel.: +1 510 741 4775. E-mail address: [email protected] (J.J. Flanagan).

http://dx.doi.org/10.1016/j.jim.2014.02.009 0022-1759/© 2014 Elsevier B.V. All rights reserved.

are also acute phase proteins whose levels are elevated in response to infection and inflammation (Quaye, 2008). HPs are produced as a preprotein that undergoes proteolytic cleavage in the endoplasmic reticulum prior to secretion as a mature form composed of two polypeptide chains (alpha and beta) held together covalently by disulfide bonds (Hanley et al., 1983). Due to a gene duplication event in the alpha chain, two alleles of HPs exist in humans with haptoglobin 2 (HP2) containing the larger alpha chain compared to haptoglobin 1 (HP1) (Bowman and Kurosky, 1982). HPs are mainly expressed in the liver, but have also been detected in skin (Li et al., 2005), kidney (D'Armiento

J.J. Flanagan et al. / Journal of Immunological Methods 406 (2014) 34–42

et al., 1997), lung (Abdullah et al., 2009), and adipose tissue (Friedrichs et al., 1995), and depending on phenotype are present in normal human serum at concentrations from 0.38 to 2.27 mg/mL (Langlois and Delanghe, 1996). Recently, the protein zonulin was isolated in a screen to find human analogs of the bacterial toxin Zot (Wang et al., 2000), which is capable of inducing tight junction disassembly (Fasano et al., 1991). Subsequent mass spectrometry analysis identified zonulin as pre-haptoglobin 2 (pre-HP2) (Tripathi et al., 2009). Biochemical characterization showed that pre-HP2 can modulate tight junctions in the gut through activation of EGFR, and that this activity can be inhibited by enzymatic cleavage of the preprotein into mature HP2 (Tripathi et al., 2009). The two strongest inducers of pre-HP2 secretion were found to be enteric bacteria and gliadin, a known trigger of celiac disease (El Asmar et al., 2002; Drago et al., 2006). Therefore it appears that the preprotein form of HP2 plays a role in the modulation of intestinal permeability, where defects can lead to autoimmune disorders due to exposure of the underlying gut associated lymphoid tissue to self and non-self antigens. In fact, using various methods elevated serum levels of pre-HP2 have been reported in patients with celiac disease (Fasano et al., 2000), type I diabetes (T1D) (Sapone et al., 2006), sepsis (Klaus et al., 2013), and obesity (Moreno-Navarrete et al., 2012). The apparent involvement of pre-HP2 in various diseases and its role as a possible regulator of immune tolerance make it desirable to have reagents for its detection and quantification in serum. However, the substantial sequence identity between various forms of haptoglobin, as well as the high concentrations of mature HPs normally found in serum, make it essential that these reagents be very specific. Here we describe the development and characterization of monoclonal antibodies (mAbs) specific to pre-HP2. In addition, we establish their use in an enzyme-linked immunosorbent assay (ELISA) and validate our assay for the measurement of pre-HP2 levels in human serum. 2. Materials and methods 2.1. Haptogloblin proteins Recombinant pre-haptoglobin 1 (pre-HP1; GenBank accession no. NP_001119574) and pre-HP2 (GenBank accession no. NP_005134) were produced in insect cells by Genscript Corp. (New Jersey, USA) using standard techniques. Briefly, protein coding sequences were codon optimized and synthesized with C-terminal hexahistadine tags followed by cloning into an appropriate expression vector. Baculovirus was produced and used to infect 1–5 L cell cultures, and protein was one-step affinity purified. Protein concentrations were determined using the Bradford assay with BSA as a standard. Haptoglobin protein isolated from patients with 1-1 (haptoglobin 1) or 2-2 (haptoglobin 2) genotypes was purchased from Sigma Aldrich (St. Louis, MO) 2.2. Calibrators and controls Since pre-HP2 is known to be proteolytically cleaved in normal serum (Hanley et al., 1983), calibrators for the pre-HP2 ELISA were constructed by spiking recombinant pre-HP2 protein

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into a matrix consisting of protein-depleted human serum (defibrinated, delipidated, immunodepleted, and salt precipitated). Spiked serum was diluted two-fold to create a 7-point calibration curve, and a 4-parameter logistic fit model was used to calculate the concentrations in unknown samples. An assay blank consisting of serum matrix without spiked pre-HP2 was included in the calibration curve. Controls consisted of serum from celiac patients with low, medium, and high levels of pre-HP2. Both calibrators and controls were aliquoted for single use and stored at −80 °C.

2.3. Production of anti-prehaptoglobin 2 monoclonal antibodies Experimental protocols requiring the use of mice were performed according to national regulations and experiments approved by the regional ethics committee of Languedoc-Roussillon, France (authorization #CEEA-LR0915-005). Ten micrograms of recombinant human pre-HP2 expressed in insect cells with a C-terminal hexahistidine tag (kindly provided by A. Fasano) were emulsified in equal ratio with Sigma Adjuvant System or Alum adjuvant (Thermo Scientific Pierce, Rockford, IL). Four 8-week-old CD1 mice (Charles River) were immunized by intraperitoneal injections. Two booster injections were administrated at the same doses at 2-week intervals. Mice were bled 10 days after each boost and serology was controlled by indirect ELISA to select the best responding mouse. A pre-fusion boost was administrated 4 days before fusion. Sp2/0Ag14 myeloma cell line (ATCC CRL 1581) was fused with splenocytes from selected immunized mouse according to standard protocols and fusion product was plated in culture microplates for 15 days before primary screening. For clone selection, either indirect or sandwich ELISA were used all along the process: from primary screening, confirmation to sub-cloning screenings. Antigens used were recombinant pre-HP2 as specific antigen and recombinant pre-HP1, purified mature haptoglobin 1 and 2 (Sigma Aldrich) and a non-specific his-tagged protein as control antigens. For sandwich ELISA format, microplates were first coated with a goat anti-mouse IgG antibody (Jackson Immuno Research, West Grove, PA) before adding hybridoma supernatants; antigen binding was detected with a rabbit anti-pan haptoglobin polyclonal antibody (internal source) followed by the addition of a horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody (Biosource, Grand Island, NY). For the indirect ELISA, antibody binding to adsorbed antigens was detected with a HRP-conjugated goat anti-mouse IgG antibody (Sigma). For the two formats, tetramethylbenzidine (TMB) was used as substrate, and after stopping the reaction with H2SO4, optical density (OD) was read at 450 nm in an Infinite F200 ELISA reader (Tecan, San Jose, CA). After primary screening, antibody-producing hybridomas were twice sub-cloned and then frozen in liquid nitrogen. Monoclonal antibodies were produced in vitro by collecting concentrated supernatants. Purifications were done by affinity chromatography on Protein A Sepharose (GE Healthcare, Piscataway, NJ). The mAbs were isotyped with a mouse isotyping test kit (Roche, Indianapolis, IN) according to the manufacturer's recommendations.

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2.4. Epitope mapping A total of 392 overlapping pentadecapeptides frame-shifted by one residue covering the entire amino acid sequence of human pre-HP2 were prepared by automated chemical synthesis on a cellulose membrane (Intavis) as previously described (Laune et al., 2002). Free cysteines were replaced with nonreactive acetamidomethyl cysteines. Purified mAbs were incubated on the membrane, and then antibody binding was detected by using an alkalinephosphatase conjugated goat anti-mouse IgG (Sigma Aldrich). For staining 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma Aldrich) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, Sigma Aldrich) were used as substrates. A blue precipitate was observed on peptides which the antibody was bound to. To allow the re-use of the membrane, it was sequentially treated with dimethylformamide, then 1% SDS, 0.1% 2-mercaptoethanol in 8 M urea, then 50% ethanol, 10% acetic acid and, finally, 100% ethanol in order to remove the precipitated dye and molecules bound to the peptides. 2.5. Surface plasmon resonance Immobilization of the monoclonal antibodies was performed in the vertical orientation of the ProteOn XPR36 system (Bio-Rad) using a flow rate of 30 μl/min at 25 °C on a GLC chip. Four channels were activated for 3 min (90 μl) using a mixture of 20 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride carbodiimide(EDC) and 5 mM sulfo-Nhydroxysulfosuccinimide (sulfo-NHS). This was followed by an immediate injection of 50 μl of 10 μg/ml monoclonal antibody in 10 mM acetate buffer (pH 4.5). Finally, 150 μl of 1 M ethanolamine-HCl (pH 8.5) was injected to deactivate any remaining activated carboxyl groups. This resulted in the immobilization of approximately 1200 to 2000 response units (RU) of the different antibodies in the channels. All the binding measurements were performed with PBST as the continuous running buffer at 25 °C. The different forms of haptoglobin proteins were injected using a flow rate of 50 μl/min for 3 min (150 μl). For screening experiments, single concentrations of 100 nM of analyte were injected in channels 1 to 4, and 10 nM of analyte was injected in the fifth channel as a control. Running buffer was injected in the sixth channel. For binding kinetic experiments, five different concentrations of analyte (1000 nM serially diluted 3-fold to 4 nM for pHP2; 300 nM to 3 nM for pHP1; 100 nM to 1 nM for HP1 and HP2) were injected in channels 1 to 5. Running buffer was injected in the sixth channel. All binding sensorgrams were collected, processed and analyzed using the integrated ProteOn Manager software (Bio-Rad). Binding curves were fitted using the Langmuir model. Each interaction was fitted using grouped ka (association rate constant), kd (dissociation rate constant), and a local Rmax (RU when all of the ligand binding sites are saturated by the analyte). Constant affinity KD was calculated from ka and kd for each interaction. 2.6. Pre-haptoglobin 2 ELISA Pre-HP2 capture antibody (clone 13D11-G7-B10) was diluted to 2 μg/mL in PBS (10 mM Sodium Phosphate, 150 mM NaCl,

pH 7.8) and added to a 96-well plate (100 μL/well; Maxisorp, Nunc, Roskilde, Denmark). After an overnight incubation at 4 °C, antibody solution was removed and wells were blocked with blocking buffer (PBS + 3% BSA) for 1 h at room temperature (RT). Serum samples and calibrators diluted in sample diluent (PBS + 0.1% Tween-20 + 0.1%BSA + 0.3 μg/mL mouse IgG (Meridian Life Science, Memphis, TN)) were then added to the plate (100 μL/well) after removal of the blocking buffer and incubated for 2 h at RT. The plate was then washed three times with PBS + 0.1% Tween-20 (PBST) using an automated plate washer (BioPlex Pro II Wash Station, Bio-Rad, Hercules, CA). Next, biotinylated detection antibody (clone 11G3-G9-G8) diluted to 2 μg/mL in conjugate diluent (sample diluent without mouse IgG) was added (100 μL/well) and incubated for 1 h at RT. The plate was again washed three times in PBST, and streptavidin-HRP (Thermo Fisher) diluted 1:10,000 in conjugate diluent was added (100 μL/well) and incubated for 1 h at RT. After washing, Ultra TMB ELISA substrate (Thermo Fisher) was added to the plate (100 μL/well) and color was allowed to develop for 15 min at RT. Color development was stopped using 10% v/v sulfuric acid (Ricca Chemical, Arlington, TX) and the optical density (OD) was read at 450 nm using a Benchmark Plus plate reader (Bio-Rad). Data was analyzed using Microplate Manager software (Bio-Rad). 2.7. Western blotting For native Western blotting, 2 μg of protein was run on a gradient acrylamide gel (AnyKD TGX, Bio-Rad) in the absence of SDS. For reducing conditions, β-mercaptoethanol was added to the sample prior to loading. After separation, protein was transferred to a PVDF membrane (TransBlot Turbo, Bio-Rad) and blocked in TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 8.0) with 5% non-fat dry milk. Primary antibodies were diluted to 2 μg/mL in blocking solution and incubated with the membrane for 1 h at RT. The membrane was washed three times for 5 min in TBST. Goat anti-mouse HRP secondary antibody (Jackson Immuno Research) was diluted 1/10,000 in blocking solution and incubated with the membrane for 1 h hour at RT. The membrane was again washed with TBST then developed using a chromogenic substrate (Opti 4-CN; Bio-Rad). Images were obtained using a Gel Doc EZ imager (Bio-Rad). 2.8. ELISA validation Parallelism was determined by comparing the linear regression slopes of patient serum with elevated levels of endogenous pre-HP2 serially diluted in calibrator matrix to the slope of recombinant pre-HP2-based calibrators. In order to linearize the data for regression analysis, raw ODs were converted first to proportions by dividing all data in a dilution series by the max OD of that series + 0.1 (3.8 in all cases). Then a logit transformation was applied to the converted data according to y′ = ln[y / (1 − y)]. A line was fitted to the linearized data, and a pair-wise comparison between the slopes of each fitted line was then performed by a Tukey's HSD test. Statistical analysis was performed using the software JMP Pro 10 (SAS, Cary, NC). Linearity was assessed according to Clinical Laboratory Standards Institute (CLSI) guideline EP6-A. Briefly, OD values from 2-fold serial dilutions of celiac patient serum or calibrators

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were plotted against the inverse of the dilution factor. Samples with OD values above the linear range of the plate reader (OD = 3.0) were removed from further analysis. Both firstand third-order polynomial regression models were then fitted to the data, and a t-test was applied to the non-linear coefficients of the third-order model using JMP Pro 10 software (SAS). A significant t-test (p b 0.05) indicated non-linearity of the data. The degree of non-linearity at each dilution factor was then determined by %Difference = (linear predicted OD / non-linear predicted OD)-1. OD values were then converted to concentrations by back-calculating against a standard curve, and the %Difference values were plotted against concentration to determine the lowest value for which the assay remained linear. To determine intra-assay precision, control serum samples were run in 20 replicates on a single plate. Inter-assay precision was performed by running control samples in triplicate over 5 days. Each assay run included calibrators in duplicate. Pre-HP2 concentrations were calculated for each sample from the calibrator curve, and the coefficients of variation (CVs) were determined. Recovery was assessed by spiking recombinant pre-HP2 protein at three levels (6.25, 25, and 100 ng/mL) into depleted serum. Percent recovery at each spike level was calculated as the ratio of observed concentration versus expected. Recovery between 80% and 120% was considered acceptable. The limit of detection (LoD) was determined using methods similar to those outlined in Clinical Laboratory Standards Institute (CLSI) guideline EP17-A. Briefly, OD values of the inter-assay precision study calibrators were converted first to concentrations by back-calculating against the calibrator curve. Then using the back-calculated calibrator concentrations (10 data points per calibrator level), the limit of blank (LoB) was defined according to the following equation: LoB = μB + 1.645σB, where μB is the mean calculated concentration of the blank and σB is the standard deviation of the blank concentrations. Finally, the LoD was calculated according to the following equation: LoD = (LoB + (1.645 × intercept)) / (1 − (1.645 × slope)), where slope is the regression line slope of a standard deviation versus calibrator concentration plot, and intercept is the y-intercept of the same plot.

2.9. Evaluation of healthy blood donors Sera from 76 healthy blood donors were purchased from multiple commercial vendors and aliquoted for storage at − 80 °C prior to use. Serum was diluted 1/2 in sample diluent and run in duplicate in the pre-HP2 ELISA as described above. For each plate, calibrators were also diluted 1:2 and run in duplicate. Concentrations for each sample were determined by back-calculating against the calibrator curve and multiplying the result by the dilution factor. Samples with ODs exceeding the value of the highest calibrator were 2-fold serially diluted and re-run in the assay. The lowest dilution factor giving an OD value within the calibrator curve was used to determine the concentration of pre-HP2. Samples with concentrations below the assay LoD were assigned a value of 0 for calculating statistics. Normality of the distribution of pre-HP2 concentrations was assessed using a Shapiro–Wilk W test. All statistical analyses were performed with JMP 10 Pro (SAS).

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3. Results 3.1. Development of monoclonal antibodies to pre-haptoglobin 2 Since pre-HP2 has perfect sequence identity with its processed, mature form and is only differentiated from the related pre-haptoglobin 1 by a 59 amino acid duplication region, we selected two immunization strategies to help ensure the development of a pre-HP2 specific antibody. First, sequences unique to pre-HP2 within the alpha duplication region were identified and synthesized as peptides for immunization. Second, to ensure that antibodies to conformational epitopes unique to pre-HP2 could also be produced, recombinant protein comprising the full-length coding sequence of pre-HP2 was generated and used for immunization. Primary screening by direct or sandwich ELISA (see Materials and methods) of 5434 clones from three fusions resulted in 10 subclones (0.18%) with reactivity to pre-HP2, all of which were derived from immunizations using full-length protein. Further subcloning and characterization by sandwich ELISA with all four haptoglobin proteins (pre-HP1, pre-HP2, HP1, HP2) resulted in 16 candidate antibodies with various specificities to different forms of haptoglobin. 3.2. Development of a pre-haptoglobin ELISA and antibody characterization To select antibodies suitable for a pre-HP2 specific ELISA pair-wise screening of the 16 selected clones was performed in a sandwich ELISA format for specificity against pre-HP1 and normal serum levels of mature HP1 and HP2. One pair (comprising clones 13D11-G7-B10 for capture and 11G3-G9-G8 for detection) specifically bound pre-HP2 versus pre-HP1 in the ng/mL range and did not cross-react with HP1 or HP2 (data not shown). This antibody pair was then tested for its ability to selectively detect low levels of pre-HP2 in the presence of excess pre-HP1 and normal serum levels of HP1 and HP2. The pair showed 95–121% recovery of pre-HP2 depending on which species of haptoglobin was co-incubated (Fig. 1). Further optimization of capture antibody coating concentrations, detection antibody concentrations, sample dilutions, and blockers for non-specific signals were performed (data not shown). To characterize the binding kinetics of the two monoclonal antibodies both were evaluated by surface plasmon resonance (SPR). Each clone was covalently coated to a sensor chip and pre-HP1, pre-HP2, HP1-1, or HP2-2 protein was flowed over the surface while the binding was monitored (Fig. 2A). Clone 13D11-G7-B10 showed specificity for pre-HP2 with binding affinity in the low nM range, while 11G3-G9-G8 bound preHP1 and pre-HP2 both with low nM affinities (Fig. 2B). Neither clone recognized the mature forms of haptoglobin. A third clone (15F12) that showed reactivity to all forms of haptoglobin by direct ELISA was also included as a positive control for the assay. Epitope mapping was performed to determine the specific amino acid sequence(s) recognized by the two monoclonal antibodies. The complete pre-HP2 protein with signal sequence was synthesized as overlapping peptides and spotted on a membrane for probing with each antibody (Fig. 3A). 13D11G7-B10 reacted with specific peptides having the core sequence

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Both antibodies were also tested for the ability to specifically recognize pre-HP2 protein by Western blot. Recombinant pre-HP1 or pre-HP2 and native HP1-1 and HP2-2 were run on a non-denaturing gel with or without reducing agent, and probed with 13D11-G7-B10 or 11G3-G9-B10 after transfer to a membrane. Under non-reducing conditions, both antibodies detected pre-HP1 and pre-HP2, but not the mature forms of haptoglobin. When the proteins were reduced, 11G3-G9-B10 failed to produce an immunoreactive band while 13D11-G7-B10 recognized all proteins, presumably due to the shared linear epitope (Fig. 3D). Fig. 1. ELISA interference from pre-HPl and mature HPs. Recombinant pre-HP2 protein (0.1 μg/mL) was spiked alone into serum based matrix or in combination with pre-HPl (2 μg/mL), HP1-1 (1500 μg/mL), or HP2-2 (1500 μg/mL). Samples were then assayed in the ELISA. Percentages above each bar represent the amount of pre-HP2 recovered in the presence of interfering protein versus alone. Error bars represent intra-assay standard deviations from samples run in duplicate.

GYVEHSVRY, where glycine and tyrosine appear important but not essential for recognition (Fig. 3B and C). This sequence is present as part of the alpha duplication region in pre-HP2 and HP2 and singly in pre-HP1 and HP1. The 11G3-G9-G8 antibody failed to recognize any linear peptides (data not shown).

3.3. Validation of the pre-haptoglobin 2 ELISA To study parallelism 2-fold serial dilutions of serum from two celiac patients with elevated endogenous pre-HP2 levels were compared to a calibration curve of recombinant pre-HP2 in modified human serum (Fig. 4A). After transformation of the data, linear regression curves showed a fit of R2 N 0.96 for all models, and pair-wise analysis of the regression slopes using a Tukey's HSD test showed no significant difference (p b 0.05) demonstrating parallelism between the calibrators and endogenous pre-HP2 (Fig. 4B). Linearity was then assessed at each dilution by comparing the predicted OD values derived from a

Fig. 2. PreHP2 mAb characterization. (A) Binding kinetics of the 13D11-G7-B1O (capture) antibody and 11G3-G9-G8 (detection) antibody were determined by surface plasmon resonance. Also included were an unrelated antibody and pan-haptoglobin antibody (15F12) as negative and positive controls, respectively. The type and concentration of protein applied to the antibody coated sensor chip is represented as colored lines in each graph. One response unit (RU) equals l pg/mm2 of molecules on the chip surface. (B) KD calculations for each antibody-protein pair. Dashed line indicates no binding.

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Fig. 3. Pre-HP2 mAb characterization. (A) Amino acid sequence of pre-HP2. Signal sequence is in italics. Alpha duplication region is in bold. Underlined sequence indicates core epitope of 13D11-G7-B10 antibody. (B) Dot blot of 13D11-G7-B1O epitope mapping. Purple circles indicate peptide that positively reacts with the antibody. (C) Peptide sequences from (B) that react with the 13D11- G7-B1O antibody. Numbers on left denote starting amino acid position of each peptide within pre-HP2 sequence. Relative dot blot signal intensity for each peptide is indicated on the right. (D) Non-denaturing Western blots probed with 11G3-G9-B1O or 13D11-G7-B1O antibodies. As indicated pre-HPl, HP1-1, pre-HP2, or HP2-2 purified proteins were run under non-reducing or reducing conditions. Marker sizes given in kDa.

linear fit model to those calculated from a polynomial fit. OD values corresponding to concentrations from 9.0 to 208 ng/mL differed ≤21% between the models, and this was used to define the working range of the assay. To determine assay precision, intra- and inter-assay runs were performed using controls consisting of serum from four celiac patients with undiluted pre-HP2 levels ranging from 0.21 to 2.43 μg/mL. Inter-assay precision CVs ranged from 5.3% to 7.7%, and intra-assay precision CVs ranged from 3.3% to 7.9%. Due to proteolytic cleavage of recombinant pre-HP2 protein in normal human serum, protein recovery studies were conducted by measuring spiked pre-HP2 at levels ranging from 6.25 to 100 ng/mL in depleted human serum. Recovery from modified serum was determined to be 98% to 110% of that expected. The lower limit of detection (LLOD) for the assay was defined as 1.67 ng/mL. The ELISA performance data is summarized in Table 1. 3.4. Pre-HP2 levels in normal human serum Seventy-six healthy blood donors were tested for pre-HP2 levels using the ELISA (Fig. 5A). Concentrations for 7 (9%) of the patients fell below the limit of detection for the assay, and 13 (17%) samples with high signals required further dilutions to fall within the calibrator curve. The mean pre-HP2 serum level was found to be 221.2 ng/mL (95% CI: 106.5–335.9 ng/mL) with a maximum value of 3165.6 ng/mL. The distribution of pre-HP2

concentrations in the cohort was determined to be non-Gaussian (p b 0.01) (Fig. 5B) with a median value of 23.9 ng/mL. 4. Discussion Here we describe the development of monoclonal antibodies capable of detecting pre-HP2 with high specificity and reproducibility when used in a sandwich ELISA format. Due to the near identical sequences of the various forms of haptoglobin, we chose to use both peptide and full-length protein immunization strategies in order to increase our chances of obtaining pre-HP2 antibodies. The linear peptides failed to produce specific antibodies with suitable binding affinities indicating that a conformational epitope may be required. Indeed, immunization with the full-length pre-HP2 protein yielded antibodies with various specificities for pre-HP2; however, this was from less than 0.2% of the clones screened demonstrating the difficulty in developing these antibodies. Pair-wise screening and further analysis by SPR identified a suitable ELISA antibody pair consisting of a pre-HP2 specific clone for capture (13D11-G7-B10) and a pre-HP specific clone for detection (11G3-G9-B10). Epitope mapping using overlapping peptides covering the full pre-HP2 amino acid sequence failed to identify a reactive sequence for 11G3-G9-B10, confirming that its epitope is conformational. Detection of natively folded, but not reduced or denatured, recombinant

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J.J. Flanagan et al. / Journal of Immunological Methods 406 (2014) 34–42 Table 1 Assay performance characteristics. Inter-assay precision was determined from five separate runs using four OC samples with pre-HP2 levels ranging from 5.1 ng/mL to 60.9 ng/mL. Intra-assay precision was determined from running 20 replicates of the same OC samples on a single day. Recovery from a serum-based matrix was tested at three levels and is calculated as the ratio of observed versus expected concentration. Parameters

Performance

Inter-assay precision (%CV) Intra-assay precision (%CV) Recovery (%) Working range (ng/mL) Detection limit (ng/mL)

5.3–7.7 3.3–7.9 98–110 9–200 1.7

pre-HP2 by Western blot confirmed that proper folding of the protein through cysteine bonds is required for recognition by the 11G3-G9-B10 antibody. For the 13D11-G7-B10

Fig. 4. Determination of parallelism and linearity. (A) ODs of 2-fold serial dilutions from two patient sera with elevated levels of pre-HP2 were compared to a calibrator curve consisting of recombinant pre-HP2 spiked into protein depleted serum. (B) Linear regression was performed on logit transformed raw OD values of serially diluted calibrators and patient sera from (A). Slopes of the regression lines were compared pair-wise using a Tukey's HSD test with no significant differences found. AU: arbitrary units derived from data transformation. (C) Ratio of pre-HP2 concentration predicated by linear modeling versus a non-linear, best-fit model plotted at known concentrations. Differences between models were b21% at pre-HP2 concentrations above 9.0 ng/mL for the endogenous analyte and above 3.1 ng/mL for the recombinant protein used in the calibrators.

Fig. 5. Pre-HP2 levels in healthy blood donors. (A) Pre-HP2 concentration was determined in 76 blood donor samples using the ELISA. Mean serum level is 221.2 ng/mL (95% Cl: 106.5–335.9 ng/mL) with a range of b1.7 ng/mL (limit of detection) to 3165.6 ng/mL. Top and bottom of box represent the 75th and 25th quantiles, respectively. Horizontal line within the box represents the median value of 23.9 ng/mL. (B) Histogram showing the non-Gaussian distribution of pre-HP2 levels among all patients.

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antibody a core linear epitope of nine amino acids in the alpha duplication region of pre-HP2 was defined. This sequence is present in all forms of haptoglobin, and the 13D11 antibody was able to detect pre-HP1, HP1-1, and HP2-2 by Western blot under reducing conditions. However, under native conditions only pre-HP2 was strongly bound, which is consistent with the specificity of 13D11-G7-B10 for pre-HP2 as seen by SPR. It is likely that the linear epitope is shielded when the proteins are properly folded, but may still be critical in forming a conformational epitope on pre-HP2 that is recognized with good affinity by 13D11-G7-B10. Weak binding of pre-HP1 by 13D11-G7-B10 in Western blotting but not by SPR is likely due to the high concentration of protein present in the membrane. While the endogenous levels of pre-HP1 have not been reported, it is anticipated that protein expression would be similar to that of pre-HP2 since they share the same regulatory elements, and therefore concentrations in serum should be comparable. In fact, mature HP serum concentrations in HP1-1 and HP2-2 individuals is similar, indicating similar levels of the preproteins (Langlois and Delanghe, 1996). Our testing of healthy blood donors indicates that the upper levels of pre-HP2 in normal serum are 3–4 μg/mL, which is similar to the amount of pre-HP1 tested for cross-reactivity by SPR. Therefore, we believe that the SPR data most closely resembles physiological concentrations of elevated levels of pre-HP1 and pre-HP2, and based on these results that the 13D11-G7-B10 is specific for recognizing endogenous levels of pre-HP2 in patient serum. To further investigate the specificity of the antibodies as a pair, we tested the recovery of pre-HP2 from a serum-based matrix in the presence of pre-HP1, HP1-1 or HP2-2 by ELISA. Depending on phenotype mature haptoglobins are normally present in human serum at 0.38–2.27 mg/mL and levels may increase 3–8 folds in response to inflammation (Langlois and Delanghe, 1996), which makes them a potential source of interference due to possible cross-reactivity with the mAbs used in the ELISA. Analysis of recovery from co-incubated samples revealed that detection of pre-HP2 in the presence of physiological levels of mature haptoglobins was not significantly affected indicating that mature haptoglobins are not competing for binding in the assay. While a 20% increase in signal was detected with co-incubation of HP2-2, commercial preparations of this purified protein have been shown to be contaminated with pre-HP2 (Tripathi et al., 2009), which would result in the additive signal seen here. Co-incubation with high concentrations of pre-HP1 also did not negatively affect pre-HP2 detection. Taken together, these results indicate that the ELISA is specific for pre-HP2 and the signal is not affected by the presence of various forms of haptoglobins at elevated levels. Our ELISA validation experiments (summarized in Table 1) showed excellent assay precision with a working range greater than one order of magnitude. In regard to recovery, it has been previously reported that recombinant pre-HP2 incubated in serum is rapidly cleaved, presumably due to the presence of a circulating protease (Hanley et al., 1983). To overcome this, we generated our calibrators in a sample matrix consisting of human serum that has been defibrinated, delipidated, immunodepleted, and salt precipitated. Parallelism could be shown between our calibrators, which are composed of recombinant pre-HP2, and an authentic sample with elevated levels of endogenous pre-HP2. Testing of 76 healthy blood donors using the validated ELISA showed a mean pre-HP2 level of 221.2 ng/mL and a median of

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22.9 ng/mL with a non-Gaussian distribution. While a more sensitive antibody pair may reveal the presence of pre-HP2 at concentrations below the current detection limit of our assay, it is also possible that pre-HP2 is absent from serum except in the case of underlying systemic disease or an acute reaction. For example, under normal physiological conditions a circulating protease could act as a sink for pre-HP2 that secretes into the serum without being processed into a mature form in the cell; however, during upregulation of pre-HP2 expression (e.g. due to exposure to bacteria, gluten, or an acute protein response) the amount of pre-HP2 in circulation exceeds that of the protease, allowing for measurable levels to be present in the serum. It is also possible that secreted pre-HP2 is typically bound up in a larger protein complex, potentially those within the complement system as HPs evolved from a complement-associated protein (Kurosky et al., 1980). Again, high expression levels would overwhelm binding partners that normally mask detection and result in measurable levels by ELISA. In addition to samples with low to undetectable levels of pre-HP2, 17% of the healthy control sera tested showed elevated pre-HP2 levels presumably in the absence of disease. While elevation of pre-HP2 has been reported in established disease, increased serum concentrations can also be detected prior to symptoms. For example, elevated pre-HP2 levels were found before onset of T1D and in relatives of T1D patients (Sapone et al., 2006). Therefore pre-HP2 expression may be an early marker of asymptomatic intestinal barrier dysfunction that eventually leads to illness. Due to the variety of diseases that have been recently linked to dysregulation of the intestinal barrier, and the recent discovery of pre-HP2 as a modulator of tight junctions, a specific ELISA for pre-HP2 has become an important investigational tool. While pre-HP2 ELISAs based on cross-reacting bacterial antibodies or polyclonal antibodies in a competitive format have been described (Sapone et al., 2006; MorenoNavarrete et al., 2012; Klaus et al., 2013), our assay has the advantages of utilizing monoclonal antibodies directed against human pre-HP2 in a sandwich format and of being highly validated, especially for specificity against other forms of haptoglobin. Therefore, we believe this newly developed ELISA will establish a more sensitive and specific means of detecting pre-HP2 in human serum for research, diagnostic, and therapy monitoring purposes. References Abdullah, M., Schultz, H., Kähler, D., Branscheid, D., Dalhoff, K., Zabel, P., Vollmer, E., Goldmann, T., 2009. Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues. Pathol. Res. Pract. 205, 639. Bowman, B.H., Kurosky, A., 1982. Haptoglobin: the evolutionary product of duplication, unequal crossing over, and point mutation. Adv. Hum. Genet. 12 (189–261), 453. D'Armiento, J., Dalal, S.S., Chada, K., 1997. Tissue, temporal and inducible expression pattern of haptoglobin in mice. Gene 195, 19. Drago, S., El Asmar, R., Di Pierro, M., Grazia Clemente, M., Tripathi, A., Sapone, A., Thakar, M., Iacono, G., Carroccio, A., D'Agate, C., Not, T., Zampini, L., Catassi, C., Fasano, A., 2006. Gliadin, zonulin and gut permeability: effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scand. J. Gastroenterol. 41, 408. El Asmar, R., Panigrahi, P., Bamford, P., Berti, I., Not, T., Coppa, G.V., Catassi, C., Fasano, A., 2002. Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. Gastroenterology 123, 1607. Fasano, A., Baudry, B., Pumplin, D.W., Wasserman, S.S., Tall, B.D., Ketley, J.M., Kaper, J.B., 1991. Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions. Proc. Natl. Acad. Sci. U. S. A. 88, 5242.

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