Dual-monoclonal, sandwich immunoassay specific for glucose-dependent insulinotropic [peptide.sub.1-42], the active form of the incretin hormone.
[GIP.sub.1-42] plays a critical role in the incretin effect (1-3). It is believed that [GIP.sub.1-42] secretion by K cells of the small intestine in response to an oral glucose load may partly account for the increased insulin secretion observed compared to what occurs when a comparable glucose load is delivered intravenously (9). There have also been suggestions that [GIP.sub.1-42] may improve [beta]-cell survival and regulate [beta]-cell viability and antiapoptotic effects (10-14). Because of the recognized crucial role of GIP in augmenting glucose-induced insulin secretion, there have been efforts to develop robust immunoassays to measure [GIP.sub.1-42]. Unfortunately, because [GIP.sub.1-42] differs from the inactive form of the hormone, [GIP.sub.3-42], by the loss of only 2 amino acids at its N-terminus, it has proved difficult to develop monoclonal antibodies selective for [GIP.sub.1-42]. As a result, existing ELISAs measure both [GIP.sub.1-42] and [GIP.sub.3-42] and thus give an indication of total GIP concentrations (8, 15, 16), whereas a previously described RIA for GIP 1-42 used a polyclonal antibody (8).
LC-MS assays specific for [GIP.sub.1-42] have been described, but these assays have relied on either direct LC-MS analyses or the use of immunoprecipitation of total GIP followed by LC-MS quantification of [GIP.sub.1-42] (8, 15, 16). Although these assays have been shown to be both accurate and precise, their complexity and expense, and the high level of operator expertise required each time the assay is performed, have prevented their implementation into most laboratories. In addition, low concentrations of [GIP.sub.1-42] observed during the fasting state ([less than or equal to]20 ng/L) have made quantification a challenge for these assays (8, 15, 16). To develop a highly specific and robust immunoassay for active GIP, we generated a monoclonal antibody that specifically targeted the N-terminus of GIP 1-42. Afterward, in vitro-directed evolution was applied to improve the affinity of the antibody while maintaining specificity for [GIP.sub.1-42]. We then paired this monoclonal antibody with an anti-total GIP monoclonal antibody to develop a dual monoclonal sandwich ELISA that specifically measures [GIP.sub.1-42].
Materials and Methods
SAMPLES AND REAGENTS
Blood samples from 16 healthy volunteers were collected in P800 tubes (Becton Dickinson) containing EDTA, aprotinin, and DPP4 inhibitor. All volunteers were enrolled in the Eli Lilly volunteer blood donor program and gave informed consent. The degradation of GIP by DPP4 is not as rapid as for GLP-1, but it is still appreciable; hence the inclusion of the DPP4 inhibitor. After plasma was separated from cells, plasma samples were stored at -70 [degrees]C before analysis of [GIP.sub.1-42] concentrations. Blood samples were collected from volunteers under fasting conditions and at specific time points after they consumed a mixed meal challenge consisting of approximately 400 fat calories, 400 carbohydrate calories, and 100 protein calories. Synthesized GIP 1-42 and [GIP.sub.3-42] peptides were purchased from Ana Spec. Anti-total GIP monoclonal antibody was purchased from Millipore. The exact binding region of this antibody is unknown, and it recognizes both [GIP.sub.1-42] and [GIP.sub.3-42]. We measured total GIP concentrations using a sandwich ELISA kit (Millipore). This total GIP ELISA uses the same anti-total GIP monoclonal antibody described above as the capture antibody and rabbit polyclonal anti-total GIP antibody as the detection antibody. The manufacturer states that this kit is specific for GIP and does not recognize GLP-1, GLP-2, or glucagon.
Anti-[GIP.sub.1-42] antibody generation and affinity maturation. The primary amino acid sequence of [GIP.sub.1-42] is YAEG TFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKH NITQ and differs from that of [GIP.sub.3-42] only by tyrosine and alanine residues at the N-terminus. To generate a monoclonal antibodyspecific for this N-terminus, we obtained anti-GIP hybridoma clones after fusing splenocytes from mice immunized with a peptide corresponding to the first 6 residues of human [GIP.sub.1-42] conjugated to keyhole limpet hemocyanin. Next, IgGs from individual clones were screened for reactivity to both [GIP.sub.1-42] and [GIP.sub.3-42]. An anti-[GIP.sub.1-42]-specific antibody was selected and optimized by using yeast cell surface display (17).We extracted mRNA and amplified the Ig heavy chain variable region (VH) and Ig light chain variable region (VL) genes from cDNA using a mouse Ig primer set, and determined sequences after insertion into a TA TOPO (vaccinia DNA topoisomerase I) cloning vector (Invitrogen). The wild-type single-chain fragment variable (scFv) gene, in which the antigen-specific VH and VL domains are linked into a single polypeptide, was created by single overlap extension-PCR and ligated into a display vector after digestion with SfiI restriction enzyme. Spiked mutagenesis with degenerate oligonucleotides was used for saturation mutagenesis of the VH and VL complementarity-determining regions (CDR). Individual CDR libraries were constructed by recombining the mutated scFv DNA pool into display vector digested with SfiI after cotransformation into EBY100 yeast (a kind gift from Dr. Dane Wittrup, Massachusetts Institute of Technology) by using lithium acetate as described (18,19) and propagated in synthetic dextrose casamino acid media at 30 [degrees]C with shaking. The scFv expression was induced by transferring midlog growth cells into synthetic galactose casamino acid media (2% galactose) and grown at 20 [degrees]C for 12-24 h with shaking. An overrepresentation of each CDR library or sort output from a previous round of selection was incubated with [GIP.sub.1-42]-biotin peptide (CPC Scientific) and anti-V5 monoclonal antibody (1 mg/L) (Invitrogen) in selection buffer [PBS (157 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L [Na.sub.2]HP[O.sub.4], 1.8 mmol/L K[H.sub.2] P[O.sub.4]), pH 7.4, 0.5% BSA] at 25 [degrees]C for 30 min before transfer to ice and washing with cold selection buffer. Bound antigen was detected by using streptavidin-Rphycoerythrin (Invitrogen) at a 1:200 dilution, and binding was normalized to the amount of scFv expression by using Alexa Fluor 488 goat antimouse antibody (Invitrogen) at a 1:100 dilution. Clones of interest were enriched using fluorescence-assisted cell sorting on a FACSAria cell sorter (BD Biosciences). Individual clones from CDR libraries with improved binding were sequenced.
A combinatorial library combining the CDR regions of clones from the individual libraries with improved binding was constructed by single overlap extension-PCR, and subjected to further rounds of selection with increased stringency. Individual combinatorial clones were sequenced, and the relative binding affinity determined after antigen titration (20, 21). The resulting VH and VL genes were ligated into separate murine expression vectors, respectively, to create a final optimized variant, which was purified by protein A after large-scale transient transfection using HEK (human embryonic kidney) 293 cells.
Kinetic analysis and binding specificity of the anti-[GIP.sub.1-42] specific antibody were determined by use of a Biacore T100 instrument. A Series S Sensor Chip CM5 (Biacore) with approximately 6000 response units of goat antimouse k capture antibody (Southern Biotech) covalently attached was used to capture approximately 1500 response units of IgG diluted to 10 mg/L in running buffer (HEPES buffered saline containing 3 mmol/L EDTA and 0.05% Tween, Biacore). Increasing concentrations of [GIP.sub.1-42] were injected for 5 min at a flow of 30 [micro]L/min followed by a 10-min dissociation phase. The surfaces were regenerated following each dissociation phase with two 15-[micro]L injections of glycine-HCl, pH 1.5, at a flow rate of 100 [micro]L/min. Kinetic constants were determined using the T100 Evaluation software. Specificity for [GIP.sub.1-42] was tested by injections of [GIP.sub.3-42] peptide under the described assay conditions.
After the final purification process, a dual monoclonal [GIP.sub.1-42] MesoScale Discovery (MSD) ELISA was constructed using the N-terminal-specific anti [GIP.sub.1-42] monoclonal antibody and the anti-total GIP monoclonal antibody. Standard MSD 96-well plates were incubated for 1 h with 50 [micro]L of anti-total GIP antibody (5 mg/L). Afterward, wells were aspirated and washed 3 times with TBST (Tris buffered saline containing 10 mmol/L Tris pH 7.40, 150 mmol/L NaCl with 1 mL Tween 20/L). Wells were blocked with 200 [micro]L of TBS-casein (Thermo Fisher). Next, 50 [micro]L of [GIP.sub.1-42] calibrators (varying concentrations of [GIP.sub.1-42] protein in assay buffer consisting of 50 mmol/L HEPES, pH 7.40, 150 mmol/L NaCl, 10 mL/L Triton X-100, 5 mmol/L EDTA, and 5 mmol/L EGTA) were added to the wells to generate a calibration curve. Plasma samples were diluted 1:4 in assaybuffer and added to their respective wells, and the ELISA plate was incubated for 1.5 h at room temperature. Following aspiration, wells were washed 3 times with TBST, and 50 u L of conjugate antibody (biotin-labeled N-terminal anti-[GIP.sub.1-42] specific antibody, 1 mg/L) were added to the wells for a 1-h incubation at room temperature. After aspiration, wells were washed 3 times with TBST, followed by the addition of 50 u L of 1:5000 diluted streptavidin-ruthenium conjugate (Sulfo-Tag, MSD) for a 1-h incubation at room temperature. After the final aspiration, wells were washed 3 times with TBST, 150 [micro]L of 2X MSD read buffer were added to the wells, and plates were developed using an MSD reader, which recorded ruthenium electrochemiluminescence.
MSD software and SigmaPlot version 8.0 were used for fitting ELISA calibration curves. Data were plotted and analyzed with Fig. P (version 2.98, Biosoft). In each case, a P value [less than or equal to]0.05 was considered to indicate statistical significance.
We first obtained an anti-[GIP.sub.1-42] monoclonal antibody by immunizing mice with a peptide corresponding to the first 6 residues of human GIP conjugated to keyhole limpet hemocyanin. Initial Biacore analysis of this parental antibody (Fig. 1A) indicated that although it was promising, its affinity was likely not suitable for our ultimate goal. The parental antibody did, however, demonstrate specificity for [GIP.sub.1-42] vs [GIP.sub.3-42] (Fig. 1B). To improve the affinity of the antibody, the VH and VL genes were obtained from the parental hybridoma cell line, and mutations were introduced into the CDRs.
Three rounds of selection with decreasing concentrations of [GIP.sub.1-42] were performed, and sequences of individual variants with improved binding were determined. A combinatorial library, in which the CDR regions of clones from the individual CDR libraries with improved binding were combined, was constructed and subjected to further rounds of selection with increased stringency to identify additive or synergistic mutational pairings. Individual combinatorial clones were sequenced, and binding characteristics were determined. Biacore analysis (Fig. 1C) indicated that the optimized antibody had much improved affinity for GIP 1-42. Importantly, the optimized antibody was also specific for [GIP.sub.1-42] and did not recognize [GIP.sub.3-42] (Fig. 1D). Table 1 summarizes the characteristics of the parental and optimized antibodies and demonstrates that the optimized anti-[GIP.sub.1-42]-specific antibody resulted in a > 100-fold improvement in the dissociation rate and 10-fold improvement in overall affinity for [GIP.sub.1-42] compared to the parental antibody.
This optimized antibody, together with an anti-total GIP monoclonal antibody, was then investigated for pairing in a sandwich ELISA. The optimal pairing was found to be anti-total GIP antibody as the capture antibody and anti-N-terminal specific [GIP.sub.1-42] antibody as the conjugate antibody. Fig. 2 shows a typical calibration curve obtained with this ELISA orientation, in which [GIP.sub.1-42] peptide was prepared at a concentration of 1 [micro]g/L and serially diluted to create a calibration curve. Based on a 3-SD evaluation of the zero calibrator, the limit of detection of the ELISA was determined to be 1 ng/L. The lower limit of quantification was determined to be 5 ng/L, and the analyte measurement range of the assaywas determined to be 5 ng/L to 4000 ng/L.
[FIGURE 1 OMITTED]
The sandwich ELISA was specific for [GIP.sub.1-42] and did notrecognize [GIP.sub.3-42] (Fig. 2). Specificity of the ELISA for other members of the peptide superfamilywas also tested by using glucagon, GLP-1, GLP-2, secretin, and vasoactive intestinal peptide, each at concentrations up to 10 [micro]g/L. No cross-reactivity was observed with any of these other peptides, further suggesting that the ELISA was specific for [GIP.sub.1-42]. ELISA dilution curves for the synthetic standard and actual human plasma samples were determined to be parallel, and the ELISA demonstrated acceptable dilutional linearity. When observed vs expected results were compared for the dilution studies performed, the r value was 0.99 and the regression equation was: y = 1.17x-4.71.
[FIGURE 2 OMITTED]
We next evaluated the ELISA using human plasma samples to assess overall robustness. Freeze-thaw stability was evaluated by testing 4 different plasma samples. Results showed acceptable freeze-thaw stability with 80%-120% recovery after 5 freeze-thaw cycles. Individual results for freeze-thaw cycles were as follows: sample A, 23,25,24,24, and 23 ng/L, respectively; sample B, 173, 171, 168, 158, and 153 ng/L, respectively; sample C, 470, 486, 477, 459, and 451 ng/L, respectively; and sample D, 738, 790, 740, 737, and 747 ng/L, respectively. We assessed precision of the ELISA using human plasma samples containing 24, 360, and 754 ng/L of endogenous [GIP.sub.1-42]. Intraassay (n = 20) imprecision results (CVs) were 3.3%, 3.1%, and 3.5%, respectively. Interassay imprecision was also determined by having 2 different operators analyze the above samples in quadruplicate on each of 3 different days. Interassay (n = 24) imprecision results (CVs) were 11.5%, 8.7%, and 6.1%, respectively.
To assess recovery, [GIP.sub.1-42] peptide was added to pooled fasting human plasma (containing 10 ng/L of endogenous [GIP.sub.1-42]), at concentrations of 500, 250, and 125 ng/L, and samples were analyzed using the ELISA. Mean (SD) results were 592 (43) ng/L, 287 (28) ng/L, and 139 (8) ng/L, indicating 80%-120% recovery at all concentrations of [GIP.sub.1-42] tested.
We next used our assay to measure [GIP.sub.1-42] concentrations following a mixed meal test in healthy individuals. To do this, 16 healthy volunteers had blood drawn at 0800 following an overnight fast. Study participants were fed a mixed meal breakfast and had their blood drawn 1 and 2 h afterward. [GIP.sub.1-42] increased dramatically (Fig. 3A) in the postprandial state compared to the fasting state. At baseline in the fasting state, the median plasma [GIP.sub.1-42] concentration was 18 ng/L with a 25%-75% range of 14-29 ng/L. At 1 and 2-h postprandial time points, [GIP.sub.1-42] concentrations increased dramatically (median 552 ng/L, 25%-75% range 439-922 ng/L; and median 470 ng/L, 25%-75% range 278-644 ng/L), respectively (P < 0.001 compared to fasting for both postprandial time points). Total GIP concentrations were also measured and were also observed to increase dramatically when the study participants went from a fasted to fed state. At baseline in the fasting state, the median total GIP concentration was 33 ng/L with a 25%-75% range of 20 -62 ng/L. At 1 and 2-h postprandial time points, total GIP concentrations increased dramatically (median 766 ng/L, 25%-75% range 594-1195 ng/L; and median 773 ng/L, 25%-75% range 501-965 ng/L, respectively (P < 0.001 compared to fasting for both postprandial time points).
[FIGURE 3 OMITTED]
We next examined the correlation between total GIP and GIP 1-42 (Fig. 3B). During the fasted state, GIP 1-42 values were modestly but not significantly correlated with total GIP values (r = 0.42; P = 0.09). In the postprandial state, [GIP.sub.1-42] concentrations were more highly correlated with total GIP concentrations (r = 0.93; P < 0.001). However, the ratio of [GIP.sub.1-42] to total GIP was not the same in all study participants (Fig. 3B), suggesting large interindividual variability in the relative concentrations of [GIP.sub.1-42] to total GIP in the fasting and postprandial states. In the fasting state, the range of the ratios of total GIP/[GIP.sub.1-42] was 0.51-4.62. At the 1-h postprandial time point, the range of the ratios was 0.96-1.90, and at the 2-h postprandial time point, the range of ratios was 0.76-3.00.
To develop a sandwich ELISA for measuring [GIP.sub.1-42], we generated a first-in-class monoclonal antibody targeting the N-terminus of the incretin peptide. Although this parental antibody possessed the required specificity, it had a dissociation rate that would ultimately limit the assay limit of quantification. The recent advent of in vitro display technologies (22-24) provided us the flexibility to use CDR saturation mutagenesis to increase its affinity. The optimized version of the antibody had a 100-fold improvement in the dissociation rate for [GIP.sub.1-42] peptide compared to the parental antibody. At the same time, specificity for [GIP.sub.1-42] was maintained after optimization, and no binding was observed to [GIP.sub.3-42].
We were then able to use this antibody to build a dual monoclonal sandwich ELISA for [GIP.sub.1-42] and show that the sandwich ELISA was capable of specifically measuring [GIP.sub.1-42] concentrations. This work builds upon previously reported assays for [GIP.sub.1-42] (8, 15, 16). Compared to previously reported assays, however, which are LC-MS-based or rely on the use of a polyclonal antibody in an RIA format (8, 15, 16), our approach uses a monoclonal antibody specific for the N-terminus of [GIP.sub.1-42] in a sandwich format to measure only active GIP. Using this assay, we were able to demonstrate that healthy individuals show dramatic increases in [GIP.sub.1-42] in the postprandial state compared to the fasting state. Although [GIP.sub.1-42] concentrations were correlated overall with total GIP concentrations, there was large interindividual variation in the ratio of active [GIP.sub.1-42] to total GIP. In addition, this correlation was higher in the fed state than the fasted state, possibly due to the lack of uniformity among the study participants during their overnight fasting.
As a result of the 2 monoclonal antibodies used, this sandwich ELISA is highly specific for [GIP.sub.1-42]. From a practical standpoint, the advantage of a sandwich ELISA method over an LC-MS type method for measuring [GIP.sub.1-42] concentrations is that the ELISA can be implemented in most laboratories that may have neither the complex equipment nor the highly specialized operator expertise required to routinely perform LC-MS type assays. In addition, the ELISA has the potential for higher throughput than an LC-MS assay and therefore provides the basis for the first sandwich ELISA method that can specifically measure [GIP.sub.1-42] concentrations. Another advantage of this ELISA is its lower limit of quantification of 5 ng/L. This may be especially important because we observed that 50% of healthy individuals had plasma [GIP.sub.1-42] concentrations <20 ng/L in the fasted state.
For many years, there has been great interest in the role of GIP in regulating [beta]-cell function and postprandial insulin secretion (25, 26). It has long been observed that the concentration of circulating insulin measured after administration of an oral glucose load is significantly greater than the circulating insulin concentration measured after a comparable amount of glucose has been administered intravenously (9). Secretion of GIP from small intestine K cells following an oral glucose load is increasingly being recognized as contributing to these differences in insulin response (25, 26).
Recent publications have further drawn attention to the relationship that GIP may have with the risk for developing impaired glucose tolerance and/or type 2 diabetes. Renner and coworkers created a transgenic pig with a dominant negative GIP receptor (GIPR) and demonstrated that this animal model showed a normal response to an IV glucose tolerance test, but an impaired response to an oral glucose tolerance test (27). These observations were particularly interesting in light of previous studies, suggesting that an impaired response to an oral glucose tolerance test (OGTT) is associated with increased risk of developing type 2 diabetes and increased cardiovascular events (28). Very recently, Saxena and colleagues demonstrated that mutations in the GIPR were associated with decreased insulin secretion and an increase in the 2-h post-OGTT glucose value of 0.15 mmol/L, and they concluded that GIPR mutations may increase the risk of developing type 2 diabetes (29).
It has also become clear that [GIP.sub.1-42] is the main GIP species to have activity through the GIPR. Deacon and colleagues demonstrated that [GIP.sub.3-42] bound the receptor with much less affinity than did [GIP.sub.1-42] and had no effect on cAMP accumulation (30). The same researchers also demonstrated that although [GIP.sub.3-42] weakly antagonized [GIP.sub.1-42]-induced cAMP accumulation and insulin output in vitro, it did not act as a physiological antagonist in vivo (30).
In light of these findings, it is logical to expect that there will be an increased need for robust assays to measure [GIP.sub.1-42]. By providing a relatively straightforward and robust method to do so, our [GIP.sub.1-42] dual-monoclonal sandwich ELISA should help increase our understanding of the role of GIP in regulating glucose-dependent insulin secretion. Because our assay provides specificity for the active form of the hormone, a lower limit of quantification of 5 ng/L, and an extremely broad dynamic range, it is possible that this ELISA or one similar to it could eventually be adopted for measurement of [GIP.sub.1-42]. This may be particularly helpful in evaluating GIP-based therapies or therapeutic molecules that work through the GIP pathway.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: Eli Lilly and Company
Consultant or Advisory Role: None declared.
Stock Ownership: J.S. Troutt, Eli Lilly and Company; R.W. Siegel, Eli Lilly and Company; J. Chen, Eli Lilly and Company; J.H. Sloan, Eli Lilly and Company; M.A. Deeg, Eli Lilly and Company; G. Cao, Eli Lilly and Company; R.J. Konrad, Eli Lilly and Company.
Honoraria: None declared.
Research Funding: Eli Lilly and Company.
Expert Testimony: None declared.
Role of Sponsor: The sponsor played a direct role in the design of the study, the review and interpretation of data, the preparation of the manuscript, and the final approval of the manuscript.
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Jason S. Troutt, ([dagger]) Robert W. Siegel, ([dagger]) Jinbiao Chen,  John H. Sloan,  Mark A. Deeg,  Guoqing Cao,  and Robert J. Konrad  *
 Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN.
([dagger]) J.S. Troutt and R.W. Siegel contributed equally to the work, and both should be considered as first authors.
* Address correspondence to this author at: Eli Lilly and Company, Indianapolis, IN 46285, USA. Fax 317-276-5281; e-mail email@example.com.
Received November 23, 2010; accepted March 24, 2011.
Previously published online at DOI: 10.1373/clinchem.2010.159954
 Nonstandard abbreviations: GIP, glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide-1; DPP4, dipeptidyl peptidase 4; VH, Ig heavy chain variable region; VL, Ig light chain variable region; scFv: single chain fragment variable; CDR, complementarity-determining region; MSD, MesoScale Discovery; TBST, Tris buffered saline + tween; GIPR: GIP receptor; OGTT, oral glucose tolerance test.
Table 1. Affinities of parental and optimized N-terminal-specific anti-[GIP.sub.1-42] monoclonal antibodies. [MATHEMATICAL EXPRESSION NOT Clone REPRODUCIBLE IN ASCII] [k.sub.d] (1/s) Parental 2.11 x [10.sup.7] 1.19 X [10.sup.2] Optimized 2.22 x [10.sub.6] 6.59 X [10.sup.5] Clone [DELTA][k.sub.d] [K.sub.D] (M) [DELTA][k.sub.d] Parental 1x 5.63 X [10.sup.10] 1x Optimized 180x 2.97 X [10.sup.11] 20x [sup.a][k.sub.a], association rate constant; [k.sub.d], dissociation rate constant; [DELTA][k.sub.d], change in dissociation rate constant relative to parental clone; [K.sub.D], equilibrium dissociation constant; [DELTA][K.sub.D], change in equilibrium dissociation constant relative to parental clone.
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|Title Annotation:||Endocrinology and Metabolism|
|Author:||Troutt, Jason S.; Siegel, Robert W.; Chen, Jinbiao; Sloan, John H.; Deeg, Mark A.; Cao, Guoqing; Kon|
|Date:||Jun 1, 2011|
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