Novel ELISA for the specific detection of protease NEXIN-1 in human biological samples.

Introduction: Serpin E2 or protease nexin-1 (PN-1) is a glycoprotein belonging to the serpin superfamily, whose function is closely linked to its ability to inhibit thrombin and proteases of the plasminergic system. Objectives: In the absence of specific quantitative methods, an ELISA for the quantification of human PN-1 was characterized and used in biological fluids. Methods: The ELISA for human PN-1 was developed using two monoclonal antibodies raised against human recombinant PN-1. PN-1 was quantified in plasma, serum, platelet secretion from controls and patients with hemophilia A and in conditioned medium of aortic tissue. Results: A linear dose-response curve was observed between 2 and 35 ng/mL human PN-1. Intra- and interassay coefficients of variation were 6.2% and 11.1%, respectively. Assay recoveries of PN-1 added to biological samples were ≈95% in plasma, ≈97% in platelet reaction buffer, and ≈93% in RPMI cell culture medium. Levels of PN-1 secreted from activated human platelets from controls was similar to that of patients with hemophilia A. PN-1 could be detected in conditioned media of aneurysmal aorta but not in that of control aorta. Conclusion: This is the first fully characterized ELISA for human serpin E2 level in biological fluids. It may constitute a relevant novel tool for further investigations on the pathophysiological role of serpin E2 in a variety of clinical studies.

Therapeutic drug monitoring in dermatology: the way towards dose optimization of secukinumab in chronic plaque psoriasis.

BACKGROUND: Despite the favourable efficacy profile of secukinumab, clinicians encounter varying clinical responses among patients potentially associated with under- and overdosing. As biologics are expensive, their rational use is crucial and evident. Therapeutic drug monitoring could guide clinicians in making decisions about treatment modifications. AIM: In this multicentre, prospective study, we aimed to develop and validate a secukinumab immunoassay and searched for the therapeutic window in patients with psoriasis. METHODS: We determined secukinumab concentrations at trough in sera from 78 patients with psoriasis at multiple timepoints (Weeks 12, 24, 36, 48 and 52; after Week 52, measurements could be taken at an additional three timepoints) during maintenance phase, using an in-house secukinumab immunoassay consisting of a combination of MA-SEC66A2 as capture antibody and MA-SEC67A9, conjugated to horseradish peroxidase, as detecting antibody. At each hospital visit, disease severity was assessed using the Psoriasis Area and Severity Index (PASI). RESULTS: After quantification, 121 serum samples were included for dose-response analysis. Based on a linear mixed-effects model, secukinumab trough concentrations were found to decrease with increasing body mass index (BMI). Based on receiver operating characteristic (ROC) analysis, we concluded that the minimal effective secukinumab threshold was 39.1 mg/L in steady state, and that this was associated with a 92.7% probability of having an optimal clinical response (PASI ≤ 2 or reduction in PASI of ≥ 90%). CONCLUSIONS: Monitoring and targeting a secukinumab trough concentration of 39.1 mg/L may be a viable treatment option in suboptimal responders. In patients with higher BMI, weight-based dosing may be needed in order to prevent underdosing.

Endoscopic remission can be predicted by golimumab concentrations in patients with ulcerative colitis treated with the changed label.

BACKGROUND: In 2018, the European Medicines Agency (EMA) replaced a fixed 50 mg every 4-week maintenance regimen of golimumab for ulcerative colitis (UC) patients weighing <80 kg with new, flexible dosing that allows reactive dose optimization to 100 mg if clinically needed. We analyzed the endoscopic remission rates and pharmacokinetics of this new dosing regimen in real-life settings. METHODS: We prospectively recruited 30 consecutive (17 with body weight <80 kg) patients with UC who received golimumab with the new EMA label. The primary endpoint was endoscopic remission (Mayo ≤1) assessed by centrally-read endoscopy at week 14 and year 1. Golimumab concentrations, measured at nine prespecified timepoints, were correlated with endoscopic remission and identified cut-offs. RESULTS: Endoscopic remission was achieved in 15/30 (50%) and 10/30 (33%) patients at week 14 and year 1, respectively. Reactive dose optimization to 100 mg maintenance was needed in 13/17 (76%) patients. Golimumab concentrations at week 6 predicted week 14 and year 1 endoscopic remission. Week 6 concentrations >10.7 µg/ml were a strong predictor for achievement and maintenance of endoscopic remission during the first year of treatment, while concentrations <5.1 µg/ml identified the opposite. CONCLUSION: One-third of the patients reached and maintained endoscopic remission during the first year of golimumab treatment, but the need for dose optimization to 100 mg every 4 weeks of maintenance was high in patients weighing <80 kg. Golimumab concentrations <5.1 µg/ml at week 6 identified patients who are unlikely to reach and maintain endoscopic remission with the new, flexible EMA label.

Development and validation of immunoassays for monitoring of guselkumab and anti-guselkumab antibodies in patients with moderate-to-severe psoriasis.

Therapeutic drug monitoring, which is the measurement of drug concentrations in the blood, is a useful tool to guide clinical decision-making and treatment adjustments, on the condition that drug concentrations are correlated with treatment response. For guselkumab, an anti-IL-23 monoclonal antibody for the treatment of moderate-to-severe psoriasis, such a concentration-response relationship could not yet be determined as no commercial assays for the quantification of this drug or antibodies against this drug are available. Therefore, the aim of this study was to develop and validate immunoassays for the quantification of guselkumab and anti-guselkumab antibodies according to the guidelines of the European Medicines Agency (EMA). A diverse panel of 20 highly specific anti-guselkumab monoclonal antibodies (MA-GUS) was generated of which eight revealed a neutralizing capacity of ≥65 %. At least seven different antibody clusters were identified based on their epitope binning profile. Using MA-GUS9F6 as the capture antibody and MA-GUS12G12 as the detection antibody, an ELISA was developed with a dose-response curve ranging from 0.08 to 5 ng/mL. The assay was specific, selective and could accurately and precisely quantify guselkumab concentrations in spiked healthy control serum and serum from guselkumab-treated psoriasis patients with a cut-off for quantification of 0.014 µg/mL. The presence of IL-23 in physiological concentrations or of non-neutralizing antibodies did not impact the quantification of guselkumab, while the presence of neutralizing antibodies did. Using MA-GUS12A9 as a calibrator, two anti-guselkumab antibody assays were developed to detect anti-guselkumab antibodies, which differ in the threshold for detection and quantification and the tolerance to the presence of guselkumab. Together, these validated immunoassays are essential to establish a concentration-response relationship and will allow the future implementation of therapeutic drug monitoring in moderate-to-severe psoriasis patients receiving guselkumab treatment.

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin.

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples.

Validation of a sample pretreatment protocol to convert a drug-sensitive into a drug-tolerant anti-infliximab antibody immunoassay.

A meta-analysis revealed that up to 51% of patients treated with infliximab develop anti-drug Abs (ADA) which are associated with loss of response. Detection of ADA is strongly influenced by assay technique since drug-sensitive ADA assays are not able to detect ADA in the presence of drug and therefore underestimate ADA development. In addition, the lack of a calibrator antibody that can be used in a drug-sensitive and drug-tolerant assay hampers an adequate comparison among different assays. Here we present a sample pretreatment protocol to convert the bridging assay, originally developed as a drug-sensitive assay, into a drug-tolerant assay, allowing use of the same assay and calibrator antibody MA-IFX10F9. Using the sample pretreatment protocol, the bridging assay detects antibodies towards infliximab in samples containing up to 5-fold infliximab over anti-infliximab. Analysis of consecutive serum samples from infliximab treated patients revealed that the drug-tolerant assay detects ADA development up to 40 weeks earlier compared to the drug-sensitive assay. In conclusion, the sample pretreatment protocol can be implemented in various assay formats and allows determination of ADA in the presence of drug, providing the possibility for early treatment optimization. Copyright © 2016 John Wiley & Sons, Ltd.

Generation and characterization of a unique panel of anti-adalimumab specific antibodies and their application in therapeutic drug monitoring assays.

A number of assays are currently available to support therapeutic drug monitoring of adalimumab. A complete characterization of the assays and comparison of different assays has not been performed. The aim of this study, therefore, is to generate and characterize of a panel of monoclonal antibodies towards adalimumab (MA-ADM); to use this panel to develop novel assays to determine adalimumab concentrations; to assess the impact of tumor necrosis factor (TNF) and (non-)neutralizing antibodies on adalimumab detection and to compare the performance of assays. In total, ten specific MA-ADM were generated of which four revealed a neutralizing potency of >78%. At least six different clusters were identified using principal component analysis. MA-ADM40D8 was selected as detecting antibody to determine adalimumab in the TNF-coated ELISA (A) and the MA-ADM28B8/MA-ADM40D8 antibody pair was chosen for use in the MA-coated ELISA (B). The impact of TNF and (non-) neutralizing antibodies was similar in both ELISAs. Finally, serum samples of adalimumab-treated Crohn's disease patients were collected and used for an external validation using the assay of Sanquin (C) and the apDia kit (D). All adalimumab assays showed excellent Pearson correlation: r=0.96 for A versus B, 0.96 for A versus C, 0.94 for A versus D, 0.97 for B versus C, 0.95 for B versus D and 0.94 for C and D. The excellent agreement with the two commercially available ELISAs allows harmonization of treatment algorithms in and between different hospitals/infusion centers.

Characterization and Application of a Unique Panel of Monoclonal Antibodies Generated against Etanercept.

The clinical response in ankylosing spondylitis (AS) patients treated with biologic agents can be influenced by pharmacokinetic variability among and within these patients. Therapeutic drug monitoring is seen as a valuable tool to improve patient care. The aim of this study was to generate a panel of mAbs toward etanercept (ETN) and to determine ETN and anti-ETN concentrations in AS patients. mAbs against ETN (MA-ETN) were generated using hybridoma technology. For quantification of ETN concentrations, a mAb-based TNF-coated ELISA and a mAb/mAb-based sandwich-type ELISA were developed. For evaluation of the anti-ETN Ab response, a bridging ELISA, as well as a functional cell-based assay, were constructed. Disease activity of the AS patients was measured with the AS Disease Activity Score (ASDAS). Active disease was defined as ASDAS ≥ 2.1. A total of 59 of 76 generated mAbs were ETN specific and were characterized further. Fifty-one mAbs revealed inhibitory properties in a cell-based assay. Analysis of serum concentrations of 21 ETN-treated AS patients with the TNF/MA-ETN68C5-HRP ELISA and the MA-ETN63C8/MA-ETN61C1-HRP ELISA revealed a good Pearson's r (+0.974) but a poor intraclass correlation coefficient (+0.528) as the result of underestimation of the values in the former ELISA. At 24 wk, ETN concentrations were similar in patients with ASDAS < 2.1 and ≥ 2.1. Anti-ETN Abs were not detected in any of the patient samples tested. In conclusion, highly sensitive mAb-based immunoassays were developed for quantification of ETN and anti-ETN concentrations. The impact of these methods needs to be evaluated further in clinical practice.

Variability in Golimumab Exposure: A 'Real-Life' Observational Study in Active Ulcerative Colitis.

BACKGROUND AND AIMS: Golimumab has been approved recently to treat refractory moderate-to-severe ulcerative colitis [UC]. To date it is not clear why a considerable fraction of patients do not respond, or lose initial response, to golimumab therapy. Our aim was to investigate whether a low golimumab serum concentration and/or a positive anti-golimumab antibody status reduces the efficacy of this drug in patients with UC. METHODS: Serum samples of 21 patients with moderate-to-severe UC were collected during the first 14 weeks of golimumab therapy. For measurement of golimumab serum concentrations, both a tumour necrosis factor [TNF]-coated enzyme-linked immunosorbent assay [ELISA] and a sandwich-type ELISA were developed. Anti-golimumab antibodies were measured using a bridging ELISA and a newly-developed drug-tolerant immunoassay. Clinical response and mucosal healing were assessed 14 weeks after start of treatment. RESULTS: Out of 21 patients, 10 [48%] reached partial clinical response at Week 14. Median [interquartile range] serum golimumab concentration was significantly higher in partial clinical responders than in non-responders: 10.0 [7.8-10.5] µg/ml versus 7.4 [4.8-8.3] µg/ml at Week 2 [p = 0.035] and 5.1 [4.0-7.9] µg/ml versus 2.1 [1.8-4.2] µg/ml at week 6 [p = 0.037]. Four out of 21 UC patients developed anti-golimumab antibodies, detectable only using a drug-tolerant immunoassay, and three had a partial clinical response at that time. Clinical non-responders had a significantly more severe colitis, indicated by a higher endoscopic Mayo score at baseline compared with partial clinical responders [p = 0.048]. CONCLUSION: Adequate exposure to golimumab drives clinical response. A worse disease at baseline influences clinical response rate negatively.

An Optimized Anti-infliximab Bridging Enzyme-linked Immunosorbent Assay for Harmonization of Anti-infliximab Antibody Titers in Patients with Inflammatory Bowel Diseases.

BACKGROUND: The formation of anti-infliximab antibodies (ATI) is associated with loss of response and adverse events in patients with inflammatory bowel diseases, leading to the introduction of ATI monitoring for guiding treatment adjustments. However, a lack of standardization among current available assays exists, hampering comparison of results from different studies. This study aimed to improve the harmonization of clinically validated ATI enzyme-linked immunosorbent assays (ELISAs) by introducing a monoclonal anti-infliximab antibody (MA-IFX). METHODS: A panel of MA-IFX was evaluated as calibrator in the first generation ATI ELISA. After selection of 1 MA-IFX, assay conditions were optimized and biotin-streptavidin-enhanced detection of bound infliximab was introduced. The novel second generation ELISA was used for reanalysis of 127 serum samples from a cohort of 12 patients with inflammatory bowel disease, previously identified as ATI positive. RESULTS: Of 55 MA-IFX, MA-IFX10F9 was selected as calibrator in the ATI ELISA. After optimization of the assay conditions, a 4-fold improvement in sensitivity was obtained. Reanalysis of 127 serum samples revealed that in 5 of 12 patients (46%), ATI were detected at least 1 time point earlier with the second generation ELISA compared with the first generation ELISA. In 1 patient, the second generation ELISA allowed to detect ATI before the reinitiation of IFX after a drug holiday. CONCLUSIONS: In addition to the improved sensitivity and specificity of the second generation ATI ELISA, MA-IFX10F9 can serve as a universal calibrator to achieve assay harmonization. Moreover, the superiority of the second generation assay in analyzing serum of restarters was demonstrated.

Generation of a Highly Specific Monoclonal Anti-Infliximab Antibody for Harmonization of TNF-Coated Infliximab Assays.

BACKGROUND: Determination of infliximab (IFX) serum concentrations has been used for treatment optimization of patients with inflammatory bowel disease. A wide range of enzyme-linked immunosorbent assays (ELISA) exists to quantitate IFX. Most of these assays lack specificity and cross-react with other anti-tumor necrosis factor (TNF) agents. The ability of these IFX assays to detect IFX in complex with antidrug antibodies is not known. The objective of our study was to develop an IFX-specific immunoassay to monitor IFX serum concentrations and to evaluate the impact of antidrug antibodies on the assay performance. METHODS: A panel of monoclonal antibodies toward IFX (MA-IFX) was generated by hybridoma technology and evaluated to replace the polyclonal antibody in a TNF-coated IFX assay. The selected monoclonal antibody-based (MA-based) IFX ELISA was benchmarked to a clinically validated, reference polyclonal antibody-based (pAb-based) IFX ELISA using 209 inflammatory bowel disease serum samples. RESULTS: Fifty-five MA-IFX were generated and grouped into 9 clusters. Of the 22 monoclonal antibodies tested, MA-IFX6B7 was selected for use in the IFX ELISA and the assay was further optimized. MA-IFX6B7 is a high-affinity (KD = 1.40E-09 mol/L), noninhibitory IgG1 antibody that binds to the Fab fragment of IFX and exhibits no cross-reactivity with other anti-TNF drugs. The linearity of an IFX dose-response curve was demonstrated in the range of 1.2-37.5 ng/mL (R = 0.988). The MA-based assay showed a good Pearson correlation (R = 0.986) and agreement (intraclass correlation coefficient = 0.985) with the pAb-based assay. The MA-based assay detects IFX in complex with nonneutralizing anti-IFX antibodies but not when complexed with neutralizing anti-IFX antibodies. CONCLUSIONS: In this study, a highly specific MA-IFX was developed as detection antibody in an ELISA to quantify IFX serum concentrations. The assay was benchmarked to the clinically validated reference pAb-based IFX ELISA.

Development of a universal anti-adalimumab antibody standard for interlaboratory harmonization.

BACKGROUND: Therapeutic drug monitoring of adalimumab (ADM) has been introduced recently. When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated. A variety of assays to measure the occurrence of ADA have been developed. Results are expressed as arbitrary units or as a titration value. The aim was to develop a monoclonal antibody (MA) that could serve as a universal calibrator to quantify the amount of ADA in ADM-treated patients. METHODS: Hybridoma technology was used to generate a MA toward ADM. The functionality of the MA was tested in a bridging enzyme linked immunosorbent assay (ELISA) setup and in a cell-based assay. Sera from 25 anti-tumor necrosis factor naive patients with inflammatory bowel disease were used to determine the cutoff values. Sera from 9 ADM-treated patients with inflammatory bowel disease, with undetectable serum concentrations of ADM were used to quantify the ADA response. RESULTS: In this study, MA-ADM6A10, an IgG1 that can be used as a calibrator in both an ELISA to quantify the amount of binding antibodies and in a cell-based assay to quantify the amount of neutralizing antibodies, was generated. Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity. CONCLUSIONS: The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.

Characterization of a panel of monoclonal antibodies toward mouse PAI-1 that exert a significant profibrinolytic effect in vivo.

INTRODUCTION: PAI-1 is the main physiological inhibitor of t-PA and u-PA. Elevated PAI-1 levels have been implicated in the pathogenesis of several thrombotic and non-thrombotic diseases. The effect of PAI-1 inhibition can be studied in mouse models, when appropriate immunological tools are available. The majority of the available monoclonal antibodies against PAI-1 have been raised against human PAI-1. Even though some of these antibodies cross-react with non-glycosylated PAI-1 from different species, these antibodies often do not cross-react sufficiently with glycosylated mouse PAI-1. Moreover, the antibodies that cross-react with glycosylated mouse PAI-1 often have decreased inhibitory properties in the presence of vitronectin. Our objective was the generation of a panel of monoclonal antibodies reacting with vitronectin-bound glycosylated mouse PAI-1. RESULTS: Five monoclonal antibodies revealed binding to glycosylated mouse PAI-1 and exerted a strong (i.e., 58-80% inhibition of PAI-1 activity) inhibitory effect toward mouse PAI-1. Similar inhibitory effects were seen in the presence of a 33-fold molar excess of vitronectin. The PAI-1 inhibitory potential of the antibodies in vivo was demonstrated in a thromboembolism model, in which the evaluated antibodies significantly increased the percentage of mice with normal physical activity in comparison to mice treated with negative control antibody. CONCLUSIONS: To the best of our knowledge this is the first panel of monoclonal antibodies that can inhibit mouse PAI-1 in the presence of vitronectin and that show a profibrinolytic effect in vivo. Therefore these antibodies provide excellent immunological tools to further investigate the role of PAI-1 in mouse models.

Development and evaluation of monoclonal antibodies as probes to assess the differences between two tomato pectin methylesterase isoenzymes.

The enzyme pectin methylesterase (PME) was purified from red ripe tomatoes (Lycopersicon esculentum) and through affinity chromatography two isoenzymes were fractionated (t1PME and t2PME). Further analysis of these two isoenzymes, both having a molar mass of 34.5kDa, revealed a difference in the N-terminal sequence and in amino acid composition. t1PME was identified as the major isoenzyme of PME in tomato fruit. In this study the aim was to develop a toolbox, consisting of monoclonal antibodies, that allows to gain insight into the in situ localization of PME in plant based food systems like tomatoes. A panel of six interesting monoclonal antibodies was raised against both isoenzymes, designated MA-TOM1-12E11, MA-TOM1-41B2, MA-TOM2-9H8, MA-TOM2-20G7, MA-TOM2-31H1 and MA-TOM2-38A11. The differences in epitopes between these monoclonal antibodies were determined using affinity tests towards denatured PME, cross-reactivity tests and inhibition tests. Characterization of these antibodies indicated an immunological difference between t1PME and t2PME, also revealing a conserved epitope on t2PME, carrot PME and strawberry PME. Different epitopes are recognized by the generated antibodies making them excellent probes for immunolocalization of PME by tissue printing. In tomato, t1PME and t2PME showed a pronounced co-localization, especially in the pericarp and the radial arms of the pericarp. Three of the generated antibodies could be used for immunolocalization of PME in carrots (Daucus carota L.), which was only present in the cortex and not in the vascular cylinder of carrots.

Development of ELISAs measuring the extent of TAFI activation.

OBJECTIVE: To date, quantitation of TAFI antigen levels has been mainly focused on "total" antigen levels and has been shown to yield ambiguous results because of the existence of different isoforms and various degrees of activation. Our objective was to develop assays that allow measuring the extent of TAFI activation. METHODS AND RESULTS: A variety of enzyme-linked immunosorbent assays (ELISAs) were evaluated for their preferential reactivity toward TAFI before and after activation, and toward the recombinantly expressed activation peptide. Three ELISAs with distinct reactivities were selected: recognizing either exclusively nonactivated TAFI, the released activation peptide, or exclusively TAFIa (activated TAFI). Evaluation of TAFI activation during clot lysis revealed that decreases of TAFI levels are associated with increases of the released activation peptide and TAFIa levels. In addition, antigenic measurement of TAFIa parallels activity measured by chromogenic assay. Analyzing plasma samples revealed that subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; P<0.001) and TAFIa (112.1 versus 103.3; P=0.03), and not of TAFI antigen (92.5 versus 87.9; P=0.07) (results in % of plasma pooled from normolipidemic subjects). CONCLUSIONS: ELISAs that allow to measure the extent of TAFI activation were developed. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular diseases.

Development of a genotype 325-specific proCPU/TAFI ELISA.

OBJECTIVE: A Thr/Ile polymorphism at position 325 in the coding region of proCPU has been reported. Immunological assays, fully characterized (including genotype dependency), are required for the quantitation of proCPU levels. METHODS AND RESULTS: We have generated a panel of monoclonal antibodies against human, plasma-derived proCPU. Two combinations exhibiting distinct reactivities were selected for measurement of proCPU in plasma. T12D11/T28G6-HRP yielded values of 10.1+/-3.1 microg/mL (mean+/-SD, n=86; normal donors), and T32F6/T9G12-HRP yielded values of 5.4+/-3.0 microg/mL. Grouping according to the 325 genotype demonstrated that T12D11/T28G6-HRP was independent to this polymorphism whereas T32F6/T9G12-HRP revealed a complete lack of reactivity with the Ile/Ile genotype (ie, 0.0+/-0.0, 4.2+/-1.7, and 7.3+/-2.9 microg/mL for the Ile/Ile, Ile/Thr, and Thr/Thr isoforms, respectively). Commercially available antigen assays appeared to be partially dependent on the 325 genotype (eg, 44+/-8.9% and 100+/-30% for the Ile/Ile and Thr/Thr isoforms, respectively). CONCLUSIONS: Our data demonstrate that great care should be taken when evaluating proCPU antigen values as a putative causative agent or as a diagnostic risk marker for cardiovascular events.