Sensitive and quantitative detection of cardiac troponin I with upconverting nanoparticle lateral flow test with minimized interference.

Measurement of cardiac troponin I (cTnI) should be feasible for point-of-care testing (POCT) to diagnose acute myocardial infarction (AMI). Lateral flow immunoassays (LFIAs) have been long implemented in POCT and clinical settings. However, sensitivity, matrix effect and quantitation in lateral flow immunoassays (LFIAs) have been major limiting factors. The performance of LFIAs can be improved with upconverting nanoparticle (UCNP) reporters. Here we report a new methodological approach to quantify cTnI using UCNP-LFIA technology with minimized plasma interference. The performance of the developed UCNP-LFIA was evaluated using clinical plasma samples (n = 262). The developed UCNP-LFIA was compared to two reference assays, the Siemens Advia Centaur assay and an in-house well-based cTnI assay. By introducing an anti-IgM scrub line and dried EDTA in the LFIA strip, the detection of cTnI in plasma samples was fully recovered. The UCNP-LFIA was able to quantify cTnI concentrations in patient samples within the range of 30-10,000 ng/L. The LoB and LoD of the UCNP-LFIA were 8.4 ng/L and 30 ng/L. The method comparisons showed good correlation (Spearman's correlation 0.956 and 0.949, p < 0.0001). The developed UCNP-LFIA had LoD suitable for ruling in AMI in patients with elevated cTnI levels and was able to quantify cTnI concentrations in patient samples. The technology has potential to provide simple and rapid assay for POCT in ED setting.

Glycovariant-based lateral flow immunoassay to detect ovarian cancer-associated serum CA125.

Cancer antigen 125 (CA125) is a widely used biomarker in monitoring of epithelial ovarian cancer (EOC). Due to insufficient cancer specificity of CA125, its diagnostic use is severely compromised. Abnormal glycosylation of CA125 is a unique feature of ovarian cancer cells and could improve differential diagnosis of the disease. Here we describe the development of a quantitative lateral flow immunoassay (LFIA) of aberrantly glycosylated CA125 which is widely superior to the conventional CA125 immunoassay (CA125IA). With a 30 min read-out time, the LFIA showed 72% sensitivity, at 98% specificity using diagnostically challenging samples with marginally elevated CA125 (35-200 U/mL), in comparison to 16% sensitivity with the CA125IA. We envision the clinical use of the developed LFIA to be based on the substantially enhanced disease specificity against the many benign conditions confounding the diagnostic evaluation and against other cancers.

Skeletal troponin I cross-reactivity in different cardiac troponin I assay versions.

OBJECTIVES: To study the skeletal troponin I (skTnI) cross-reactivity of four different commercially available antibodies in four cardiac troponin I (cTnI) research assay versions having the same epitope specificity as evidenced by peptide mapping. DESIGN AND METHODS: The four research assays all use two solid phase antibodies and one detection antibody attached to intrinsically fluorescent nanoparticles. Two alternative antibodies were used for one capture antibody and two for the detector antibody. The assays were evaluated in terms of analytical sensitivity and by determining assay cross-reactivity to skTnI. Additionally, regression analysis was performed by measuring a sample panel (n=101) with all of the four assay versions. RESULTS: A false-positive cTnI concentration of >7000ng/L was measured with one of the assay versions, when serum was spiked with 500,000ng/L skTnI. The corresponding observed cTnI values for the other three assay versions varied from 616ng/L to 727ng/L. Out of the 101 clinical samples assayed, five showed spuriously (3- to 148-fold) elevated cTnI values with the skTnI interference prone assay setup, but not with the other assay versions. According to our investigational skTnI assay, all five samples contained measurable amounts of skTnI (range: 5500-702,000ng/L). CONCLUSIONS: Two out of four cTnI antibodies tested cross-reacted vastly with skTnI but did not cause any notable interference unless paired together. Therefore, skTnI cross-reactivity should be carefully assessed when cTnI assay antibodies claimed to be cTnI specific are selected.

Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

OBJECTIVES: To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. DESIGN AND METHODS: A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. RESULTS: The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to

Extension of dynamic range of sensitive nanoparticle-based immunoassays.

Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000 ng/ml, and with cTnI from 25 to 1000 ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07-0.11) and a y-intercept of -7.79 (-17.87-2.29)ng/L (n=65, r=0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.

A comparison of capture antibody fragments in cardiac troponin I immunoassay.

OBJECTIVES: To compare cardiac troponin I (cTnI) values measured from 32 normal plasma specimens with a two-site cTnI research assay exploiting different molecular forms of a capture antibody. DESIGN AND METHODS: The current research assay consists of two capture antibodies immobilized on streptavidin-well surface and one detection antibody attached to highly fluorescent europium(III)-chelate-doped nanoparticles. Four different molecular forms of one of the capture antibodies (intact monoclonal (Mab), F(ab')2 fragment, Fab fragment and chimeric Fab fragment (cFab)) were tested. The developed immunoassays were evaluated in terms of their analytical sensitivities and assay kinetics. Furthermore, cTnI concentrations were measured from 32 heparin plasma samples from apparently healthy donors (mean age 32; range 24-60 years). RESULTS: The differences in the measured cTnI concentrations (corrected for the buffer-based zero calibrator) between the Mab and the three fragmented forms were highly significant (P<0.0001). Replacing the intact Mab with the antibody fragments also reduced the required antibody amount from 100 ng to 66 ng (F(ab')2) and 16.5 ng (Fab and cFab). Furthermore, the limit of detection was improved when Fab fragments were employed (Mab: 0.90 ng/L, Fab: 0.69 ng/L and cFab: 0.41 ng/L). The apparent normal range median (minimum/maximum) of the 32 healthy subjects was reduced from 7.28 ng/L (2.64/116 ng/L) with Mab to 1.80 ng/L (0.746/10.6 ng/L) for the cFab. CONCLUSIONS: Eliminating the Fc-part from one of the two capture antibodies in an immunofluorometric cTnI assay substantially reduced the measured cTnI concentrations, simultaneously improving the assay sensitivity and reducing the reagent consumption.

Development of an immunoassay for the detection of cystatin C dimers.

Human cystatin C (CysC) is a reversible cysteine protease inhibitor, which is abundantly secreted to body fluids. It is a potential marker of kidney dysfunction, but has been suggested to be of diagnostic importance in a number of neurodegenerative diseases, as well. The amyloid formation by a L68Q variant CysC accounts for the hereditary CysC amyloid angiopathy (HCCAA). Also, the wild type CysC forms inactive dimers at partly denaturing conditions through a domain swapping mechanism. Here, we have developed an immunoassay for the detection of dimeric CysC consisting of either a full length or an N-terminally truncated form. A codon optimized gene encoding a full length CysC was expressed in Escherichia coli, where the product was directed to the periplasmic space. Two different forms of CysC were isolated, a full length product and a form proteolytically truncated by 8 N-terminal amino acid residues. In vitro dimerization experiments were conducted in order to enable the selection of monoclonal antibodies for the construction of an immunoassay being able to primarily recognize the dimers. The analytical detection limit of the assay was 0.043 microg/l, with assay imprecision below 16%. The assay was linear in the range of 5-100 microg/l (R(2)=0.997). The dimer assay was employed for the measurement of serum and cerebrospinal fluid (CSF) sample panel of 20 multiple sclerosis (MS) and 22 non-MS patients. A dimer signal was observed in both serum and CSF samples. The dimer signals from CSF were approximately 2-22 times higher (average 13) than the corresponding signals from serum samples. However, the measured signal levels between the different patient groups showed no statistically significant difference in serum or in CSF (P=0.07 and P=0.98 respectively). In conclusion, the immunoassay provides direct means for detecting CysC dimers in serum and CSF in respect to the amount of total CysC.