Specific Capture Antibody Research Articles

Voltammetric Detection of Cancer Biomarkers Exemplified by Interleukin-10 and Osteopontin with Silica Nanowires

We report real-time voltammetric detection of potential lung cancer biomarkers, namely, interleukin-10 (IL-10) and osteopontin (OPN), using localized silica nanowires as templates, through an electrochemical alkaline phosphatase (AP) based assay. The high surface to volume ratio associated with nanowires enhances the loading of a specific capture antibody toward a particular cancer antigen. An AP enzyme attached to the cancer antigens (IL-10 and OPN) via detector antibodies hydolyses (dephosphorylates) the p-nitrophenyl phosphate substrate to a yellow, water-soluble product, p-nitrophenol (pNP). This was detected in a small test volume of 3 μL, using a microelectrode cell fabricated in Au. The electro-oxidation of pNP occurs between 0.85 and 0.9 V with anodic peak current showing linear dependence with the capture antibody and antigen concentrations, respectively. The detection of IL-10 down to 1 fg/mL in ideal pure solution and 1 pg/mL in clinically relevant samples was achieved reproducibly. Moreover, both the concentration of the capture antibody and the cancer antigen were found to have a strong influence on the anodic peak current, as demonstrated considering specific example of OPN.

Stable Competitive Enzyme-Linked Immunosorbent Assay Kit for Rapid Measurement of 11 Active Beta-Lactams in Milk, Tissue, Urine, and Serum

Beta-lactams are used as veterinary drugs for the treatment of food-producing animals. For consumer protection, legislation is in place to set limits for their residues. An enzyme-linked immunosorbent assay (ELISA) was developed which allowed, in a single reaction, the class-specific measurement of 11 beta-lactams, with limits of detection below European maximum residue limits. Determinations were feasible in milk, tissue, urine, and serum with simple and rapid sample preparation. In this format, the specific capture antibodies were precoated on the microtiter plate and horseradish peroxidase-labeled conjugate was used to compete with free beta-lactams. The stability of the precoated microtiter plate and conjugate was at least 1 year when stored at 2 to 8 degrees C; upon reconstitution, the conjugate was stable for 6 days at 2 to 8 degrees C. The stability of lyophilized ampicillin standards was at least 6 months when stored at 2 to 8 degrees C and at least 1 year when stored at -20 degrees C. A low cross-reactivity, 3.6%, was observed with ampicillin with open beta-lactam ring relative to 100% for intact ampicillin. Generic recognition was shown by relative cross-reactivity values ranging from 22 (penicillin V) to 144% (nafcillin). Cross-reactivity for cephalosporins was <0.1%. Intra- and interassay precisions expressed as coefficient of variation were typically 2-8%. The inhibitory concentration with 50% binding for ampicillin was typically 2 ppb. Recovery for different spiked levels was >70% with all the matrixes.

Gastric reflux and acute posttympanostomy otorrhea

Objectives: Acute posttympanostomy otorrhea (APTO) is a common complication of tympanostomy tube placement. APTO has been related primarily to viral upper respiratory infections and external ear contamination. Recently, elevated levels of gastric enzymes have been found in a large proportion of chronic middle ear effusions, implicating gastric reflux (GR) in its pathogenesis. The purpose of this study was to determine if GR may be a contributing factor in the development of APTO. Methods: Otorrhea samples were collected from children with APTO. Total pepsinogen concentrations were measured using a commercial, enzyme-linked immunosorbent assay (ELISA) kit, utilizing a pepsinogen I specific capture antibody, and horseradish peroxidase detection antibody. Results: Twenty-six samples from 24 patients were collected and analyzed. Only 17 of 26 samples demonstrated measurable pepsinogen I, and the concentration ranged from less than 0.002–0.017 mg/mL, well below the normal serum reference ranges for pepsinogen I (0.044–0.086 mg/mL). Conclusions: GR does not seem to play an important role in the etiology of APTO in children.

Optical chip immunoassay for hCG in human whole blood

We report on the development of an integrated optic chip sensor for performing rapid and sensitive immunoassays with human whole blood using human chorionic gonadotropin (hCG) as the model system. The optical chip is based on the Hartman interferometer, which uses a single planar lightbeam to address multiple interferometers, each comprising a signal/reference pair of sensing regions. The binding of antigen to specific capture antibodies on the signal sensing region causes a change in the refractive index of the surface layer, which is detectable by its effect on the evanescent field of the guided lightbeam. The reference-sensing region is coated with an irrelevant antibody, which optically cancels a large fraction of the non-specific adsorption that occurs on the specific-sensing region when the sensor is tested with clinical specimens. This work extends previous experiments with buffer and human serum to measurements in undiluted whole human blood. Optical chips were stored dry after surface functionalization, and rehydrated with blood. Colloidal gold nanoparticles conjugated to a second anti-hCG monoclonal antibody were used to provide signal amplification, thereby enhancing assay sensitivity, in a one-step procedure with the gold conjugate added to the test sample immediately prior to measurement. Background signals due to non-specific binding (NSB) in blood were found to be higher than those previously reported with human serum. In addition, a high level of background signal was found with the gold conjugate, which had not been observed in experiments with either buffer or serum. Nevertheless, hCG could be detected at 0.5 ng/ml within 10 min of sample application. The sensor response was linear over the concentration range 0.5–5 ng/ml hCG, as compared with the clinically-relevant range 0.3–1.5 ng/ml. Detection at higher concentrations was affected by scattering from large amounts of bound gold nanoparticles. However, initial binding rate measurements could be used to maintain assay quantitation.

The development of a sialic acid specific lectin-immunoassay for the measurement of human chorionic gonadotrophin glycoforms in serum and its application in normal and Down's syndrome pregnancies.

We have developed and validated a lectin-immunoassay for the recognition of sialic acid residues on hCG glycoforms in serum. This assay employs a hCG specific capture antibody and a sialic acid specific lectin (Wheat Germ Agglutinin) labelled with horse radish peroxidase. The standard curve covered hCG concentrations of 0-4000 IU/l (3rd IS for hCG, 75/537) with an analytical sensitivity of 1.0 IU/l. The within and between batch coefficient of variation was < 7% for all doses. Cross-reactivity of < 1% with TSH, LH, FSH, hCGalpha, hCGbeta and desialylated hCG confirmed assay specificity. Dilutions of serum of < 10% final concentration were parallel to the standard curve (within and between batch CV, < 6%). The assay working range was 100 - > 500 000 IU/l and the recovery of hCG from serum was in the range of 94.5% to 115.4%, with a mean value of 102.1%. The assay detected a time dependent change in hCG sialylation during normal pregnancy with the relative abundance of sialylated hCG declining after week 9 and increasing after week 15 of gestation. In addition preliminary studies showed that maternal serum hCG concentrations measured with the lectin-immunoassay were elevated in high risk Down's pregnancies (as defined by conventional screening tests between weeks 16-18 gestation, median multiple of median, 3.14; range 1.81-19.12, P < 0. 001) and low risk (1.57, 0.49-6.14, P = 0.034) compared to normal (1. 00, 0.32-3.20) pregnancies. Furthermore, the lectin immunoassay had greater discriminatory power compared to conventional immunoassay of hCG and hCGbeta between normal and both low and high risk Down's pregnancies. This assay will allow analysis of serum samples for the investigation of sialylated variants of hCG glycoforms in various pathological and physiological situations.

A Novel Specific Immunoassay for Plasma Two-Chain Factor VIIa: Investigation of FVIIa Levels in Normal Individuals and in Patients With Acute Coronary Syndromes

AbstractWe report the development of an enzyme-linked immunosorbent assay (ELISA) that is specific for factor VIIa (FVIIa). This assay uses a neoantigen specific capture antibody directed to the amino acid peptide sequence N terminal to the FVII cleavage activation site. The antibody exhibits ∼3,000-fold greater reactivity to FVIIa than FVII on a molar basis. Experiments using plasma with added (exogenous) human FVIIa gave quantitative recovery in the ELISA over a range of 0.20 to 3.2 ng/mL of FVIIa. The intra- and inter-assay coefficient of variation (CVs) of the ELISA are 4.5% and 9.8%, respectively. The ELISA shows excellent correlation (r = .99) with a functional assay (using recombinant soluble tissue factor) in detecting FVIIa added to plasma over the range 0.05 to 18.0 ng/mL. However, a major discrepancy exists between the two assays when normal endogenous plasma concentrations of FVIIa are measured. Using normal plasma (n = 14) the functional assay reported 3.10 ± 0.30 ng/mL (mean ± SE) whereas only 0.025 ± 0.010 ng/mL was detected in the same samples by the immunoassay. Patients (n = 43) presenting with acute coronary syndromes (myocardial infarction and unstable angina) exhibited elevations (P < .05) in immunologically detected FVIIa, 0.093 ± 0.013 ng/mL (mean ± SE) compared to patient controls (n = 20) contemporaneously admitted with noncardiac chest pain, 0.048 ± 0.007 ng/mL (mean ± SE). These elevations in the acute coronary syndromes were accompanied by increased (P < .05) and correlating prothrombin fragment F1 + 2 levels (Spearman correlation coefficient rs = .4, P < .01), demonstrating that thrombin generation is certainly associated with, and may even be caused by, extrinsic pathway activation.

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of staphylococcal protein A (SpA) present as a trace contaminant of murine immunoglobulins purified on immobilized protein A

A specific non-competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detecting and quantifying protein A (SpA), present as a trace contaminant of therapeutic murine monoclonal antibodies (McAb) purified on immobilized SpA preparations. The assay employs a microtitration plate system, in which affinity-selected chicken anti-SpA antibodies from the egg yolks of immunized hens provide a specific capture antibody, followed by the addition of standards or sample with a McAb concentration of 1 mg/ml, in conditions unfavourable for Fc binding, and finally an affinity-selected rabbit anti-SpA peroxidase label. The working range of this assay is between 0.5 and 10.0 ng/ml (CV less than 5%), with a lower limit of detection of 0.2 ng/ml (CV less than 10%). This assay was used to evaluate SpA leakage when purifying a serum-free murine IgG1 cell culture supernatant using SpA immobilized on agarose (Protein A-Sepharose CL-4B) or controlled pore glass (Prosep A, high capacity). These gave average antibody SpA contamination levels of 6.7 +/- 1.6 and 2.4 +/- 0.5 (mean +/- SD) parts per million respectively.

Fibrin fragment D-dimer and fibrinogen B beta peptides in plasma as markers of clot lysis during thrombolytic therapy in acute myocardial infarction

The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the “Thrombolysis in Myocardial Infarction II” (TIMI II) trial. Cross- linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1–42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15–42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1–42 in the fibrinopeptide beta 15–42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15–42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1–42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin- specific tag ELISA may provide more accurate information when correlated with clinical endpoints.

Fibrin Fragment D-Dimer and Fibrinogen Bß Peptides in Plasma as Markers of Clot Lysis During Thrombolytic Therapy in Acute Myocardial Infarction

The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the "Thrombolysis in Myocardial Infarction II" (TIMI II) trial. Cross-linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1-42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15-42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1-42 in the fibrinopeptide beta 15-42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15-42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1-42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin-specific tag ELISA may provide more accurate information when correlated with clinical endpoints.