Unbound Conjugate Research Articles

Enzyme Linked Immunosorbent Assay (ELISA)

Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are both widely used as diagnostic tools in medicine and as quality control measures in many industries. ELISA has been the system of choice when testing soluble antigens and antibodies. EIA / ELISA uses the basic immunological concept of antigen binding to specific antibodies, which allows the detection of small amounts of antigens such as proteins, peptides, hormones or antibodies in fluid samples. In all protocols, solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled with the enzyme. The unbound conjugate is washed and a chromogenic or fluorogenic substrate is added.

On-Chip Enzyme Immunoassay of a Cardiac Marker Using a Microfluidic Device Combined with a Portable Surface Plasmon Resonance System

This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable surface plasmon resonance (SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction with acetylcholine esterase-labeled antibody (conjugate), and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction with the trapped conjugate was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We achieved a detectable concentration range of 5 pg/mL-100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This success resulted from the use of a T-shaped microfluidic device structure, which prevents the sample solution from flowing over the gold film used for SPR detection. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device. We employed the procedure to detect serum BNP by using spiked samples in human serum and achieved satisfactory recovery for heat-treated samples to denature the esterase in the serum before the immunoreaction.

Radiation doses to non-Hodgkin's lymphoma cells and normal bone marrow exposed in vitro. Comparison of an α-emitting radioimmunoconjugate and external γ-irradiation

Purposes : The α -emitting radionuclide 211 At conjugated to the CD20 targeting chimeric monoclonal antibody rituximab was studied to: (a) Estimate radiation dose components to lymphoma and bone marrow (BM) cells exposed in vitro. (b) Calculate the mean absorbed radiation doses in various normal tissues of mice following intravenous injection. Materials and methods : B-lymphoma cells (RAEL) and normal human BM cells were incubated with increasing concentrations of the radioimmunoconjugate. Based on binding kinetics and on measured cellular and nuclear diameters, the radiation doses were calculated using microdosimetric methods. Results : Targeting of 211 At-rituximab to RAEL cells was extensive and stable compared with the binding to BM cells. The absorbed radiation doses from cell-bound activity at an initial activity concentration of 10 kBq ml -1 were 0.645 and 0.021 Gy to RAEL and BM cells, respectively. In comparison, the contribution from unbound conjugate in the medium during 1h exposure was 0.042 and 0.043 Gy. The D 0 value for RAEL cells was 0.55 Gy, but only 0.34 Gy for BM cells, whereas corresponding D 0 values were 0.72 and 1.21 Gy after a single exposure to external 60 Co γ-rays. Mean absorbed doses of 1.31, 0.48 and 0.36 Gy for blood, lungs and heart were calculated in mice injected with 5.4kBq g -1 of 211 At-rituximab. Conclusion : Despite the higher inherent sensitivity of the BM cells to the α -irradiation, there was, related to the radioactivity concentrations of 211 At-rituximab, several logs greater cell kill in RAEL cells, illustrating the tumour-specific nature of the targeting.

In vitro cytotoxicity following specific activation of amygdalin by beta-glucosidase conjugated to a bladder cancer-associated monoclonal antibody.

We describe a novel version of antibody-directed enzyme prodrug therapy (ADEPT), with the use of amygdalin as prodrug. Amygdalin is a naturally occurring cyanogenic glycoside, which can be cleaved by sweet almond beta-glucosidase to yield free cyanide. If amygdalin could be activated specifically at the tumour site, then malignant cells would be killed without the systemic toxicity usually associated with chemotherapy. To this end, we have conjugated beta-glucosidase to a tumour-associated monoclonal antibody (MAb) (HMFG1) and the conjugate has been tested in vitro for specificity and cytotoxicity subsequent to activation of amygdalin. Amygdalin was cytotoxic to HT1376 bladder cancer cells only at high concentrations, whereas the combination of amygdalin with HMFG1-beta-glucosidase enhanced the cytotoxic effect of amygdalin by 36-fold. When 2 concentrations of HMFG1-beta-glucosidase were compared, the toxic effect was dose dependent. The cytotoxicity of amygdalin was also enhanced by the MAb-enzyme conjugate even when the unbound conjugate was removed from the medium prior to exposure to amygdalin and the cells were washed. In addition to the cytotoxic effect, we also demonstrated specificity, using a MAb-enzyme conjugate that does not recognise the HT1376 bladder cancer cells. Finally, we studied the cytotoxic effect of the conjugate in co-culture of HMFG1-positive and-negative cell lines (HT 1376 and U87MG cells). We demonstrated that the rate of surviving cells corresponds well to the percentage of U87MG (HMFG1-negative) cells in the flask. Our findings indicate that ADEPT is more effective than non-directed enzyme activation of a prodrug and can result in a non-toxic cancer therapy.

Vitros Digoxin Immunoassay Evaluated for Interference by Digoxin-like Immunoreactive Factors

Serum digoxin concentrations are routinely monitored because of the narrow therapeutic range of digoxin. Endogenous digoxin-like immunoreactive factors (DLIFs) may cause a false analytic increase in the measured digoxin concentration of patients undergoing digoxin treatment (1)(2). DLIF interference is most commonly found in the sera of neonates, and cord blood and neonatal sera frequently produce false positive results for digoxin (3)(4). Here we examine the newly introduced Vitros digoxin immunoassay (DGXN, Johnson & Johnson Clinical Diagnostics) performed on the Vitros 250 analyzer and compare it to a method (OnLine, Roche Diagnostic Systems) substantially free from DLIF interference (5) and to a method (Digoxin II, Abbott Laboratories) where DLIF interference is known to be present (1). These particular methods are not unique with regard to interference by DLIFs, and methods vary considerably in the impact of DLIF interferences (1)(2)(6)(7). Serum samples used in this study were aliquots of samples that had been sent to St. Louis Children’s Hospital or Barnes-Jewish laboratories (BJC Health System) for the routine measurement of serum digoxin or aliquots of samples received for other routine chemistry analyses. Samples were from newborns (<2 weeks of age) and from adults. The study followed a protocol approved by the Human Studies Committee of Washington University. All samples were analyzed immediately or stored at −20 °C until use, with no more than three freeze-thaw cycles preceding analysis. The Vitros DGXN digoxin assay is an enzymatic heterogeneous, competitive immunoassay that uses dry slide technology. Digoxin-peroxidase conjugate and sample digoxin compete for a limited number of binding sites on the immobilized antibody reagent. After the unbound conjugate is eluted and …

Competitive nonseparation electrochemical enzyme binding/immunoassay (NEEIA) for small molecule detection

A nonseparation electrochemical enzyme binding/immunoassay (NEEIA) for the detection of small molecules via a competitive format is described. The NEEIA concept is based on the use of a microporous gold electrode, which serves as both the working electrode, and solid phase for the immobilization of binding protein/antibody through a chemisorbed layer of thioctic acid. Competitive assays are performed by incubating the small molecule of interest and an alkaline phosphatase (ALP) labeled analyte competitor (conjugate) with the modified electrode. Surface bound conjugate is spatially resolved from unbound conjugate by introducing the substrate ( p-aminophenyl phosphate) through the backside of the microporous gold electrode. The substrate diffuses rapidly through the microporous gold electrode where it first encounters surface bound conjugate. The enzymatically generated product ( p-aminophenol) is subsequently oxidized at the electrode (+190 mV vs. Ag/AgCl). Due to the competitive nature of the assay, the magnitude of the amperometric signal is inversely proportional to the concentration of analyte in the sample. Detection of biotin, digoxin and digitoxin in buffer is demonstrated with detection limits of 1, 0.1 and 10 nM, respectively. In addition, it is shown that digoxin can be measured in undiluted sheep serum with a detection limit of 1 nM, demonstrating that the proposed competitive NEEIA format can be employed for the detection of small molecules directly in complex matrices without any discrete separation or washing steps.

Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten.

The structural basis of the binding of phenylphosphocholine haptens to antibodies was studied. This was done by preparing antibodies and testing binding to conjugates of phenylphosphocholine. The choice of haptens was made in order to evaluate the contribution of the carrier to binding, and its effect on hapten conformation in the active site. Thus, phosphocholine (PC) was diazophenyl-linked to tyrosine or histidine as single amino acid carriers and to tripeptides or octapeptides containing tyrosine or histidine as central amino acids to which PC was attached. Relative affinity was assessed by inhibition enzyme-linked immunosorbent assay (ELISA) and binding constants were determined by fluorescence quenching. Fluorinated haptens were used to determine the kinetics of binding using 19F nuclear magnetic resonance. The transferred nuclear Overhauser effect was used to characterize conformation of the bound hapten. We had previously shown that nitrophenylphosphocholine unlinked to carrier is bound in the active site as a bent structure [Bruderer, U., Peyton, D. H., Barbar, E., Fellman, J. H., & Rittenberg, M. B. (1992) Biochemistry 31, 584-589]. We show here that this same bent conformation is retained in the active site regardless of the neighboring carrier or the conformation of the hapten in the unbound conjugate. The presence of the carrier residues in the bound state does, however, influence affinity.

Hybridisation techniques on gridded high density DNA and in situ colony filters based on fluorescence detection.

Hybridisation techniques on gridded high density DNA and in situ colony filters based on fluorescence detection.

Detection of total DNA with single-stranded DNA binding protein conjugates

We have developed a rapid and sensitive method for total DNA measurement using single-stranded DNA binding protein from E. coli conjugated with horseradish peroxidase or urease. To detect DNA, the sample is heated or alkali treated to denature the DNA and then filtered through nylon or nitrocellulose membranes. After the single-stranded DNA is bound to the membrane, single-stranded DNA binding protein enzyme-conjugate is incubated with the membrane. Next, the unbound conjugate is washed off the membrane and the bound conjugate detected colorimetrically. The assay can detect 10 pg of DNA in <3 hr. This method can be applied to the detection of DNA contamination in therapeutic proteins produced by recombinant DNA or hybridoma techniques.

Automated quantification of thyrotropin by radial partition immunoassay.

We describe a radial partition enzyme immunoassay in which fully automated quantification of human thyrotropin (hTSH) takes less than 11 min. This "sandwich"-type assay involves two monoclonal antibodies, both specific for the intact hTSH molecule. The solid phase consists of tabs of glass-fiber filter paper containing a pre-immobilized monoclonal anti-hTSH antibody complexed with a goat antibody specific for the Fc region of mouse IgG. The patient's sample is first applied to the central "reaction zone" of the tab, wherein hTSH binds to the immobilized antibody. Application of a buffered solution containing enzyme-labeled Fab' fragments of the second monoclonal anti-hTSH antibody initiates "sandwich" formation. A wash buffer containing a fluorogenic substrate elutes unbound conjugate to the tab periphery. The bound enzyme conjugate is quantified by measuring the rate of increase in fluorescence in the reaction zone of the tab, then converting the rate to clinical units by comparison with a stored calibration curve. The clinical utility and performance of the present assay compare favorably with those of other sensitive assays for hTSH.

Quantitative determination of phenobarbital and phenytoin by dry-phase apoenzyme reactivation immunoassay system (ARIS).

We assessed the performance of the apoenzyme reactivation immunoassay system (ARIS) reagent strip tests for determination of phenobarbital (PB) and phenytoin (PHT) with the Seralyzer reflectance photometer. In the assay, the drug of the sample competes with a flavine adenine dinucleotide (FAD)-drug conjugate for binding to a specific antibody; the unbound conjugate then activates apoglucose oxidase to reconstitute glucose oxidase, whose activity is kinetically monitored by a coupled chromogenic reaction. Within-run coefficients of variation (CVs) were less than or equal to 5.0% for PB and less than or equal to 5.6% for PHT; between-run CVs were less than or equal to 6.1% for PB and less than or equal to 6.5% for PHT. Mean analytical recoveries were 100.3% for PB and 100.2% for PHT. Test results were not significantly affected by bilirubin (5 mg/dL), hemoglobin (25 mg/dL), triglycerides (500 mg/dL), uric acid (15 mg/dL), or elevated levels of other antiepileptic drugs. Reagent strip tests correlated very well with substrate-labeled fluorescent immunoassay (r = 0.9923 and 0.9944 for PB and PHT, respectively), enzyme multiplied immunoassay technique (r = 0.9941 and 0.9919), and gas-liquid chromatography (r = 0.9980 and 0.9960). These homogeneous competitive colorimetric immunoassays are particularly suitable for emergency use, for testing small batches of samples, wherever prompt results are needed.

Radial partition immunoassay applied to automated quantification of human choriogonadotropin with use of two monoclonal antibodies.

We describe a novel application of radial partition immunoassay to quantification of human choriogonadotropin (hCG). In this "sandwich"-type assay, two monoclonal antibodies, specific for different sites on the intact molecule are used. The solid phase consists of tabs of glass-fiber filter paper containing a pre-immobilized antibody specific for the alpha subunit of hCG. The patient's sample is first applied directly to the central "reaction zone" of the tab, allowing hCG to bind to the solid-phase antibody. Then a buffered solution containing enzyme-labeled Fab' fragments of a monoclonal antibody specific for the beta subunit of hCG is applied, initiating "sandwich" formation. Finally, a wash buffer containing a fluorogenic substrate is applied, eluting unbound conjugate to the tab periphery. Bound enzyme conjugate is quantified by measuring the rate of increase in fluorescence. Rates are converted to clinical units by comparison with a stored calibration curve. Elapsed time from sample application to results is less than 8 min. Specific performance characteristics of this assay are reported.

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.