Development and validation of a particle-enhanced turbidimetric immunoassay for C-reactive protein

The development of a particle-enhanced turbidimetric immunoassay for C-reactive protein is described. The method demonstrates excellent precision, with the calibration curve remaining stable for at least 16 weeks. The method compares well with established techniques and there is no interference from a variety of autoantibodies, endogenous serum constituents or commonly used drugs.     

A rapid and robust particle-enhanced turbidimetric immunoassay for serum beta 2 microglobulin

A rapid particle-enhanced turbidimetric immunoassay (PETIA), for the measurement of serum beta 2-microglobulin is described. The method has a working range of 0.2-40 mg/l, with good precision and a correlation coefficient of 0.97 when compared with an established radioimmunoassay method. One of the major advantages of this assay is the stability of the calibration curve (up to at least 20 months). This, and the fact that no pretreatment of serum samples is necessary, makes the assay ideally suited for all types of routine determination.     

Residues determination of carbofuran in vegetables based on sensitive time-resolved fluorescence immunoassay

Using direct competitive time-resolved fluorescence immunoassay (TRFIA), a rapid, highly selective and sensitive method was developed for the determination of carbofuran residues in lettuce and carrot. The method was based on a direct competitive immunoassay using europium-labelled anti-carbofuran monoclonal antibody and carbofuran-ovalbumin as coated antigen. The sensitivity, estimated as the I ₅₀ value, was 34.54 ng/mL, with a detection limit (I ₁₀) of 1.94 ng/mL and a practical working range between 1.33 and 341.6 ng/mL. The average recoveries of carbofuran from lettuce and carrot were 81.26–108.0% and 113.9–124.8%, respectively. Eventually, confirmation test between TRFIA and enzyme-linked immunoassay (ELISA) was performed. It confirmed that the results given by the TRFIA method were in agreement with those of the ELISA method.     

Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach

We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Ab1). In this format the primary Ab1 was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab1. Fluorescence was proportional to the concentration of oestradiol over the range 0-8 ng/ml. The sensitivity of the assay was 80 +/- 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand (n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays.     

Forces acting on particle-enhanced immunoassays

The aim of the present work is to study the role of the different forces involved in the agglutination of immune γ-globulin (IgG) covered latex particles due to antigen-antibody reaction. An experimental investigation on the adsorption of IgG molecules on three latexes with different surface charge densities is described. Photon correlation spectroscopy was used to determine the hydrodynamic layer thickness of the IgG molecules adsorbed on the latexes. In order to get an insight into the forces acting between two antibody-covered particles approaching each other, the colloidal stability and immunoreactivity of these biocomplexes were studied. They can be stabilized by electrostatic or hydration forces. The immunological agglutination of IgGimmobilized latex particles due to the addition of the antigen was quantified through scattered light intensity measurements. The immunoresponse increases with ionic strength of the medium until a maximum value is achieved. Above this maximum, the immunoreactivity decreases.     

Antibody immobilization on to polystyrene substrate—on-chip immunoassay for horse IgG based on fluorescence

A simple microfluidic immunoassay card was developed based on polystyrene (PS) substrate for the detection of horse IgG, an inexpensive model analyte using fluorescence microscope. The primary antibody was captured onto the PS based on covalent bonding via a self-assembled monolayer (SAM) of thiol to pattern the surface chemistry on a gold-coated PS. The immunosensor chip layers were fabricated from sheets by CO₂ laser ablation. The functionalized PS surfaces after each step were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antibody–antigen interaction as a sandwich immunoassay with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, the intensity of fluorescence was measured on-chip to determine the concentration of the target analyte. The present immunosensor chip showed a linear response range for horse IgG between 1 μg/ml and 80 μg/ml (r = 0.971, n = 3). The detection limit was found to be 0.71 μg/ml. The developed microfluidic system can be extended for various applications including medical diagnostics, microarray detection and observing protein–protein interactions.     

Forces acting on particle-enhanced immunoassays

The aim of the present work is to study the role of the different forces involved in the agglutination of immuno gamma-globulin (IgG) covered latex particles due to antigen-antibody reaction. An experimental investigation on the adsorption of IgG molecules on three latexes with different surface charge densities is described. Photon correlation spectroscopy was used to determine the hydrodynamic layer thickness of the IgG molecules adsorbed on the latexes. In order to get an insight into the forces acting between two antibody-covered particles approaching each other, the colloidal stability and immunoreactivity of these biocomplexes were studied. They can be stabilized by electrostatic or hydration forces. The immunological agglutination of IgG-immobilized latex particles due to the addition of the antigen was quantified through scattered light intensity measurements. The immunoresponse increases with ionic strength of the medium until a maximum value is achieved. Above this maximum, the immunoreactivity decreases.     

Double-label fluorescence immunoassay of bacteria.

Fluorescence of sensitized bacterial suspensions, reactive with either fluorescein-labeled or rhodamine-labeled antiglobulins, could be quantitatively distinguished in dual-labeled preparations by fluorescence immunoassay.     

A novel bead-based fluorescence immunoassay for aldosterone

Aldosterone quantification helps evaluate the rennin-angiotensin-aldosterone system. The new bead-based mul-tiplex platform has not been applied in aldosterone detection to achieve simultaneous measurements of multiple hormones. A new sensitive competitive bead immunoassay based on Luminex technology for detecting aldoster-one in small sample volumes was developed using two-antibody coupled beads and biotinylated aldosterone as tracer in combination with an extraction step. The assay was validated in human and mouse samples and exhibited a linear working range from 10 to 1,000 pg/mL. The assay was reproducible and precise with intra-assay coeffi-cient of variations (CVs) from 6.0% to 11.2%, inter-assay CVs from 8.0% to 13.0% and good recovery [(90-110)%] and linearity [(89-107)%]. Excellent correlation was found between this new assay and the reference method (r = 0.96, P < 0.000,1). The successful establishment of this assay provides high possibility for carrying out bead-based multiplex assay measuring aldosterone and other parameters simultaneously in one 50 μL sample so that the efficiency can be improved and precious samples can be saved.     

R—藻红蛋白固相免疫荧光检测中洗涤条件的筛选

目的 筛选R-藻红蛋白免疫荧光检测中适宜的洗涤条件。方法 采用二步法改变洗涤液离子强度、pH值,比较弱阳性血清的检出率及阴性假阳性率,由此筛选最佳洗涤条件。结果 洗涤液离子强度的增加有助于降低阴性血清假阳性现象,在本实验所涉及样品范围内,摩尔浓度大于100mmol/L时,假阳性率可降低到0;120mmol/L时,阳性检出率最高值达93.3%。中性、偏碱性的pH值对检出率影响较小。结论 以120mmol/LPBS（pH7.5)为洗涤液有利于提高R-藻红蛋白荧光免疫法的特异性和灵敏度。     

A blocking-free microfluidic fluorescence heterogeneous immunoassay for point-of-care diagnostics

In this article, a rapid, sensitive, and disposable microfluidic immunosensor is presented for point-of-care (POC) testing and clinical diagnosis. For the first time, the blocking process is eliminated from a microfluidic heterogeneous immunoassay by using protein A functionalized polydimethylsiloxane microchannels. The nonspecific binding of the assay is maintained around the chip background level by using a pair of antibodies with different affinity to protein A under optimized experimental conditions. C-reactive protein (CRP), a biomarker for inflammation and cardiovascular disease risk assessment, is selected as a model analyte to demonstrate the sensitivity of this blocking-free microfluidic heterogeneous immunoassay. A four parameter logistic function is used to model and assess the data. The limit of detection obtained is 0.54 μg/mL, which is lower than the cut-off value for clinical diagnosis. The overall assay is completed in 5 min. The protein A modified PDMS chips wet-stored at 4°C can maintain biofunctionality up to 14 months. The developed blocking-free microfluidic heterogeneous immunoassay will immediately provide benefits to most immunosensing microdevices targeted for POC diagnostics by shortening analysis time, simplifying fluid transportation, reducing sample consumption, and lowering waste generation.     

Laboratory evaluation of five assay methods for vancomycin: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay, radioimmunoassay, and fluorescence immunoassay

We compared the precision and accuracy of five methods used to measure the concentration of vancomycin in serum: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay (FPIA [TDX; Abbott Laboratories, North Chicago, Ill.]), radioimmunoassay (RIA), and fluorescence immunoassay. Based on an analysis of seven standards and of 106 patient samples, all five methods were accurate, and four (bioassay, high-pressure liquid chromatography, FPIA, and RIA) were also precise. The FPIA was the most precise and the fluorescence immunoassay was the least precise of the methods tested; intrarun coefficients of variation for these two methods were 0.9 to 3.0% versus 8.9 to 14.5%, and interrun coefficients of variation were 2.8 to 8.1% versus 12.2 to 16.2%, respectively. The RIA was inconvenient because it required an extra dilution of the specimen being tested and an additional (64 micrograms/ml) vancomycin standard for specimens with 32 to 64 micrograms of vancomycin per ml. Based on its rapid turnaround time and the stability of its standard curve, we believe that the FPIA is the best method currently available to quantitate vancomycin in the clinical laboratory.     

上转换荧光材料的化学修饰及在免疫检测的应用

上转换纳米材料是近年来发展起来的新兴稀土荧光材料。在近红外光的激发下可将其转变成可见光这一光学特性,使其具有抗光漂白能力强、宽的反斯托克斯位移、低毒性以及无自体荧光干扰等优点,在免疫检测中可提高灵敏度和信噪比。上转换稀土纳米颗粒水溶性和分散性较差,因此要通过化学修饰成水溶性和分散性较好的上转换稀土颗粒,以进一步与生物分子偶联。本文主要在合成上转换纳米颗粒的基础上,对化学修饰的方法及荧光共振能量传递机制、基于磁分离富集的免疫检测技术、固相微孔板荧光标记技术、免疫传感技术和免疫层析技术的应用研究做一综述。上转换技术的成功应用解决了传统的纳米材料应用中低灵敏度的问题,在医学检测领域具有很大的应用潜力。     

Comparison of fluorescence polarization immunoassay and bioassay of vancomycin.

Human serum samples were analyzed for vancomycin concentrations by two different methods: the fluorescence polarization immunoassay and the disk plate bioassay. Each assay method offered acceptable precision. The correlation between both assay methods was excellent (correlation coefficient = 0.985). Excluding technical time, the bioassay was the least expensive method to perform but was more labor intensive than the fluorescence polarization immunoassay.     

Particle concentration fluorescence immunoassay (PCFIA): a new, rapid immunoassay technique with high sensitivity

A new solid-phase fluorescence immunoassay technique is described and is exemplified by the detection of murine monoclonal antibodies to human IgG in hybridoma culture supernatants and the detection of murine IgG. The assay is performed in a specially designed 96-well plate. For antibody detection, antigen bound to submicron polystyrene particles is bound to its specific antibody, which is in turn reacted with fluorescein-labeled affinity-purified goat anti-mouse IgG. The reaction is complete in 10 min at ambient temperature. The solid phase is separated from the reaction mixture by filtration, washed and the total particle-bound fluorescence is determined by front-surface fluorimetry. The sensitivity of the technique for antibody detection is equivalent to enzyme-linked immunoabsorbent assay and 2-4 ng/ml for murine IgG detection. It is readily amenable to automation.     

Solid-phase Clq-binding fluorescence immunoassay for detection of circulating immune complexes.

A fluorescence immunoassay for detection of immune complexes bound to solid-phase C1q was developed. The method was standardized by using human aggregated immunoglobulin G (IgG) to simulate immune complexes. A linear relationship existed between the concentrations of the aggregated IgG standards and the resulting fluorescent intensity. The method was found to be reproducible and capable of detecting as little as 10 micrograms of aggregated IgG per ml of heat-inactivated human serum. Antigen-antibody complexes prepared in vitro were detectable from equivalence to moderate antigen excess. Endogenous serum C1q inhibited the binding of aggregated IgG to solid-phase C1q. Pretreatment of test sera with EDTA was ineffective in eliminating this competitive effect. Heating the sera at 56 degrees C alleviated, but did not abolish, interference of endogenous C1q. Elevated levels of immune complexes were detectable in sera fro seven of nine patients wit systemic lupus erythematosus, provided the samples were heat inactivated before testing. Heparin and DNA were also found to interfere with the detection of aggregated IgG added to human serum. Assay values were falsely decreased due to competitive inhibition by these anions. Lipopolysaccharides from a variety of bacterial preparations produced no detectable interference. A comparative study was conducted on samples that had previously been tested by fluid-phase C1q-binding radioimmunoassay. The two methods were concordant in assigning normal or elevated levels of immune complexes in 70% of the samples tested. This solid-phase fluorescence immunoassay is proposed as a possible alternative to radioimmunoassay for the detection of circulating immune complexes.     

Evaluation of fluorescence excitation transfer immunoassay for measurement of specific proteins

Fluorescence excitation transfer immunoassay is a suitable technique for the measurement of serum IgG and CRP. The reagents, once reconstituted, are stable for at least 3 months. The method shows no interference due to bilirubin, lipaemia or haemolysis up to high levels. The assay is simple to perform, reliable and offers considerable advantages over manual techniques such as radial immunodiffusion or electroimmunoassay.     

An immunoassay for histamine based on monoclonal antibodies.

Monoclonal antibodies with high specificity for histamine as well as for 1-methylhistamine were obtained after immunization of mice with a conjugate where the histamine was coupled via its ring 1-nitrogen to dog serum albumin. An immunoassay was developed for the quantitation of histamine release from basophils and 1-methylhistamine release from mast cells after provocation. The test method is based on competitive inhibition between histamine and a labelled histamine conjugate for the antigen binding sites of the antibodies. The separation step is performed by the addition of solid phase bound anti-mouse subclass specific antibodies. The sensitivity of the assay is 2 micrograms/l for histamine and 0.1 micrograms/l for 1-methylhistamine. No cross-reactivity was obtained with other metabolites of histamine or with histidine. Serotonin and dopamine were detectable, but only in doses (mg/l) well above the normal concentration found in the circulation. The immunoassay has been evaluated for its capacity to measure histamine release in vitro. A good correlation with the conventional fluorometric assay was obtained when histamine released from allergen stimulated leucocytes from allergic patients was tested. Urinary samples from patients undergoing hyposensitization showed a mean excretion of 1-methylhistamine at a level of 131 mumol MeHi/mol creatinine. The release of histamine and 1-methylhistamine in vivo was examined in plasma samples taken during a bronchial provocation test. A significant elevation above the basal analyte level occurred ten minutes after provocation.