Increased sensitivity of autoantibody determination by coupled-particle light-scattering assay by poly(ethylene glycols)-modified beads

New kinds of coatings by using polyethylene glycols were studied and were applied to a qualitative homogeneous immunoassay that exploits the agglutination reaction for the detection of autoimmune antibodies in a complex matrix. We used a piece of new technology, the Copalis^™, which uses a special optical-sizing flow particle analysis and a semiconductor laser as a light source.

Polystyrene microbeads coated with the antigen were used as markers and were put in contact with the serum sample. Different polyethylene glycols were synthesised and tested, optimising the experimental parameters. Human serum specimens were evaluated and we obtained a higher sensitivity with good discrimination between negative and positive samples. All the experimental steps are easy, rapid and enable us to process many samples in a short period of time.     

A Direct Competitive Homogeneous Immunoassay for Progesterone – the Redox Quenching Immunoassay

A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti‐ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation‐free progesterone immunoassay with a lower detection limit of 1 ng mL^?1 (3.18 nmol L^?1) in 1 : 2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium‐PEG‐progesterone tracer and a bioconjugate of one anti‐progesterone and one anti‐ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.     

Homogeneous Amperometric Immunoassay for Digoxin

Abstract

A homogeneous spectrophotometric EMIT immunoassay for the measurement of digoxin has been converted to an amperometric immunoassay format capable of analysing digoxin in diluted serum. NADH produced by glucose-6-phosphate dehydrogenase (G6PDH) labelled digoxin was detected amperometrically by its rapid, efficient oxidation at platinized activated carbon electrodes (PACE) poised at +150 mV vs Ag/AgCl. The new amperometric procedure resulted in a simplified assay protocol with a decreased overall assay time, enhanced NADH detection and minimal sample pretreatment.     

A sensitive and rapid immunoassay for quantification of testosterone by microchip electrophoresis with enhanced chemiluminescence detection

A sensitive and rapid approach to perform testosterone (T) competitive immunoassay by microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed. The assay is based on the competitive immunoreactions between T and N-(4-aminobutyl)-N-ethylisoluminol-labeled T (ABEI-T) with a limited amount of antibody (Ab), and the rapid electrophoretic separation of an equilibrated mixture of ABEI-T-Ab complex and free ABEI-T, followed by CL detection using horseradish peroxidase-catalyzed ABEI-H(2)O(2) system. Free ABEI-T and the ABEI-T-Ab complex are well separated within 30 s under the assay conditions. The developed method could be used to determine T with good precision and a detection limit lower than 1.0 nM. This method was applied for the quantification of T in human serum. The results demonstrated that the current MCE-CL-based competitive immunoassay maybe served as an alternative tool for clinical analysis.     

心衰标志物可视化联合检测方法研究

基于夹心免疫原理和表面免疫吸附模型,采用Image Pro Plus软件对硅片表面磁颗粒分布图像进行定量化处理,建立了硅片表面心衰标志物可视化联合检测方法。研究表明,该方法从抗体包被到获得检测结果不超过1 ｈ,操作简单,在单张硅片上可实现心衰标志物NT-proBNP和cTnI的联合检测。NT-proBNP和cTnI的最低检出限分别为21.8 pg/mL和0.034 ng/mL,检测范围分别为21.8~40000 pg/mL和0.034~50 ng/mL。83例不同程度心血管疾病患者的血清样本的检测结果与商品化免疫分析仪检测结果一致,相关系数r>0.98。因此,该快速免疫检测方法可以满足临床检测心衰标志物的需要。     

A novel mimetic enzymatic fluorescence immunoassay for hepatitis B surface antigen by using a thermal phase separating polymer

Iron tetrasulfonatophthalocyanine (FeTSPc), a peroxidase mimic, was used as a labeling reagent and poly(N-isopropylacrylamide) (PNIP) as the separation support of the immune complex for the mimetic-enzymatic immunoassay of hepatitis B surface antigen (HBsAg). PNIP was precipitated from aqueous solution when the ambient temperature was higher than its lower critical solution temperature of 31 degrees C. In a sandwich immunoassay, the antigen (HBsAg) first reacted with mouse anti-human HBsAg antibody immobilized on PNIP (PNIP-antibody) and then further reacted with FeTSPc-labeled mouse anti-HBsAg antibody (antibody-FeTSPc) at room temperature in a homogeneous format. After changing the temperature to separate the PNIP-antibody-HBsAg-antibody-FeTSPc conjugate moiety, it was re-dissolved and determined by coupling with the fluorogenic reaction of hydrogen peroxide and p-hydroxyphenylpropionic acid. The sensitivity of this method (3 ng mL-1) was close to that of the traditional ELISA using the same reactants. However, the assay was much faster (the assay time decreased from 100-120 to 45 min). This method was applied to determine HBsAg in human serum with satisfactory results.     

Novel solution-phase immunoassays for molecular analysis of tumor markers

at 3 x 10(9) M(-1) and a step-wise binding process with PSA-free MAB. Thus, this solution-phase quantitative ECL immunoassay allowed measurement of the affinity of serum PSAs with their MABs and screening of PSAs based upon their affinity to MABs. Unlike other immunoassays, this immunoassay demonstrated one-step rapid analysis while simultaneously eliminating immobilization, separation and washing steps and detected PSA at a level of 1.7 pg mL(-1), which is 1000-fold more sensitive than current PSA immunoassays. Furthermore, single-molecule (SM) phosphorescence microscopy was developed to detect single serum PSA-free and PSA-complex molecules in solution with no use of antibody showing that PSA-free molecules diffused faster than PSA-complex molecules in solution. This finding is consistent with ECL measurements and implies the possibility of screening individual analytes in a complex mixture using their distinct SM diffusion distance. This is the first report describing the detection of single protein molecules labeled with a metal-complex using phosphorescence microscopy and also the screening of serum tumor markers using ECL and SM phosphorescence solution-phase assays.     

A novel piezoelectric immunosensor for detection of carcinoembryonic antigen

A simple, rapid, and highly sensitive immunosensor for the direct determination of carcinoembryonic antigen (CEA) in human serum using a piezoelectric crystal has been developed and optimized. In order to improve sensitivity of the immunosensor, a protein A-based orientation-controlled immobilization method for antibodies was adopted together with an immunoreactive accelerant of polyethyleneglycol (PEG) used to amplify the signal response of frequency. Human normal serum was utilized as a reference background. The linear range for CEA concentration obtained by the end-point method was 66.7-466.7 ng/mL. Clinical samples from cancer patients were analyzed by the proposed piezoelectric immunoassay, and the analytical results were reasonably comparable with those obtained by the chemiluminescence immunoassay (CLIA). The proposed immunosensor provides a new promising method for the highly sensitive immunoassay of CEA in clinical laboratory.     

Gas chromatography-tandem mass spectrometry analysis of gabapentin in serum

A new highly sensitive analytical method for determining gabapentin [1-(aminomethyl) cyclohexaneacetic acid; Neurontin] in serum using gas chromatography/tandem mass spectrometry (GC-MS/MS) was developed. GC-MS/MS was applied to determine the levels of gabapentin in serum samples of mice at 1 and 6 h after oral or intraperitoneal treatment (300 mg/kg). At 1 h, the concentrations of the drug were 4.02 +/- 0.42 and 4.32 +/- 0.28 microg/mL in mice treated orally and intraperitoneally, respectively. At 6 h, drug levels decreased by about 66% in both groups. The method, coupling two stages of mass analysis, could be very useful in identifying the drug in complex mixtures such as blood and urine. Moreover, it is easy and rapid to perform, and sensitive enough to allow the presence of the drug to be determined at very low detection limits. It is a very reliable method for both clinical and experimental monitoring of gabapentin.     

Immune Complex Transfer Enzyme Immunoassay for Anti-Thyroglobulin IgG Using 2,4-Dinitrophenyl-thyroglobulin,Biotinyl-thyroglobulin and Streptavidin-β-D-galactosidase Conjugate

Abstract

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in serum is described. Anti-thyroglobulin IgG in serum was reacted simultaneously with 2,4-dinitrophenyl-thyroglobulin and biotinyl-thyroglobulin. The immune complex formed of the three components was trapped onto polystyrene balls coated with (anti-2,4-dinitrophenyl group) IgG and, after washing, reacted with streptavidin-β-D-galactosidase conjugate. After washing, the immune complex was eluted from the polystyrene balls with εN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with (anti-human IgG γ-chain) IgG. β-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Biotinylthyroglobulin and streptavidin-β-D-galactosidase conjugate could be prepared more easily than thyroglobulin-β-D-galactosidase conjugate used in the previous immunoassay. Inactive β-D-galactosidase, used to eliminate interference by anti-β-D-galactosidase antibodies in the previous immunoassay, was not required. The present immunoassay was 300-fold more sensitive than the conventional enzyme immunoassay, although 10-fold less sensitive than the previous immunoassay. Antithyroglobulin IgG was demonstrated in all patients with autoimmune thyroid diseases and 54% of healthy subjects.     

A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood

We describe an automated, self-powered chip based on lateral flow immunoassay for rapid, quantitative, and multiplex protein detection from pinpricks of whole blood. The device incorporates on-chip purification of blood plasma by employing inertial forces to focus blood cells away from the assay surface, where plasma proteins are captured and detected on antibody "barcode" arrays. Power is supplied from the capillary action of a piece of adsorbent paper, and sequentially drives, over a 40 minute period, the four steps required to capture serum proteins and then develop a multiplex immunoassay. An 11 protein panel is assayed from whole blood, with high sensitivity and high reproducibility. This inexpensive, self-contained, and easy to operate chip provides a useful platform for point-of-care diagnoses, particularly in resource-limited settings.     

Homogeneous fluorescence-based immunoassay via inner filter effect of gold nanoparticles on fluorescence of CdTe quantum dots

Homogeneous immunoassays are becoming more and more attractive for modern medical diagnosis because they are superior to heterogeneous immunoassays in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. This assay relies upon the inner filter effect (IFE) of gold nanoparticles (AuNPs) on CdTe QDs fluorescence. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the IFE of AuNPs on CdTe QDs fluorescence is greatly enhanced, resulting in a decrease of fluorescence intensity in the system. Based on this phenomenon, a wide dynamic range of 1-100 pg mL(-1) for determination of IgG can be obtained. The proposed method shows a detection limit of 0.3 pg mL(-1) for human IgG, which is much lower than the corresponding absorbance-based approach and compares favorably with other reported fluorescent methods. This immunoassay method is simple, rapid, cheap, and sensitive. The proposed method has been successfully applied to measuring IgG in serum samples, and the obtained results agreed well with those of the enzyme-linked immunosorbent assay (ELISA).     

Capillary electrophoresis immunoassay based on an on-column immunological reaction

An on-column immunological reaction was employed to achieve simple and rapid analysis in an immunoassay based on capillary electrophoresis using semiconductor laser-induced fluorescence detection. Human serum albumin (HSA) labeled with sulfoindocyanine succinimidyl ester (Cy5), a fluorescent compound with an absorption maximum at 649 nm, was used as a fluorescent probe for the immunoassay. In a binding assay, with anti-HSA as the analyte molecule, Cy5-HSA was injected in a capillary column followed by the injection of anti-HSA so as to form individual zones. By applying a potential, the anti-HSA reacted with Cy5-HSA at the boundary between Cy5-HSA and anti-HSA zones, since anti-HSA has a higher electrophoretic mobility than Cy5-HSA. Furthermore, the on-column method enhances the sensitivity by injecting a large volume of the sample. Free Cy5-HSA and its immunocomplex with anti-HSA were separated with less degradation in resolution than that predicted from the injection time of anti-HSA, even when the injection time for anti-HSA was increased. The ratio of the peak area of the complex to that of the total Cy5-HSA (free Cy5-HSA and the complex) increased in proportion to the injection time of anti-HSA. As a result, the detection limit was improved up to eight-fold (the concentration detection limit, 0.007 mg mL(-1), for an injection time of 240 s, compared to that obtained using an off-column sample preparation. Furthermore, the on-column reaction method was applicable to an immunoassay to determine native HSA, in which native HSA and Cy5-HSA react with anti-HSA stepwise. The detection limit in the stepwise reaction immunoassay was 0.005 mg mL(-1), which is 14 times lower than that in an off-column method, with the analysis time less than 10 min as the result of increasing the injection time of native HSA. In addition, the present on-column immunoassay was applied to the sample containing a high concentration of salts for investigating the effect of salts in the sample solution.     

Enzyme immunoassay for the drug of anti-ulcer using avidin-biotin system.

We have developed a competitive enzyme immunoassay for a drug, which was a newly synthesized anti-ulcer agent, using an enzyme immunoassay. The polyclonal anti-drug antibody coupled to biotin, peroxidase labeled drug derivatives as a tracer, and a small column of Sepharose 4B covalently bound to avidin were used in the assay. This assay is simple and rapid, and the sensitivity and the measuring range can be controlled by the flow rate of the substrate solution. The correlation between serum drug concentrations (0.1-10 micrograms/ml) measured by gas chromatography and this assay was good (r = 0.991). This principle for the assay is very practical and applicable to the enzyme immunoassay for small and large molecules.     

Studies of interacting biochemical systems by flow injection analysis

Biochemical interactions of analytical importance can be investigated by heterogeneous and homogeneous techniques. In the former, small ligans are physically separated from macromolecular receptor molecules. This is technically complex but removes unwanted interferences. Homogeneous methods avoid the separation step and are rapid and convenient, but suffer more from background interferences. They also require the presence of suitable probe molecules. It is shown that flow injection analysis can be used advantageously with both heteregeneous and homogeneous techniques. Packed reactors containing group-specific receptors effectively reduce the background fluorescence of blood serum. Gradient flow-injection techniques are used with fluorescence probes in the rapid determination of ligand/protein binding parameters; the results agree well with those of slower, conventional procedures.     

Biofunctionalized dendritic polyaniline nanofibers for sensitive electrochemical immunoassay of biomarkers

Multi-armed dendritic polyaniline nanofibers (MPANFs) were first synthesized and functionalized with horseradish peroxidase (HRP) and carcinoembryonic antibody (anti-CEA) for highly efficient electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte here) in this work. Transmission electron microscope (TEM) and scanning electron microscope (SEM) techniques were employed to characterize the synthesized MPANFs. By using anti-CEA-conjugated core-shell gold-Fe(3)O(4) nanocomposites (GoldMag) as immunosensing probes and biofunctionalized MPANFs as molecular tags, a new sandwich-type homogeneous immunoassay strategy was developed for the determination of CEA by coupling with a home-made flow-through magneto-controlled microfluidic device. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of four orders of magnitude from 1.0 pg mL(-1) to 50 ng mL(-1) with a low detection limit of 0.1 pg mL(-1) CEA at 3σ. Intra- and inter-assay coefficients of variation were below 10%. The assayed results for clinical serum specimens with the electrochemical immunoassay were received in good accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.     

热敏相分离荧光免疫分析乙肝表面抗原的新方法

以N-异丙基丙烯酰胺为单体,将水溶性热敏高分子聚异丙基丙烯酰胺(PNIP)和抗体偶联,用异硫氰酸荧光素标记羊抗人乙肝表面抗原抗体,建立了夹心型热敏相分离荧光免疫分析乙肝表面抗原(HBsAg)的新方法.评价了抗体在PNIP上的固定效率和非特异性吸附情况.HBsAg在0.5～100μg/mL范围内与体系荧光强度呈良好的线性关系,检出限为10ng/mLHBsAg.该方法既具有均相免疫分析的快速性,又有固相免疫分析的高灵敏度.用于乙肝病人血清中HBsAg水平的测定,结果令人满意.     

Homogeneous time-resolved fluoroimmunoassay of 3,5,3'-triiodo-l-thyronine in human serum by using europium fluorescence energy transfer

A sensitive homogeneous time-resolved fluoroimmunoassay method for 3,5,3'-triiodo-l-thyronine (T3) based on the fluorescence resonance energy transfer (FRET) from a fluorescent Eu(3+) complex, {[(4,6-dichloro-1,3,5-triazin-2-yl)amino-biphenyl-4'-yl]-2,2':6',2'-terpyridine-6,6'-diyl}bis(methylenenitrilo) tetrakis(acetate)-Eu(3+) (DTBTA-Eu(3+)) (lambda(ex,max)=335nm, lambda(em,max)=615nm), to an organic fluorescence dye Cy5 has been developed. The new assay system combined the use of DTBTA-Eu(3+)-labeled T3-bovine serum albumin (BSA) conjugate and Cy5-labeled anti-T3 monoclonal antibody for a competitive-type immunoassay. After the competitive reactions of DTBTA-Eu(3+)-labeled T3-BSA and T3 sample with Cy5-labeled anti-T3 antibody, the T3 concentration was measured with a time-resolved mode by monitoring the sensitized emission of Cy5 derived from FRET in a homogeneous format. The method gives the detection limit of 0.26ng/ml. The coefficient variations of the method are less than 2.0% and the recoveries are in the range of 80-111% for serum sample measurement. The concentrations of T3 in 30 human serum samples were determined, and the results were compared with those of the independently determined by a radio-immunoassay method. A good correlation was obtained with a correlation coefficient of 0.989.     

Phase-resolved fluoroimmunoassay of human serum albumin

In the homogeneous immunoassay of human serum albumin described, a difference in fluorescence lifetime is used along with a small difference in fluorescence intensity to discriminate between the free and the antibody-bound labelled antigen. The immunoassay is based on the use of phase-resolved fluorescence measurements, in which sinusoidally-modulated excitation is combined with phase-sensitive detection to generate time-dependent signals which are integrated over a pi-interval to produce phase-resolved intensities. Texas Red was used as the fluorescent label. Negligible matrix effects were observed from serum, and a comparison of values determined by using the phase-resolved fluoroimmunoassay with values provided by the hospital from which the samples were obtained yielded a correlation coefficient of 0.996.     

Homogeneous immunoassay based on aggregation of antibody-functionalized gold nanoparticles coupled with light scattering detection

A universal platform of homogeneous noncompetitive immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. The assay is based on aggregation of antibody-functionalized gold nanoparticles directed by the immunoreaction coupled with light scattering detection with a common spectrofluorimeter. In phosphate buffer (pH 7.0) solution, the light scattering intensity of the gold nanoparticles functionalized with goat-anti-human IgG can be greatly enhanced by addition of the human IgG. Based on this phenomenon, a wide dynamic range of 0.05-10 microg ml(-1) for determination of human IgG can be obtained, and the detection limit can reach 10 ng ml(-1). The proposed immunoassay can be accomplished in a homogeneous solution with one-step operation within 10 min and has been successfully applied to the determination of human IgG in serum samples, in which the results are well consistent with those of the enzyme-linked immunosorbent assay (ELISA), indicating its high selectivity and practicality. Therefore, the gold nanoparticle-based light scattering method can be used as a model to establish the general methods for protein assay in the fields of molecular biology and clinical diagnostics.