Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs-a type 1 RIP (dianthin) and a type 2 RIP (ricin)-that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.     

A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.     

Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain

The ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G (IgG), was displayed on the cell surfaces of yeast Saccharomyces cerevisiae by cell-surface engineering using the C-terminal half of alpha-agglutinin under control of the 5'-upstream region of the isocitrate lyase gene from Candida tropicalis (UPR-ICL). Display of ZZ on the cell surface was confirmed by immunofluorescence microscopy. Enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA using the S. cerevisiae cells displaying ZZ detected IgG and antigen (human serum albumin) down to a concentration of 1-10 ng/ml in both cases. The detection range covered by these assay systems was wide and could be varied by adjusting the amount of cells and reaction times with horseradish peroxidase (HRP) substrate. Moreover, yeast cells displaying ZZ were successfully used for repeated affinity purification of IgG from serum. These results indicate that S. cerevisiae displaying ZZ may constitute novel and genetically renewable whole-cell immunoadsorbents widely applicable to immunoassays and affinity purification.     

Reliable enzyme-linked immunosorbent assay systems for pathogenic factors of Pseudomonas aeruginosa alkaline proteinase, elastase, and exotoxin A: a comparison of methods for labeling detection antibodies with horseradish peroxidase

Sensitive sandwich enzyme-linked immunosorbent assay (ELISA) systems for the quantification of 3 pathogenic factors of Pseudomonas aeruginosa-alkaline proteinase (aeruginolysin), elastase (pseudolysin ), and exotoxin A-were developed. The maleimide-pyridyl disulfide method was applied for the labeling of rabbit anti-each antigen IgG with horseradish peroxidase (HRP) and the conjugates were used as secondary antibodies (detection antibodies) in the ELISA systems. The EDTA, a chelating agent, was added to the buffers for sample and detection antibody, which inhibited the degradation of IgG by elastase derived from P. aeruginosa for improving the assay precision. The ELISA systems using the HRP-labeled detection antibodies produced by the maleimide-pyridyl disulfide method exhibited higher sensitivity than previously reported methods. The detection limits for alkaline proteinase, elastase, and exotoxin A were 18 pg/ml, 34 pg/ml, and 22 pg/ml, respectively. The intra-assay coefficients of variation for alkaline proteinase, elastase, and exotoxin A were 3.4%-5.0%, 1.9%-3.5%, and 1.3%-5.4%, respectively. These ELISA systems exhibited good inter-assay precision, non-cross-reactivity, dilution linearity, and recovery . Employing these ELISA systems, we revealed that pathogenic factor concentrations were different among the P. aeruginosa strains tested, which may relate to the different pathogenicity of each strain.     

Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.     

A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification

In this study, we report a simple, low-cost surface plasmon resonance (SPR)-sensing cartridge based on a loop-mediated isothermal amplification (LAMP) method for the on-site detection of the hepatitis B virus (HBV). For LAMP detection, a SPR based LAMP sensing system (SPRLAMP) was constructed, including a novel SPRLAMP sensing cartridge integrating a polymethyl methacrylate (PMMA) micro-reactor with a polycarbonate (PC)-based prism coated with a 50 nm Au film. First, we found that the change of refractive index of the bulk solution was approximately 0.0011 refractive index (RI) units after LAMP reaction. The PC-based prism's linearity and thermal responses were compared to those of a traditional glass prism to show that a PC-based prism can be used for SPR measurement. Finally, the HBV template mixed in the 10 μl LAMP solution could be detected by SPRLAMP system in 17 min even at the detection-limited concentration of 2 fg/ml. We also analyzed the correlation coefficients between the initial concentrations of HBV DNA templates and the system response (ΔRU) at varying amplification times to establish an optimal amplification time endpoint of 25 min (R(2)=0.98). In conclusion, the LAMP reaction could be detected with the SPRLAMP sensing cartridge based on direct sensing of the bulk refractive index.     

Dirofilaria immitis: performance and standardization of specific antibody immunoassays for filariasis

The factors associated with the development, optimization, and validation of immunoassays for the detection of parasite-specific antibody in filariasis infections were investigated using the dog heartworm, Dirofilaria immitis as a model. We examined two assays, the Protein A solid-phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent assay (ELISA), for quantitation of specific antibody to the parasite in canine serum. Precision, reproducibility, and parallelism were examined using response-error relationships and precision profile analyses. A staphylococcal Protein A saturation analysis was applied to the standardization of IgG anti-parasite antibody reference sera in weight per volume units (microgram/ml). Using the mean minimal detectable dose + 3 SD and an intraassay precision profile less than 10% coefficient of variation (CV) as criteria for assay sensitivity, the SPRIA and ELISA displayed comparable positive thresholds of 1 microgram/ml IgG anti-parasite antibody/ml of serum. Both assays demonstrated good reproducibility (less than 15% interassay CV) and parallelism (less than 20% interdilutional CV) over their working ranges (SPRIA: 1-40 micrograms/ml; ELISA: 1-5 micrograms/ml). Specificity of each assay was enhanced by preadsorption of cross-reacting antibodies in canine serum (i.e., specific for Toxocara canis antigens) with solid-phase antigen prior to assay. Methods for comparing different immunoassay designs are considered in relation to the variables that influence the assays' performance characteristics.     

酶联免疫吸附试验检测A型产气荚膜梭菌肠毒素

本实验建立了双抗体夹心ＥＬＩＳＡ检测产气荚膜梭菌肠毒素的实验体系。以家兔单特异ＩｇＧ包被酶标板,辣根过氧化物酶标记的绵羊ＩｇＧ作为指示物,可检测出１．２５ｎｇ／ｍｌ（０．１２５ｎｇ）的产气荚膜梭菌肠毒素,线性范围大,重复性及稳定性好,对培养上清及粪便滤液检查无非特异反应。是产气荚膜梭菌食物中毒实验室诊断的一种快速可靠的检测方法。     

Comparison of different carbon ink based screen-printed electrodes towards amperometric immunosensing

The aim of the present work was to make amperometric immunosensors based on the principle of enzyme-linked immunosorbent assay (ELISA). For this purpose, screen-printed electrodes (SPEs) were fabricated using various carbon inks (commercially available inks Gwent, Acheson, Eltecks and two homemade inks PSG & PVCG) to determine the best ink in realizing immunosensors. Amperometric immunosensors made by different carbon inks were compared with standard ELISA in terms of total assay time, amount of biological materials used and sensitivity of detection. A model system containing rabbit anti-mouse immunoglobulin G (RαMIgG) as the capturing antibody, mouse IgG (MIgG) as antigen and alkaline phosphatase conjugated RαMIgG as revealing antibody was used. In these studies, 1-naphthyl phosphate was used as substrate. The experiments done include electrochemical characterization of electrodes, optimization of dilutions of antibodies, immobilization of antibody on the electrode were carried out. The minimum detection limit for the best results of MIgG determination were obtained on screen-printed electrode made by Gwent carbon ink and PSG carbon ink, with a detection limit of 1.0 and 2.0 ng/ml respectively. The time required for detection of mouse IgG was 40 min for SPEs. By using the conventional spectrophotometric method (ELISA method), the minimum detection limit for the MIgG (antigen) detection was 50 ng/ml and the time required for analysis was found to be 140 min.     

A PCR-ELISA Method for Direct Detection of the Oyster Pathogen Haplosporidium nelsoni

A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), was developed for detection of oyster MSX disease. The technique included using Haplosporidium nelsoni pathogen-specific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle tissues), and cloned H. nelsoni rRNA plasmid DNA (for use as a capture probe). Digoxigenin was incorporated into the pathogen-specific PCR products, which were captured by the coated probe in a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification on cloned plasmid DNA was 10 fg for detection by stained agarose gel, and increased to 0.01 fg for ELISA. Positive signals were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection. Received April 14, 1998; accepted September 30, 1998.     

Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

CRP determination based on a novel magnetic biosensor

The c-reactive protein (CRP) is a very significant human blood marker for inflammatory processes and is routinely determined for many clinical purposes. The widespread and well established detection method for this approximately 115 kDa hepatic protein is the high-sensitivity ELISA assay (hsCRP-ELISA) in blood serum. New approaches in medical CRP diagnosis (e.g. for CVD, inflammatory bowel disease) require rapid quantification in native matrices. A novel CRP determination method based on magnetic detection is described and tested for human blood serum, saliva and urine. The detection principle is based on two different anti-CRP antibodies (monoclonal, IgG) for CRP trapment and labelling. The linear detection range of this immunosensor ranged from 25 ng/ml to 2.5 microg/ml and is therefore much more sensitive than typical hsCRP-ELISA-assays.     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and anatoxins

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).     

Detection of mucosal and serum antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b by enzyme-linked immunosorbent assay

A sensitive enzyme-linked immunosorbent assay (ELISA) with rough-surfaced glass beads used as the solid phase was developed for detection of IgG, IgM, and IgA antibodies to Haemophilus influenzae type b capsular polysaccharide (HITB-CP). A successful method for indirect coating of glass bead surfaces with HITB-CP, and parameters affecting the specificity and sensitivity of the assay are described. This ELISA system proved to be 100 times as sensitive as the standard indirect fluorescent-antibody assay. The assay was applied to the measurement of antibodies to HITB-CP in serum and nasal secretions and proved to be a useful tool in the evaluation of immunological response to HITB infection.     

牛血清蛋白酶联免疫检测试剂盒的研制及验证

为研制酶联免疫试剂盒以检测病毒性疫苗中残余牛血清蛋白(BSP)含量,制备高效价高纯度的兔抗BSP多克隆抗体作为包被抗体和酶标抗体,建立了ELISA双抗体夹心法并组建试剂盒,通过标准剂量曲线可对样品中所含BSP、BSA及B-IgG进行定量,经验证该方法标准曲线线性范围内r≥0.98,对BSP的检测限量为3ng/ml;分别检测5、10、20ng/ml含量的BSP时,试验内(n=12)和试验间(n=3)测定的变异系数在3.71%到7.29%之间,回收率在93.4%~106.3%,未见该方法与人血清白蛋白、卵清蛋白以及疫苗复合保护剂之间有交叉反应。该法敏感度高,准确性、重复性和稳定性好,可用于疫苗牛血清残余蛋白的质量控制。     

Highly stable electrochemical immunosensor for carcinoembryonic antigen

The long-term stability of sensing interfaces is an important issue in biosensor fabrication. A novel stable gold nanoparticle (AuNP)-modified glassy carbon (GC) electrode interface (GC-Ph-AuNP)-based biosensor for detecting carcinoembryonic antigen (CEA) was developed. GC electrodes were modified with 1,4-phenylenediamine to form a stable layer, and then AuNPs were bound onto the GC electrodes through CAu bonds. Anti-CEA was directly adsorbed on AuNPs fixed on the GC electrode. The linear range of the immunosensor was from 10 fg to 100 ng mL(-1) with a detection limit of 3 fg mL(-1) (S/N=3). The current of the immunosensor was increased by 4% after one month. The GC-Ph-AuNP immunosensor showed high sensitivity, a wide linear range, low detection limit, and good selectivity and stability. The immobilization method of the immunosensor could be widely applied to construct other immunosensors.     

Graphene-assisted dual amplification strategy for the fabrication of sensitive amperometric immunosensor

A sensitive electrochemical immunosensor with graphene-assisted signal amplification has been developed. In order to construct the base of the immunosensor, a novel hybrid architecture was initially fabricated by combining poly (diallyldimethylammonium chloride) functionalized graphene nanosheets (PDDA-G) and gold nanoparticles (AuNPs) via a simple sonication-induced assembly. The formed hybrid architecture provided an effective matrix for antibody immobilization with good stability and bioactivity. Subsequently, a smart, multilabel, and graphene-based nanoprobe that contains gold nanoparticles functionalized exfoliated graphene oxide and horseradish peroxidase-secondary antibodies was designed for constructing a novel sandwiched electrochemical immunosensor. Enhanced sensitivity was obtained by combining the advantages of high-binding capability and excellent electrical conductivity of hybrid architecture with the multilabel signal amplification. On the basis of the dual signal amplification strategy of graphene-based architecture and the multilabel, the immunosensor displayed excellent analytical performance for the detection of human IgG (HIgG) range from 0.1 to 200 ng/mL with a detection limit of 0.05 ng/mL at 3σ. Moreover, the proposed method showed good precision, acceptable stability and reproducibility, and could be used for the detection of HIgG in real samples. Therefore, the present strategy definitely paves a way for the wide application of graphene in clinical research.     

Multiplexed detection of cardiac biomarkers in serum with nanowire arrays using readout ASIC

Early detection of cardiac biomarkers for diagnosis of heart attack is the key to saving lives. Conventional method of detection like the enzyme-linked immunosorbent assay (ELISA) is time consuming and low in sensitivity. Here, we present a label-free detection system consisting of an array of silicon nanowire sensors and an interface readout application specific integrated circuit (ASIC). This system provides a rapid solution that is highly sensitive and is able to perform direct simultaneous-multiplexed detection of cardiac biomarkers in serum. Nanowire sensor arrays were demonstrated to have the required selectivity and sensitivity to perform multiplexed detection of 100 fg/ml troponin T, creatine kinase MM, and creatine kinase MB in serum. A good correlation between measurements from a probe station and the readout ASIC was obtained. Our detection system is expected to address the existing limitations in cardiac health management that are currently imposed by the conventional testing platform, and opens up possibilities in the development of a miniaturized device for point-of-care diagnostic applications.     

An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.     

An immunoassay for detection of heat-stable proteases from thermoduric psychrotrophic Bacillus spp. of dairy origin

A homogeneous preparation of a thermostable protease from Bacillus sp. B-17 was used to raise an antiserum in rabbits. IgG of this antiserum was used to study the antigenic relationship of proteases in cell-free extracts of 21 bacilli of milk origin. Based on immunological cross reactivity, the 21 bacilli were divided into 3 serological subgroups. To raise antibodies of broader specificity, protease from Bacillus sp. B-11 (group II) and B-3 (group III) were purified, mixed with purified B-17 protease, and an antiserum was raised against this mixture. IgG of this antiserum was purified (IgG anti-bacilli protease). A sandwich ELISA was standardized using IgG anti-bacilli protease as capture antibody. The assay could detect 1.2 ng ml(-1) of protease in milk or buffer, but the assay failed to detect 4 of 21 bacilli proteases. The results suggest that this assay is useful for the detection of proteases of Bacillus spp. in dairy industry.