Evaluation of a preclinical blood test for scrapie in sheep using immunocapillary electrophoresis

An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.     

Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.

Transmissible spongiform encephalopathies in humans and in animals are fatal neuro-degenerative diseases with long incubation times. The putative cause of these diseases is a normal host protein, the prion protein, that becomes altered. This abnormal prion protein is found mostly in the brains of infected individuals in later stages of the disease, but also can be found in lymphoid and other tissues in lower amounts. In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl terminal region, amino acid positions 218-232, was labeled with fluorescein during the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, from sheep infected with scrapie and elk infected with chronic wasting disease. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the capillary electrophoresis immunoassay for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals. This assay makes a pre-clinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for manufacture of pharmaceuticals and products for human consumption.     

Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation

The development of capillary electrophoresis (CE)-based competitive immunoassay for prion protein (PrP) using carboxymethyl beta-cyclodextrin (CM-beta-CD) as a buffer additive is described here. The assay was based on the competitive binding of PrP and a fluorescein-labeled peptide from the prion protein with a limiting amount of specific antibody. The amount of both free and fluorescein-labeled peptide bound to antibody (immunocomplex) were determined by CE with laser-induced fluorescence detection. In the presence of PrP, the peak height ratio of the immunocomplex and the free peptide was altered compared to the control. These changes were directly proportional to the amount of PrP present. The fluorescently labeled peptide spanning amino acid positions 140-158 of the PrP and its corresponding monoclonal antibody is reported here. The reaction times of the antibody with either the peptide or the recombinant PrP was less than 1 min and is a large improvement over the 16-18 h required to achieve equilibrium for polyclonal antibodies. CM-beta-CD was explored as a buffer additive to suppress analyte adsorption and enhance separation selectivity in the CE analysis. A fast (1.1 min), selective (resolution 4.7), and reproducible (relative standard deviations of migration time for free and bound fluorescein isothiocyanate (FITC)-peptide 0.56% and 0.64%, respectively) separation was obtained with 0.6% CM-beta-CD in 25 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) at pH 8.8. The concentration detection limit of the assay for recombinant PrP was determined to be 80 ng/mL (or mass detection limit 1 pg). When blood samples from scrapie-infected sheep and from normal sheep were tested, the results of the blood assay were consistent with scrapie status of the sheep as determined post mortem by Western blot analysis. Development of this assay will lead to a potentially robust, rapid, and specific preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) in animals and humans.     

Immunonanogold-catalytic resonance scattering spectral assay of trace human chorionic gonadotrophin

Gold nanoparticles in diameter of 10 nm were used to label rabbit anti-human chorionic gonadotrophin (RhCG) antiserum to obtain a resonance scattering spectral probe (AuRhCG) for human chorionic gonadotrophin (hCG). The immunoreaction between AuRhCG and hCG take place to form hCG–AuRhCG immunocomplex in pH 5.0 citric acid–Na₂HPO₄ buffer solution. The immunocomplex solutions were centrifuged to obtain the supernatant solution. The AuRhCG in the supernatant solution exhibited strong catalytic effect on the particle reaction between Ag⁺ and hydroquinone to produce gold–silver composite particles in pH 3.4 citric acid–trisodium citrate buffer solution. There is a stronger resonance scattering (RS) peak at 423 nm for the particles. With the addition of hCG, the AuRhCG in the supernatant solution decreased, and the RS intensity at 423 nm decreased. The decreased RS intensity ΔI_423 nm was proportional to the concentration of hCG in the range of 2.5–208.3 mIU/mL with a detection limit of 0.83 mIU/mL. This method has been applied to the determination of hCG in urine samples, with satisfactory results.     

RESISTANCE OF THE SCRAPIE AGENT TO INACTIVATION BY PSORALENS

–In searching for a nucleic acid within the scrapie agent, we employed psoralens which penetrate the coats of most conventional viruses, form photoadducts with their genomes, and block replication of the viruses. Five psoralens, at concentrations up to 500 times greater than those required to inactivate conventional viruses, did not influence scrapie agent titers in partially purified preparations from murine spleen and hamster brain. ³H-psoralens were used to monitor the formation of photoadducts within nucleic acid standards added to preparations of the scrapie agent. Since no inhibition of psoralen photoadduct formation was observed in these preparations, one of three possibilities seems likely: the scrapie agent is devoid of nucleic acid, the psoralens failed to penetrate the protein coat of the agent, or its nucleic acid is unreactive with psoralens.     

Electrochemical Detection of Schistosoma Japonicum Antibody Using Biocatalytic Deposition

Abstract

An ultrasensitive electrochemical immunoassay based on biocatalytic deposition has been proposed for the detection of Schistosoma japonicum antibody (SjAb) in infected rabbit serum. Schistosoma japonicum antigen (SjAg) was immobilized on the gold electrode surface via glutaraldehyde crosslink and then incubated with infected rabbit serum containing SjAb; finally, the goat anti-rabbit IgG labeled with alkaline phosphatase was sandwiched to form the immunocomplex on the gold electrode surface. The alkaline phosphatase converted nonelectroactive substrate into the reducing agent and the latter, in turn, reduced metal ions to form electroactive metallic product on the electrode surface. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for SjAb. Assay conditions such as the antigen concentration and enzymatic silver deposition time were optimized. The electrochemical immunosensor was able to realize a reliable determination of SjAb in the dilution range from 1:5000 to 1:100 with a detection limit of 1:6457 of dilution ratio. The feasibility of the proposed immunosensor for possible clinical applications was also investigated by analyzing real serum samples.     

Analysis of the performance of antibody capture methods using fluorescent peptides with capillary zone electrophoresis with laser-induced fluorescence

A method to analyze the performance of an antibody capture method using fluorescent peptides by capillary zone electrophoresis using laser-induced fluorescence (CZE-LIF) for detection has been developed. Fluorescent peptides from the prion protein were synthesized and the corresponding antibodies were produced in rabbits against these peptides. The antibodies were used to capture the fluorescent peptides. The antibodies were then bound to protein A Sepharose. After elution, the amount of fluorescent peptide that was captured vs. the total amount placed in the assay was evaluated by CZE-LIF. Of the three peptides used in this evaluation, it was found that the recovery was approximately 25-35%. When the abnormal prion protein was prepared from scrapie-infected brain samples from hamsters and a sheep using the previously described extraction method and this method, the amount of abnormal prion protein that was measured in the fluorescence immunoassay correlated with amounts estimated from Western blot. We conclude that this method can be used to detect abnormal prion protein in a tissue sample.     

Development of an enzyme-linked immunosorbent assay for pentachlorophenol

Pentachlorophenol (PCP) is a hazardous pollutant with toxicity and potential carcinogenic properties being a serious threat to the environment. In this work, the development of an immunoassay for PCP is presented. A hapten was synthesised and conjugated to protein for rabbit immunisation. Three polyclonal antibodies were obtained and the best results were achieved in the antibody-coated format using antiserum R3. Calibration range was 0.3–30.5 ng ml⁻¹, with an average I₅₀ value of 2.9 ng ml⁻¹ and a detection limit of 0.1 ng ml⁻¹. The specificity of the assay was tested against PCP structurally related compounds. The method is highly specific for PCP and shows low cross-reactivity (CR) for chlorine-containing phenols, nitrophenols, benzenic and piridinic compounds. The good recoveries achieved with different water samples indicate that this assay can be a good alternative method for the determination of PCP in this kind of samples.     

Supramolecular Amperometric Immunosensor for Detection of Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) is a hormone produced in high concentrations through the placental trophoblasts and is used for the detection of pregnancy and certain diseases. Here we explored a supramolecular strategy for the potentially substrateless amperometric detection of hCG. A carboxymethylcellulose (CMC) carrier was synthesised and trifunctionalised with anti‐βhCG antibody, horse radish peroxidase (HRP) and ferrocene (Fc) moieties. The ferrocene was used to house the functionalised CMC within the cavities of electrode surface immobilised cyclodextrin, via host‐guest interactions. hCG was detected via a sandwich format, forming an immunocomplex between the surface immobilised antibody and a glucose oxidase/lactate oxidase labelled secondary antibody. Following formation of the immunocomplex, lactate/glucose, which would typically be present in serum/urine samples, was added and the hydrogen peroxide formed detected at the electrode surface via the HRP‐Fc enzyme‐mediator couple. The work reported demonstrates a potential supramolecular platform for the detection of targets in blood/urine samples using endogenous substrates.     

Ultrasensitive and accelerated detection of ciguatoxin by capillary electrophoresis via on-line sandwich immunoassay with rotating magnetic field and nanoparticles signal enhancement

A sensitive and rapid on-line immunoassay for the determination of ciguatoxin CTX3C was developed based on a capillary mixing system, which was integrated with capillary electrophoresis (CE) separation and electrochemical (EC) detection. In the sandwich immunoassay system, anti-CTX3C-functionalized magnetic nanoparticles were used as immunosensing probes, and horseradish peroxidase (HRP) and anti-CTX3C antibody were bound onto the surface of gold nanoparticles (AuNPs) and used as recognition elements. Online formation of immunocomplex was realized in capillary inlet end with an external rotating magnetic field. Compared with classical HPLC-MS and ELISA, the assay adopting AuNPs as multienzyme carriers and online sandwich immunoassay format with rotating magnetic field exhibited higher sensitivity and shorter assay time. The linear range of the assay for CTX3C was from 0.6 to 150 ng/L with a correlation coefficient of 0.9948 (n = 2), and the detection limit (S/N = 3) was 0.09 ng/L. The developed assay showed satisfying reproducibility and stability, and it was successfully applied for the quantification of CTX3C in fish samples.     

超高效液相色谱-串联质谱法测定阿胶中马、牛、羊、猪、骆驼、鹿皮源成分

建立了阿胶中马、牛、羊、猪、骆驼、鹿皮源成分的检测方法。通过比对驴皮和马皮、牛皮、猪皮、羊皮、骆驼皮、鹿皮胶原蛋白的序列,采用蛋白酶切技术和高分辨质谱技术发现理论的各杂皮胶原蛋白特征肽。同时建立三重四极杆质谱筛查方法,以含0.1%（v/v）甲酸的乙腈溶液和0.1%（v/v）甲酸水溶液作为流动相,梯度洗脱,电喷雾离子源（ESI）正离子扫描模式,多反应监测。15批阿胶样品检出了杂皮源成分。该方法操作简便,专属性强,可用于阿胶中杂皮源成分的鉴别,并已成功用于阿胶日常打假监督抽验工作。     

Study of stability of variants of ovine prions with different susceptibilities to scrapie

Well-defined set of sheep PrP polymorphisms at positions 136, 154 and 171 define susceptibility to scrapie, ranging from very high susceptibility observed for V136-R154-Q171 (VRQ) variant to resistance for A136-R154-R171 (ARR).To gain insight into the mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the sheep prion protein variants were analysed by differential scanning calorimetry over a wide range of pH. Thermal unfolding occurs, in the 5.0 to 6.0 pH range, through a reversible one-step process while at pH<4.5 and >6.0 unfolding intermediates are formed, which are stable in the 65–80°C range. The observed differences correlate with ovine susceptibility to scrapie.This revised version was published online in November 2005 with corrections to the Cover Date.     

快速检测中药材中黄曲霉毒素B1的电化学生物传感器

结合新型纳米传感膜材料,以黄曲霉毒素B1(AFB1)单克隆抗体为生物识别元件,通过考察AFB1与抗体之间的相互作用对测试底液中[Fe(CN)₆]^3-/[Fe(CN)₆]^4-([Fe(CN)₆]^3-/4-)氧化还原体系的影响,构建了一种可用于中药材中AFB1快速检测的电化学生物传感器。在最佳条件下,对AFB1浓度的线性响应范围为0.1~100 pmol/L,检出限为14.7 fmol/L。应用该方法测定大枣、薏苡仁、决明子中AFB1的含量,加标回收率在95.3%~109.5%之间。     

Purification and determination of human prostate specific antigen in the serum

A human prostate specific antigen (PA) has been purified from an extract of prostatic tissue obtained during operation for benign prostatic hypertrophy (BPH). The antigen, which can be demonstrated a single component by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), has an apparent molecular weight of about 34,000 and has lower mobility for the positive pole than prostatic acid phosphatase (PAP). Double antibody radioimmunoassay (RIA) for PA in serum was developed with the antiserum raised in rabbit against partially purified PA. In normal serum of 30 controls the concentration were studied by the RIA. The normal upper limit of the serum PA levels in assay was set at 2.5 ng/ml. Elevated levels were observed in serum from 19 out of 21 untreated patients with prostatic carcinoma and 9 out of 23 patients with BPH, but latter less than 10 ng/ml. The results indicate that the PA is a potentially useful marker as well as PAP for prostatic cancer.     

A mass spectral library based on chemical ionization and collision-induced dissociation

Antibodies were purified from normal rabbit, sheep, goat, rat, human and bovine serum using preparative electrophoresis on a Gradiflow in a single-step process using an asymmetrical cartridge with three different pore size polyacrylamide membranes. Recoveries in each case were over 80% and were higher than those obtained using affinity chromatography on protein A, protein G or protein L. Degree of purity was at least comparable with these methods. These results suggest that preparative electrophoresis can be considered a general method for the purification of research quantities of antibodies from multiple serum sources and may be particularly useful where the reactivity with protein A, G or L is unknown.     

Immunogenicity Analysis of a Novel Subunit Vaccine Candidate Molecule—Recombinant L7/L12 Ribosomal Protein of <Emphasis Type="Italic">Brucella suis</Emphasis>

Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella, livestock’s reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene’s expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein.     

Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography

Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 μm I.D. columns give better separations than 50 or 75 μm I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C₁ or C₁₈ hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress. Analysis of sheep liver extracts using optimised conditions showed the expression of at least 4 MT isoforms in response to Zn injection and 3 of these forms were evident in extracts from untreated sheep. We therefore conclude that MECC is a suitable method for MT isoform analysis.     

Single-molecule immunoassay and DNA diagnosis

Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.     

Ag+存在下催化还原铬黑T及应用于Ag+检测

以铬黑T(EBT)为探针,在有Ag⁺存在的最优条件下,EBT被NaBH 4催化还原,体系在445 nm发射波长处荧光强度明显增强,基于此构建一种光学测定溶液中微量Ag⁺的方法。在最优条件下,Ag⁺浓度在(7.5×10-9~1.0×10^-6 mol·L^-1)范围内呈良好的线性关系,线性回归方程为ΔI F=5.59×10⁸ c-31(c,mol·L^-1,r=0.9966),Ag⁺的最低有效检测浓度为6.0×10^-8 mol·L^-1。在最优条件下,运用该方法对样品中Ag⁺浓度进行检测,样品中Ag⁺浓度的检测回收率在98.8%~102.4%之间,RSD=2.1%。该检测方法能够对水溶液中微量的Ag⁺进行有效定量检测,且操作简单、检测限低、灵敏度高。     

Development of a surface plasmon resonance-based assay for the detection of Corynebacterium pseudotuberculosis infection in sheep

Caseous lymphadenitis (CLA), a disease affecting sheep and goats, is caused by Corynebacterium pseudotuberculosis and is difficult to detect, especially at early stages in its development. A surface plasmon resonance-based biosensor assay for the detection of antibodies to the phospholipase D (PLD) exotoxin of C. pseudotuberculosis in sheep serum was successfully generated. It employed a recombinant form of PLD, which was immobilised, and all aspects of the assay including minimisation of non-specific binding, and the regeneration of the chip, were optimised. The applicability of the assay was initially demonstrated using sera collected from experimentally infected sheep and from sheep with no prior history of infection. The assay was then evaluated on a panel of clinical samples and the results obtained compared very favourably to those obtained by a double sandwich ELISA (over 90% similarity) and clearly verified its analytical value.