A sensitive immunochromatographic assay using gold nanoparticles for the semiquantitative detection of prostate-specific antigen in serum

In prostate cancer screening, prostate-specific antigen (PSA) has been utilized as a valuable biomarker. There are routinely used procedures based on enzyme-linked immunosorbent assay (ELISA) for PSA detection. The procedures based on ELISA, however, are time consuming, complicated, and costly. We have developed a rapid, very simple, cost effective and sensitive immunochromatographic assay using gold nanoparticles and evaluated its applications for first screening of prostate cancer in serum samples. The sensitive immunochromatographic assay requires only 40 μL of the serum sample. The assay used is rapid and simple, that it totally takes approx 15 min to complete. The method for sensitive immunochromatographic assay has the other advantage of decreasing the antibody concentration that is used for the test line. In this study, we show the advantage to decrease the antibody concentration and the evaluation of our sensitive immunochromatographic assay for the semiquantitative detection of PSA in serum. The results obtained from 163 serum samples using sensitive immunochromatographic assay are compared with the results obtained using the chemiluminescent enzyme immunoassay (CLEIA) and normal immunochromatographic assay. The results obtained in the sensitive immunochromatographic assay correlated well with the values obtained in CLEIA. We concluded that our sensitive immunochromatographic assay is applicable to the first screening test for the diagnosis of prostate cancer. Our developed sensitive immunochromatographic assay is a promising candidate for diagnosis or research use, which may become commercially available in the near future.     

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum

Current clinically assays, such as enzyme‐linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high‐throughput analysis. A novel assay based on magnetic beads and time‐resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti‐HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium‐labeled anti‐HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra‐ and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well‐established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time‐resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high‐throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential diagnosis of the infection caused by wild-type or CD2v-deleted ASFV strains by quantum dots-based immunochromatographic assay

African swine fever (ASF), a highly contagious and lethal disease, poses a tremendous threat and burden to the swine industry worldwide. Lack of available vaccines or treatments leaves rapid diagnosis as the key tool to control the disease. Quantum dots (QDs) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In this study, we developed a quantum dots-based fluorescent immunochromatographic assay (QDs-FICA) using CD2v as the diagnosis antigen to detect ASFV antibodies. The titre of the test strip was 1 : 5·12 × 10⁵. In addition, the strip was highly specific to anti-ASFV serum and had no cross-reaction with CSFV, PPV, PRRSV, PCV-2, PRV and FMDV. Moreover, a comparative test of 71 clinical samples showed that the coincidence rate was 85·92% between the test strip and the commercial ELISA kit (coated with p30, p62 and p72). The QDs-FICA can be used to detect ASFV antibodies, which is meaningful for the surveillance, control and purification of ASF.     

Development of a test strip reader for a lateral flow membrane-based immunochromatographic assay

A low-cost, simple strip reader system using a linear movement mechanism of CD-ROM deck has been developed to characterize a lateral flow membrane-based immunochromatographic assay. The test strip reader was assembled by a CD-ROM deck and home-made optical head especially designed for immunoassays. The optical head for detecting reflected light from the test strip surface consists of green light-emitting diode, large area silicon photodiode, and anodized aluminum mounting block providing a slit structure for cutting light from the LED. The stepping motor of the deck was operated in the full step mode, whose distance of each reading point is about 0.15 mm. The performance of the strip reader was tested by analysis of HBV (hepatitis B virus) antigen test kit. This strip reader can be useful for inexpensive, disposable, and membrane-based assays that provide visual evidence of the presence of an analyte in a liquid sample.     

Novel evolved immunoglobulin (Ig)-binding molecules enhance the detection of IgM against hepatitis C virus

Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases.     

Assessment of immunochromatographic test for rapid lymphatic filariasis diagnosis

Two rapid immunodiagnostic tests (ICT Filariasis test), developed for the quick diagnosis of Wuchereria bancrofti infection, have been validated in laboratory and field situation. The aim of this study was to assess the performance and usefulness of this antigen capture assay as a diagnostic method in three foci of lymphatic filariasis, located in the South Pacific (Society archipelago, French Polynesia), with different levels of endemicity. A sample of 1,595 patients was tested with this assay in parallel with a reference Og4C3 antigen capture assay and microfilariae detection. A second-generation ICT test, available for whole blood analysis, was also tested in parallel with the first generation test, developed for serum analysis, on a sample of 50 reference cases. The correspondence between the results obtained with the two rapid tests was excellent, without any influence of rheumatoid factors, but the sensitivity was in both cases slightly inferior to the one obtained with the ELISA reference test. This seems particularly true in epidemiological situation where a high proportion of amicrofilaraemic, adult worm carriers are observed.     

副结核荧光PCR试剂盒研制与应用

采用TaqMan荧光标记探针技术原理, 建立副结核分枝杆菌特异的实时荧光PCR快速检测鉴定方法并组装形成临床诊断试剂盒。试剂盒提供荧光PCR与样品核酸提取试剂, 检测全程包括样品处理可在1 d内完成。特异性试验结果表明, 试剂盒对8株副结核分枝杆菌标准菌株的检测均呈典型阳性反应, 对牛分枝杆菌、结核分枝杆菌等其它12种分枝杆菌标准菌株以及大肠杆菌、肺炎链球菌等多种常见微生物均呈阴性反应。试剂盒检测灵敏度可达单个菌细胞、15个基因拷贝, 比常规PCR检测灵敏度提高100倍。对每份添加50~100个菌细胞的20份阴性牛奶样品进行检测, 均呈阳性反应。重复性试验结果显示, 试剂盒组内变异系数为1.41%, 组间变异系数为2.42%。采用所研制的副结核分枝杆菌荧光PCR试剂盒对来自广东地区7个奶牛场的250份牛奶样品和粪便样品、来自10个养猪场的143份猪血清样品, 以及3批次进口食蟹猴共100份血清样品进行检测, 检出牛奶样品中副结核分枝杆菌阳性率为7.7%, 牛粪样品中阳性率为3.7%, 猪血清样品中阳性率为8.2%, 进口猴血清样品中阳性率为3.0%。     

Evaluation of the usefulness of the ELISA method for determining the level of enterovirus antibodies in serum and cerebrospinal fluid

The usefulness of the methods was compared: complement fixation test (CFT), neutralization test (NT) and ELISA IgG and IgM against enteroviruses for the evaluation of specific immune reaction in sera and cerebrospinal fluid (CSF) samples of patients with confirmed enterovirus infections. The criteria were established for the assessment of ELISA results in rapid diagnosis of enterovirus neuroinfections. The criteria accepted by the producer lowered the sensitivity of the method and the possibility of recognition of local synthesis of antibodies in the CNS. The use of serum negative in CFT and negative CSF as reference for the determination made possible using of that kit for rapid diagnosis of neuroinfections. The modified ELISA IgG test makes possible determination of antibodies in CSF and serum, and accepting the generally recognized criteria for local production of antibodies in the CNS the ELISA test makes possible rapid diagnosis of neuroinfections which is not possible by other methods.     

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex

The performance of the immunochromatographic assay, SD BIOLINE TB Ag MPT64 RAPID?, was evaluated in Madagascar. Using mouse anti-MPT64 monoclonal antibodies for rapid discrimination between the Mycobacterium tuberculosis complex and nontuberculous mycobacteria, the kit was tested on mycobacteria and other pathogens using conventional methods as the gold standard. The results presented here indicate that this kit has excellent sensitivity (100%) and specificity (100%) compared to standard biochemical detection and can be easily used for the rapid identification of M. tuberculosis complex.     

Evaluation of rapid test Assure Helicobacter pylori for diagnosis of H. pylori in pediatric population

Non-invasive tests are needed to assess Helicobacter pylori infection, especially to screen a pediatric population. Assure H. pylori Rapid Test (Genelabs Diagnostics, Singapore) is an immunochromatographic assay device intended for the rapid detection of antibodies to H. pylori in human serum, plasma or whole blood. The aim of this study was to evaluate the performance of the rapid test, Assure H. pylori, in the diagnosis of H. pylori infection in children, using a Portuguese pediatric population. The study group included 130 children with age ranging from 1 to 14 years old (average age 9.2+/-3.1 years). According to the gold standard, 70 of the 130 patients studied were H. pylori positive and 60 were H. pylori negative. Using Assure H. pylori Rapid Test (Genelabs Diagnostics, Singapore), 53 sera had a positive result after 15 min (resulting in 17 false negatives) and 57 sera a negative result (resulting in 3 false positives). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the test were 75.7%, 95.0%, 94.6% and 77.0% respectively. When a longer read time of 45 min is considered, the rapid test revealed a good performance (sensitivity 98.6% and specificity 95%) in the evaluation of the H. pylori infection in a pediatric population. In conclusion, the test showed a good performance, suggesting its applicability as a screening method for the H. pylori infection.     

Homogeneous amperometric immunoassay for theophylline in whole blood

A homogeneous spectrophotometric EMIT immunoassay kit for the quantitation of theophylline in serum or plasma has been modified to produce a rapid, amperometric immunoassay requiring a 50 μl whole blood sample. The basis of the detection system for the assay is the electrochemical oxidation of NADH produced by G6PDH-labelled theophylline at a potential of + 150 mV vs Ag/AgCl using platinised activated carbon (PACE) electrodes. Comparison of the amperometric whole blood method with the conventional spectrophotometric plasma assay produced a reasonable correlation: Y = 0·90x − 1·01, (r = 0·98, N = 12). The advantage of the new method is that simple and robust instrumentation can rapidly determine theophylline in whole blood with no sample pre-treatment or separation steps.     

Development of an immunochromatographic test system for the detection of human epidermal growth factor

A method was developed for the rapid detection of human epidermal growth factor based on a sandwich-format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid flow movement across the membrane components of the test strip, immunochemical reactions, and the formation of colored lines. Requirements on the configuration of the test system in order to achieve the lowest limit of detection were defined in the course of the development of the assay. It was shown that this method enables the detection of human epidermal growth factor within 5 min at concentrations as low as 10 pg/mL in aqueous solutions, urine, and the blood serum and plasma. The developed test system can be used for point-of-care diagnostics.     

Rapid detection of glycyrrhizin by immunochromatographic assay

An immunochromatographic assay was developed for detecting glycyrrhizin (1). The qualitative assay is based on a competitive immunoassay using anti-1 monoclonal antibody (MAb) and a detector reagent that contains colloidal gold particles coated with anti-1 MAb. The immunochromatographic strip test, which has a detection limit of 250 ng/mL, is useful as a rapid screening method for detecting glycyrrhizin in plants, biological fluids and food samples.     

Rapid immunoassay to detect antibodies against human T-cell lymphotropic virus type 1 (HTLV-1) by the recombinant immunodominant antigens

A rapid immunochromatographic assay, using the recombinant immunodominant antigens of HTLV-1, has been developed to detect circulating antibodies to HTLV-1. The method was compared with an enzyme-linked immunosorbent assay by evaluating 1,631 serum or plasma samples. This HTLV-1 rapid assay was easy to perform and required no special equipment which provided visual result within 5 min with an excellent sensitivity and specificity in detecting HTLV-1 infection.     

<Emphasis Type="Italic">Herpesvirus ateles</Emphasis>, a New Lymphoma Virus of Monkeys

THERE has been a marked increase of post-transfusion hepatitis associated with the increased utilization of blood and its products. The discovery of a reaction of serum from multiple transfused haemophiliacs with serum from patients suffering from hepatitis^1,2 has made it possible to analyse the hepatitis associated antigen (HAA), the agent associated with the transmittance of hepatitis. HAA is currently detected using haemophiliac serum as the antibody source and standard serological techniques, that is, immunodiffusion, complement fixation and immunoelectro-osmophoresis³. This report describes a latex agglutination procedure which is compatible with the existing technology in blood banks as well as being very rapid and sensitive. The assay is based on the agglutination of antibody-absorbed latex particles by HAA contained in the test serum.     

Estimation of total and direct serum bilirubin using modified micro assay method

Estimation of total and direct bilirubin in serum plays an important role in differential diagnosis of hyperbilirubinemia. Several direct spectrophotometric methods are commercially available for total and direct bilirubin estimation in which the amount of the sample (serum) varies from 200 ml to 800 ml. It is difficult to collect such amount of serum from infants, as neonatal jaundice is the most common problem in this age group. To overcome this problem modified micro assay method was developed using dimethylsulfoxide (DMSO). The amount of the serum sample is reduced from 100 ml to 20 ml per test for both total and direct bilirubin. A method comparison study was performed using 100 consecutive serum samples, by modified micro assay method and a reference Jendrassik-Grof method. Total bilirubin in these human serum samples ranged from 0.4-15.0 mg/dl and direct bilirubin ranged from 0.05-12.0 mg/dl. The results conclude that modified micro assay method had significant correlation with r-value of 0.99989 for total serum bilirubin and with r-value of 0.99971 for direct serum bilirubin. Linearity of the method is 20 mg/dl and 15 mg/dl for total and direct bilirubin, respectively. Monoreagent used during the assay is stable for 24 hours at 2-8 degrees C while the kit is stable for one year at 2-8 degrees C. In conclusion this micro assay method is rapid, reliable, simple and accurate for the estimation of total and direct bilirubin with small serum quantities. It is equally reliable for manual; semi automated and automated chemistry analyzers.     

Quantum-Dot-Based Immunochromatographic Assay for Total IgE in Human Serum

To rapidly quantify total immunoglobulin E levels in human serum, we developed a novel quantum-dot-based immunochromatographic assay that employs digital recording of fluorescence. It can detect IgE levels of 5–1000 kU/L, with a coefficient of variation ranging from 2.0 to 9.5%. The assay can be processed in 10 min. The developed assay was tested on 95 serum samples. The correlation coefficient between the IgE values obtained by the proposed assay and those obtained by a commercial ELISA kit was 0.9884. Our assay thus shows promise as a new diagnostic tool for IgE detection.     

Development of a one-step immunochromatographic strip test for the detection of sennosides A and B

An immunochromatographic strip test was developed to detect sennoside A (1) and sennoside B (2) using anti-1 and anti-2 monoclonal antibodies. The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of colloidal gold particles coated with the respective sennoside antibodies. The capture reagents were 1- and 2-human serum albumin (HSA) conjugates immobilised on a nitrocellulose membrane on the test strip. The sample containing 1 and 2, together with detector reagent, passed over the zone where the capture reagents had been immobilised. The analytes in the sample competed for binding to the limited amount of antibodies in the detector reagent with the immobilised 1- and 2-HSA conjugates on the membrane and hence positive samples showed no colour in the capture spot zone. Detection limits for the strip test were 125 ng/mL for both sennosides. The assay system is useful as a rapid and simple screening method for the detection of 1 and 2 in plants, drugs and body fluids.     

Liposome-based immunoaffinity chromatographic assay for the quantitation of immunoglobulin E in human serum

Immunoglobulin E (IgE)-mediated type I allergies affect over 25% of the world's population; they are among the most common diseases in developed countries. Therefore, simple and rapid in vivo and in vitro methods for diagnosing allergies are becoming increasingly important. In this paper, we demonstrate the feasibility of using sulforhodamine B, a fluorescent dye, entrapped inside immunoliposomes, the outer surfaces of which were sensitized with IgE, as a signal amplifier for the development of a simple, rapid, and inexpensive colorimetric affinity chromatographic immunoassay for the detection of total IgE in serum. This assay operates based on competition between standards (or human serum samples) containing IgE and IgE-sensitized immunoliposomes for the limited number of antigen binding sites of immobilized anti-IgE antibodies at the antigen capture (AC) zone on the nitrocellulose membranes. The color density of the AC zone is indirectly proportional to the number of IgE units present in the test sample. The detection limit of this liposome-based immunoaffinity chromatographic assay was 0.37 ng in IgE-free serum solution (equivalent to 20 μL of a 18.5 ng mL⁻¹ solution). A commercially available ELISA kit was used as a reference method to validate the proposed assay through the analysis of three human serum samples.     

乙型肝炎病毒X蛋白定量试验与临床研究

HBV X蛋白 （HBx）是由乙型肝炎病毒（HBV）基因组编码的调控蛋白,与由乙肝引起的肝癌发生具有密切的关系。血清HBx对乙型肝炎、肝癌的诊断及发病机理研究有较高的临床应用价值。在表达并纯化HBx、制备出HBx单克隆抗体与酶标抗体的基础上,研制了HBx定量检测试剂盒（增强化学发光法）,对其灵敏度、特异性等指标进行分析,并将该试剂盒应用于临床研究。结果表明,原核表达并纯化的HBx纯度≥94%;试剂盒灵敏度达0.1ng/ml;线性范围达到0.5ng/ml -600ng/ml;特异性高,与球蛋白、脂蛋白、血红蛋白、酸性糖蛋白、HBc、HBe、HBs、HBV preS2不发生交叉反应;批间CV≤6.5%;临床标本检验慢性乙型肝炎阳性率为55%、肝硬化阳性率为68%、肝癌阳性率70%,表明此试剂盒可应用于乙肝、肝硬化、肝癌的临床诊断及发病机理研究。