Serotyping of dengue viruses by an enzyme-linked immunosorbent assay

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.     

Increased sensitivity of cholera toxin detection by enzyme-linked immunosorbent assay with chemical immobilization of antibody onto nylon surface providing hydrophobicity

We developed an improved method to chemically immobilize antibodies on a nylon surface using a styrene maleic anhydride copolymer having an aryl group, which provides hydrophobicity to the nylon surface. We applied it to a modified enzyme-linked immunosorbent assay (slip-ELISA) for the detection of cholera toxin (CT). The sensitivity of slip-ELISA for CT detection was 1,000 times higher than that of conventional methods of physical adsorption using polystyrene plates, and 10 times higher than that of the method of chemical immobilization using maleic anhydride methylvinyl ether copolymer.     

Preferential detection of pro-carboxypeptidase R by enzyme-linked immunosorbent assay

We generated two monoclonal antibodies (mAbs), 2A16 and 10G1, against pro-carboxypeptidase R (proCPR), also known as thrombin activatable fibrinolysis inhibitor (TAFI). By use of these mAbs, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect proCPR. Since the amount of the antigen detectable by the ELISA was essentially the same in fresh plasma and serum incubated at 37 C for 1 hr, we concluded that the ELISA system detected not only proCPR, but also inactivated CPR generated from proCPR. However, an appreciable amount of proCPR remained unactivated in serum. For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (TTM) can be used together with CaCl2. Following extensive conversion of proCPR to CPR by T-TM and CaCl2, converting plasma to serum (T-TM serum), antigenicity became undetectable by ELISA. Further analysis revealed that 2A16 reacts only with proCPR although 10G1 reacts with proCPR, active CPR and inactivated CPR. Therefore, we concluded that the ELISA system preferentially detects proCPR and not CPR. Our sandwich ELISA system utilizing 2A16 and 10G1 provides a suitable method for detecting proCPR and can be used to determine levels of proCPR in plasma samples from patients.     

Hyaluronidase assay using fluorogenic hyaluronate as a substrate

The reducing terminal of hyaluronate was labeled with a fluorogenic reagent, 2-aminopyridine. The pyridylaminohyaluronate was incubated with testicular hyaluronidase for 1 h. After incubation, 4 vol of ethanol was added to the incubation mixture, followed by centrifugation. The fluorescence of the supernatant containing the degradation products of hyaluronidase digestion was then determined by fluorospectrophotometry (excitation wavelength, 320 nm; emission wavelength, 400 nm). It was found that the increase of the pyridylamino products was linearly correlated with enzyme concentration (up to 0.1 national formulary unit), incubation time (up to 60 min), and substrate concentration (up to 2.5 microM). The fluorogenic substrate was also applicable for the determination of crude hyaluronidase. This simple, rapid, and sensitive hyaluronidase assay was made possible by the use of pyridylaminohyaluronate as a substrate.     

Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates

Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product or the quantum yield of a fluorescent product, coupled with the efficiency of the immobilized enzyme, is the determining factor for the sensitivity and precision of a given ELISA. The enhancement of precision and sensitivity using fluorogenic substrates was demonstrated in a direct-binding ELISA in a low-analyte concentration range compared with commonly used chromogenic substrates. The enhancement in precision was demonstrated quantitatively with lower coefficients of variation in measurements of signal intensities, approximately a five- to six-fold enhancement in signal-to-noise ratio at a given analyte concentration with fluorogenic substrates. Similarly, the amplitude of the enhancement in sensitivity, as reflected by relative limits of detection or quantitation, is approximately two- to five-fold when compared with commonly used chromogenic substrates. Additional advantages of a fluorescence-based ELISA format include the continuous monitoring of initial rates of enzymatic reactions, the measurement of fluorescence changes in the presence of particulate materials, the absence of a quench step, and a larger quantifiable analyte range.     

北京地区设施草莓8种病毒的酶联免疫吸附测定检测

[背景]病毒可以随同草莓无性繁殖材料传播扩散,导致产量和品质下降.选育无病毒种苗是草莓病毒病防治的主要措施,高效、灵敏的检测技术可为草莓病毒病防治提供技术保障.[目的]为明确8种能够侵染草莓的病毒在北京地区设施草莓上的发生情况,应用酶联免疫吸附测定(Enzyme-Linked Immunosorbent Assay,E...     

Use of Clq enzyme-linked immunosorbent assay to detect plant viruses and their serologically different strains

Clq was prepared from bovine serum using a simple method involving repeated dialysis at low ionic strength in the presence of chelating agents (yield c. 3 mg/100 ml serum). It was viable when stored at -18°C for up to 2 months, and at 4°C for at least 10 wk in a storage buffer containing 10% sucrose. When used in Clq ELISA this test was as sensitive as the direct double antibody sandwich form of ELISA (direct ELISA) in detecting purified potato virus Y (PVY), with a limit of detection in both methods of c. 15 ng/ml, and slightly more sensitive in detecting purified cocksfoot mild mosaic virus (CMMV), with limits of detection of c. 15 ng/ml and c. 15–60 ng/ml respectively. Using an antiserum to one strain of each virus, Clq ELISA readily detected strains of PVY, CMMV, Andean potato latent virus (APLV) and barley yellow dwarf virus (BYDV). This included detection of APLV-Hu by APLV-Caj antibodies and CMMV(G) by PMV(S) antibodies, neither of which system gives detection in direct ELISA. Clq ELISA was therefore less specific than direct ELISA in detecting serologically different virus strains. Virus detection by Clq ELISA was inhibited when sap of tobacco, Nicotiana clevelandii and Setaria italica was used at low dilution. Inhibition by N. clevelandii sap was alleviated by using increased concentrations of virus specific antibody to detect APLV and plum pox virus. Also, extracting APLV infective N. clevelandii or CMMV infective S. italica saps in a minimum of buffer, centrifuging at low speed and diluting the supernatant before testing, partially overcame the inhibition. The inhibitory substance(s) in sap may act by preventing the binding of Clq to virus-antibody aggregates. Sap of wheat, oat and barley did not appear to have an inhibitory effect and BYDV was readily detected in naturally infected field grown plants of these species.     

An enzyme-linked immunosorbent assay for plant cadmium-binding peptide

Polyclonal antibodies raised against Cd-binding peptide from roots of Agrostis gigantea Roth were used with an enzyme-linked immunosorbent assay (ELISA). The antigen was best adsorbed to Immulon 2 “U” microtitre plates in 50 mM acetic acid. The antibodies to the antigen from Agrostis cross-reacted with Cd-binding peptides from the roots of maize and tomato, but not with glutathione nor metallothioneins I and II from rabbit liver. The antibodies reacted specifically with peptides rich in cysteine and glutamate, and having glycine or serine in the least amount. Reaction of antibodies was limited to ELISA; the antiserum did not form antigen-antibody precipitates when tested by standard diffusion and immunoelectrophoretic methods.     

The detection of three viruses of hop (Humulus lupulus) by enzyme-linked immunosorbent assay (ELISA)

The recently described technique of enzyme-linked immunosorbent assay (ELISA) was used throughout the 1976 growing season to detect hop mosaic, arabis mosaic and prunus necrotic ringspot viruses in hop plants. On each occasion virus was detected quickly, conveniently and with great sensitivity. The technique was particularly suitable for processing numerous samples collected from the field. Serious difficulties and limitations were encountered in testing comparable material by established techniques. The serology test for the hop strain of arabis mosaic virus by double diffusion in agar gels was very insensitive and only worked satisfactorily early in the growing season. Grafting sensitive Golding hop varieties to detect hop mosaic virus was inconvenient and time-consuming and symptom expression was so slow and erratic that glasshouse space was utilized for long periods. It became impossible to detect prunus necrotic ringspot virus by sap inoculations to cucumber during an exceptionally hot period in mid-summer. The possibilities are discussed of exploiting the ELISA technique for use in large scale surveys and epidemiological studies on viruses of hop and other crops. Changes in the current methods of handling and extracting leaves are considered for increasing the throughput of samples.     

A microtiter plate assay for N-acetyl-beta-D-glucosaminidase using a fluorogenic substrate

We describe a simple endpoint method for the determination of N-acetyl-beta-D-glucosaminidase (NAGase; EC 3.2.1.30). NAGase uses a fluorogenic substrate, 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide, at pH 4.6, liberating the fluorescent 4-methylumbelliferone. The method is reproducible and fast both at room temperature and at 37 degrees C. The procedure developed can be used, e.g., in the diagnosis of bovine subclinical mastitis, where elevated NAGase activities are found in raw milk samples. The assay procedure has a high capacity and high sensitivity and several hundred milk samples can be screened per hour using 96-well microtiter plates and an automated fluorescence reader. In addition to its use in mastitis diagnosis, the assay can be used in the diagnosis of some diseases of human origin.     

Real-time fluorogenic kinase assay using protein as substrate

A real-time fluorogenic kinase assay using myelin basic protein (MBP) as a substrate is reported. MBP is part of a noncovalent complex with a negatively charged, dye-labeled lipopeptide, (N-heptadecanoyl)-K(dye2)-linker-EEIYGEF-amide. The complex is approximately 20 times less fluorescent than the free lipopeptide. The MBP-lipopeptide complex serves as a protein substrate for several Ser/Thr kinases. We infer that the observed fluorescence increase on the addition of kinase and ATP is due to the phosphorylation of MBP, which decreases the affinity of MBP with the negatively charged, dye-labeled lipopeptide. Several protein kinases (protein kinase C βII, mitogen-activated protein kinase [MAPK] Erk1, and MAPK Erk2) were tested with the assay. The assay exhibited a fivefold fluorescence increase over background, provided kinetic values comparable to literature values (apparent K_m^ATP), and produced inhibitor constants comparable to literature values for a typical inhibitor, namely staurosporine.     

Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: an assay for serum ferritin

A new highly sensitive nonseparation enzyme immunoassay for human serum ferritin is described. Reagents include a beta-galactosidase-ferritin conjugate, sheep anti-ferritin, anti-sheep IgG, and dextran-linked beta-galactosylumbelliferone as enzyme substrate. The method is based on inhibition of enzyme activity when anti-ferritin binds to the enzyme-ferritin conjugate. Ferritin in the sample and enzyme-labeled ferritin compete for a limited quantity of anti-ferritin. The enzyme activity of the reaction mixture is directly related to the ferritin content of the sample. Some patients' samples caused strong interference in the assay due to the presence of antibody to beta-galactosidase. Several ways of eliminating the interference are presented. When measures were adopted to suppress sample interference, the assay results correlated well with those of other immunoassay methods.     

A substrate phage enzyme-linked immunosorbent assay to profile panels of proteases.

It is estimated that proteases comprise nearly 2% of the human genome. Given that the primary structure of all known proteases will soon be available, an important challenge is to define the structure-activity relationships that govern substrate hydrolysis. Ideally this would be accomplished on a genome-wide scale. To this end, we have developed a one-pot phage selection system that yields the substrate recognition profile of multiple proteases from a single round of selection. The system meets five key criteria: (i) multiple proteases can be analyzed simultaneously, (ii) prior knowledge of substrate preference is not required, (iii) information regarding substrate preferences on both side of the scissile bond is obtained, (iv) the system yields selective substrates that distinguish closely related proteases, and (v) semiquantitative information on substrate hydrolysis is obtained, allowing for the assignment of initial rank-order preferences. As an illustration, a phage selection with a mixture of thrombin and factor Xa (serine proteases) along with matrix-metalloproteinase-9 and atrolysin C (metalloproteinases) was performed. Peptide substrates were identified that (i) have high k(cat)/K(m) ratios, (ii) are selective for individual proteases, and (iii) match the sequences of known physiological substrates.     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

An assay of collagenase activity using enzyme-linked immunosorbent assay for mammalian collagenase

A new method was developed for the measurement of collagenase activity using the enzyme-linked immunosorbent assay (ELISA). Rabbit colon wall collagenase, pepsin-soluble rat skin type I collagen, and its antisera were used in the present experiment. After the collagenase-degraded portion of the collagen coated on the microwell was released, the immunoreaction of the residual collagen on the microwell to anticollagen sera was determined by ELISA. This method was approximately 10 times more sensitive than the conventional assay procedure using [14C]-glycine-labeled reconstituted collagen fibrils as substrate. It was suitable for screening a large number of samples without radioisotopes.     

Quantitation of apolipoprotein A-IV in human plasma using a competitive enzyme-linked immunosorbent assay

A method for measuring human apolipoprotein A-IV has been developed using the competitive enzyme-linked immunosorbent assay (ELISA) system. The assay described is relatively easy, rapid, and inexpensive to perform, uses convenient dilutions of plasma (1/8-1/32) but is sensitive enough to quantitate the apoA-IV content of lipoproteins following gel filtration of small (0.3-0.5 ml) volumes of plasma. The working range is 100-600 ng of apoA-IV per 50-microliters sample and the intra- and interassay coefficients of variations are 7.5 and 10.2% (means), respectively. The mean apoA-IV concentration of 100 subjects was found to be 16.4 +/- 5.4 mg/dl. The assay can be performed on untreated plasma samples which may be stored frozen (-20 degrees C) for up to 2 months.     

A rapid detection of circulating antigens of Dirofilaria immitis in dogs by a dot enzyme-linked immunosorbent assay

This report describes a dot enzyme-linked immunosorbent assay (Dot-ELISA) for detecting circulating antigens in the sera of dogs infected with Dirofilaria immitis (D. immitis). Circulating D. immitis antigens could be detected in 24 of 25 infected dogs. The remaining animal had two immature worms. However, non-infected dogs and dogs infected with other parasites were all negative. Few cross-reactions to different parasite antigens were observed. The advantages of the Dot-ELISA include; 1) there is no need for pretreatment and dilution of sera and samples could be immediately bound to nitrocellulose paper set into microfiltration apparatus, 2) this assay could be carried out within 2 h at room temperature, 3) the resulting enzyme-reaction could be measured by both visual observation and densitometric reading.