Culex tritaeniorhynchus and Aedes albopictus (Diptera: Culicidae) as natural vectors of Dirofilaria immitis (Spirurida: Filariidae) in Miki City, Japan

Natural infections with Dirofilaria immitis (Leidy) in a population of mosquitoes and dogs and its antibodies in a human population were surveyed in Miki City, a rural area of Japan, to reveal ecological features of this arthropod-borne zo?notic parasite. Microfilaremic and amicrofilaremic infection rates in 190 dogs were 52.6 and 9.0%, respectively, as determined by the hematocrit centrifuge method and the indirect fluorescent antibody test. An enzyme-linked immunosorbent assay (ELISA) test for antibodies to D. immitis showed that almost all of 917 inhabitants had antibodies in varying degrees, indicating frequent exposure of this parasite to the human population. Among the six species of wild mosquitoes sampled from 1985 through 1987 by attraction with dry ice, Culex tritaeniorhynchus Giles was the most abundant in two sites and Aedes albopictus (Skuse) in another two sites. Filarial infections were found every year in Cx. tritaeniorhynchus and Ae. albopictus collected between July and September, as examined by ELISA for detecting larval antigens in mosquito homogenate, suggesting that these species are important natural vectors in this survey area.     

A monoclonal antibody enzyme linked immunosorbent assay (ELISA) directed towards a fibrin binding region of tissue-type plasminogen activator

A sensitive double monoclonal antibody ELISA has been developed for measurement of total t-PA antigen. The two mouse monoclonal antibodies employed recognise distinct epitopes at or near the kringle 2 fibrin binding region of t-PA and these clones have proved stable over several years. Complete recovery of t-PA in plasma when assayed against a standard t-PA diluted in buffer was achieved by dilution of the plasma sample in buffer containing ethylenediaminetetraacetic acid. The ELISA did not detect urokinase while t-PA complexed with endothelial plasminogen activator inhibitor was measureable in addition to free active t-PA. No discrimination between 1 and 2-chain forms of t-PA was evident. The concentration of t-PA antigen in basal human citrated plasma was found to be 3.8 ± 1.5 ng ml⁻¹ (mean ± standard deviation, n =17) and good correlations were observed between t-PA antigen measured by ELISA and by radioimmunoassay or a functional assay. The ELISA provides a reliable and simple method for measuring t-PA antigen in plasma and other biological fluids and is based on stable reagents that recognise known epitopes on t-PA.     

Effects of triturated Culiseta melanura (Diptera: Culicidae) on recovery of eastern equine encephalomyelitis virus

Experiments were done to determine the effect, if any, of larval and adult Culiseta melanura (Coquillett) suspensions on the recovery of eastern equine encephalomyelitis (EEE) virus in baby hamster kidney (BHK) and Vero cell cultures. Although triturated pools of this mosquito reduced the titer of EEE virus added to suspensions, suspensions prepared from as many as 100 Cs. melanura larvae or adults did not reduce titers of virus to undetectable levels. Similarly, the titer of EEE virus in orally infected female Cs. melanura remained detectable when a single infected mosquito was triturated in a pool with 99 uninfected mosquitoes. These results show that in BHK or Vero cell culture assay systems, triturated Cs. melanura do not completely prevent the detection of EEE virus. The results therefore indicate that mosquito suspensions would not interfere with the isolation of virus from field-collected mosquitoes. This conclusion suggests that mosquito suspensions were not a factor in previous studies that were unsuccessful at detecting transovarial transmission of EEE virus by Cs. melanura.     

Specific monoclonal antibodies to Strongyloides stercoralis: a potential diagnostic reagent for strongyloidiasis

In this study, specific hybridomas secreting monoclonal antibodies (MAb) to antigen of Strongyloides stercoralis filariform larvae were produced. Specific epitopes targeted by the MAb were protein in nature and located in situ in the internal content of the filariform larvae of the parasite but not in the esophagus. The MAb reacted to the homologous antigen in an indirect ELISA but did not reveal any reaction to the SDS-PAGE separated-homologous antigen in a Western blot analysis (WB) suggesting a conformational epitope specificity. The MAb were of IgG1 isotype which is the isotype known to have high affinity to this epitope so they were used in a dot-ELISA to detect the antigen of the parasite. The assay could detect the epitopes in 78 ng or more of the crude filariform larval extract but did not reveal any positive result when applied to detect antigen in stool samples of parasitologically confirmed strongyloidiasis patients. The negative antigen test results can be explained as follows. Either the MAb were filariform stage-specific and thus did not recognize the rhabditiform larval antigen mainly contained in the patient's stool or the amounts of antigen in the stool samples were too small and/or unevenly dispersed. In the latter instance, the MAb developed in this study would have a diagnostic potential if used in an immunological test design where more volume of fresh stool sample could be accommodated in the test, e.g. a sandwich plate ELISA.     

Determination of the hepatitis B virus surface antigen (HBsAg) by immunoenzyme analysis

Studies aimed at the development of a variant of ELISA for the determination of hepatitis B virus surface antigen (HBsAg) showed the assay to be most effective when a modified periodate method of conjugation of highly purified horseradish peroxidase with the IgG-fraction of antiserum to HBsAg was used. With the resulting conjugates, the "sandwich"-test on polystyrene solid phase could detect HBsAg in concentrations up to 5 ng/ml which is several thousand-fold higher in sensitivity than counter immunoelectrophoresis, one order of magnitude more sensitive than the passive hemagglutination test, and comparable with the radioimmunoassay (RIA). The specificity of the method was confirmed by positive results of the neutralization test. By this method, HBsAg in the sera of hepatitis B patients was detected as frequently as by the RIA.     

Antigen detection immunoassay using dipsticks and colloidal dyes

A dipstick colloidal dye immunoassay (DIA) for multiple antigen detection is described. The test combines the concepts of double antibody (Ab) sandwich ELISA, dot blotting, and colloidal particle-linked Abs to produce a dipstick test for multiple antigen (Ag) detection. Dipsticks prepared from Ab coated nitrocellulose membrane mounted on acetate strips served as the assay capture matrix. Abs absorbed to colored dye particles from a family of commercially available textile dyes (Dye/Ab reagent) served as Ag detecting reagents. DIA and enzyme labelled dot blot assays showed similar Ag detection limits down to a sensitivity of 10 ng/ml. In a pilot study, an assay designed to detect species-specific IgG for use in mosquito bloodmeal identification demonstrated the feasibility of the technique. Experiments comparing bloodmeal analysis of mosquitoes using DIA and ELISA methods showed 100% agreement. This DIA method provides an inexpensive, simple, robust test for multiple Ag detection without instrumentation suitable for a wide variety of field applications.     

A simple theoretical treatment of a competitive enzyme-linked immunosorbent assay (ELISA) and its application to the detection of human blood group antigens

A theory has been developed to explain the behaviour of a competitive enzyme-linked immunosorbent assay (ELISA) in which an immobilized antigen competes with a liquid-phase antigen for a limiting amount of antibody. The binding of antibody to antigen on a solid surface (microtitre well) is described in terms of Langmuirian adsorption with a binding constant kappa. Two equations are presented to describe the behaviour of the ELISA signal as a function of competing antigen concentration; an exact equation and an approximate equation which can be used when the surface coverage of the immobilized antigen is not known. It is shown how curves of ELISA signal vs. competing antigen concentration depend on K/kappa [antibody]. The theory has been tested using several immobilized blood group A antigens competing with ovarian cyst fluid A substance and found to adequately describe these competitive ELISAs which have a detection limit of approximately 1 ng of blood group antigen.     

Development and evaluation of a dipstick assay for detection of Plasmodium falciparum and P. vivax sporozoites in mosquitoes (Diptera: Culicidae)

We developed a nitrocellulose-based, dipstick circumsporozoite (CS)-enzyme immunoassay [ELISA] for the simultaneous detection of Plasmodium falciparum and P. vivax-210 CS protein. The assay had a detection threshold of < 250 P. falciparum or 400 P. vivax sporozoites per sample, gave results concordant with dissection of salivary glands and CS-ELISA, but was slightly less sensitive than the CS-ELISA in microtiter plates. The assay consistently detected one infected mosquito in a pool of 10 or 20 mosquitoes, and was 100% specific in discriminating between species of Plasmodium when mosquito suspensions were spiked with sporozoites. The assay could be completed in 1 h, required no specialized equipment, and therefore was useful for field applications.     

An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of other innate immune cells, such as macrophages and neutrophils. It also modulates the adaptive immune response by interacting with antigen-presenting cells and T cells. Monoclonal anti-mouse-SP-D antibodies were raised from SP-D deficient mice using recombinant SP-D as antigen. Ten monoclonal antibodies were characterized and validated for use in sandwich enzyme-linked immunosorbent assay (ELISA). Based on two of these, we established an ELISA that allows for measurements of mouse SP-D in various body fluids. The final ELISA was optimized and calibrated with a standard of purified recombinant mouse SP-D, which was analyzed by quantitative amino acid analysis. The ELISA was validated with respect to parallelism, recovery, intra- and inter-assay variation. The practical working range was estimated to be 1.9–200 ng/ml. We measured SP-D in lung lavage to an average concentration of 435 ng/ml (3-ml lavage), and in mouse vaginal fluid (1-ml lavage) to an average concentration of 94 ng/ml, but could not detect SP-D in the serum or plasma of healthy mice (< 3.8 ng/ml). With this ELISA, it will be possible to study the regulation of SP-D in various mouse models and upon various stimuli.     

抗日本血吸虫SEA特异性IgY应用于循环抗原检测的研究

本研究应用抗日本血吸虫可溶性虫卵抗原(SEA)的鸡卵黄免疫球蛋白(IgY)建立敏感、特异的检测循环抗原的双抗体夹心酶联免疫吸附试验( ELISA).用SEA皮下多点注射法免疫海蓝鸡,水稀释法制备IgY抗体,以辣根过氧化物酶标记纯化的IgY抗体(IgY-E)和兔抗IgG抗体(IgG-E)分别作为检测抗体,IgY抗体和兔抗...     

Rapid detection of equine herpesvirus type-1 antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay.

An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-1 (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swabs from ponies which were experimentally infected with EHV-1. Of 72 nasal swabs collected, 32 were found to be positive by both virus isolation (VI) and ELISA, a further 15 samples were positive by VI alone, but none of the samples were positive by ELISA and negative by VI. This yielded an overall assay sensitivity of 68% and specificity of 100%. The assay proved useful for diagnosis since virus antigen was detected during the first four days post-infection which corresponded to the acute phase of disease when some clinical symptoms were apparent. In addition, the assay could be completed within one day when antibody coated plates were available.     

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis.     

Assay of factor VII antigen by enzyme-linked immunosorbent assay (ELISA)

This paper introduces a newly developed enzyme-linked immunosorbent assay (ELISA) method, using monospecific anti-human factor VII rabbit IgG, to quantify antigen levels of factor VII. The procedure is relatively simple, its method is remarkably specific and reproducible. Its sensitivity to detect factor VII antigen is as low as 0.015 U/ml, when factor VII antigen in 1 ml normal plasma is arbitrarily defined as 1 unit. The level of factor VII antigen in 4 patients with congenital factor VII deficiency and their family members was investigated through this ELISA and the results correlated well with those obtained by electroimmunoassay previously described. The significant correlation between the antigen levels and activity of factor VII was demonstrated in 4 patients with congenital factor VII deficiency and their family members, normal individuals and patients with liver disease. However, the level of factor VII antigen in patients treated with warfarin was higher than their coagulation activity.     

Inhibition ELISA for hepatitis B surface antigen (HBsAg) using monoclonal idiotype-anti-idiotype interaction

Monoclonal anti-idiotypic antibody (Anti-Id) to the common (a) epitope of hepatitis B surface antigen (HBsAg) were raised and used to detect serum HBsAg in an inhibition enzyme-linked immunosorbent assay (inhibition ELISA). HRP-labelled Id 8D-3-6 was reacted with Anti-Id 4-8H coated on the solid phase in the presence of HBsAg. The ability of the antigen to inhibit the binding of labelled Id 8D-3-6 to anti-Id 4-8H was determined and the results correlated well with those obtained by radioimmunoassay. This assay requires only one washing step, takes 2 h and covers the range 10 ng/ml to 1 microgram HBsAg/ml. The inhibition ELISA is a more convenient, rapid and relatively sensitive assay which can be used to measure the level of a wide range of serum HBsAg.     

Antibody for the development of specific immunoassays to detect nadifloxacin in chicken muscles

A convenient and specific competitive enzyme-linked immunosorbent assay (ELISA) for nadifloxacin (NDF) was developed. The modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesise the artificial antigen of NDF. The indirect competitive ELISA developed to detect NDF has a concentration at 50% inhibition (IC₅₀) of 0.72 ng/mL and a limit of detection of 0.04 ng/mL. This ELISA kit could be used as a screening method to detect and control the illegal content of NDF in food products. In this study, the kit was applied to NDF detection in chicken muscle and produced a satisfactory result with recovery rates from samples spiked with NDF of 92% (10 ng/mL) and 101% (50 ng/mL).     

Development of a sensitive monoclonal antibody-based ELISA for the detection of clenbuterol in animal tissues

A specific monoclonal antibody (mAb) against clenbuterol, with cross-reactivities less than 0.01% for all test compounds except salbutamol (6.4%), was produced with hybridoma technology. The mAb originated from immunogen of clenbuterol–human serum albumin was combined with coating antigen of salbutamol–ovalbumin to develop a heterologous enzyme-linked immunosorbent assay (ELISA). This assay shows very high sensitivity with IC₅₀ of 0.3 ng/ml and LOD of 0.1 ng/ml when it was run in 0.01 M PBS (pH 7.5). Clenbuterol was spiked in chicken and pork samples and after a simple extraction procedure the extracts at appropriate dilution were analysed by ELISA. Satisfactory results were obtained by both intra-assay, with average recoveries of 81–102% and coefficient variations (CVs) of 3–12%, and inter-assay, with average recoveries of 77–95% and CVs of 5–13%. The survey results of ELISA and HPLC for some real world tissue samples were consistent. It suggests that the mAb-based ELISA will be a feasible quantitative/screening method for clenbuterol residue in animal tissues.     

Weak larval competition between the invasive mosquito Aedes japonicus japonicus (Diptera: Culicidae) and three resident container-inhabiting mosquitoes in the laboratory

The spread of exotic mosquito species into new environments can introduce shifts in mosquito populations and potentially alter public health risks to mosquito-borne diseases. The successful establishment of exotic species may occur due to their competitive advantage over other cohabitating species. We hypothesized that the recently introduced exotic mosquito Aedes japonicus japonicus (Theobald) would be a more effective competitor than Aedes atropalpus (Coquillett) and Aedes triseriatus (Say), and an equal competitor to Culex pipiens (L.) based on larval abundance data within tire habitats. Impacts of competition were measured using the larval developmental rate and survival of larvae, adult mortality, wing length, and sex ratio. We found that intraspecific competition acted strongest against Ae. japonicus versus the other three resident mosquito species by delaying larval development and increasing adult mortality. Interspecific competition was generally weak and significant main effects were only detected for species and density. Overall, our results show that larval competition between Ae. japonicus and the three resident species was weak when present, indicating that other ecological or behavioral factors may be influencing the invasion success for Ae. japonicus in North America.     

Serum prostatic acid phosphatase (PAP): monoclonal enzyme-linked immunoassay compared to polyclonal radioimmunoassay

A new enzyme linked immunosorbent assay (ELISA) kit which utilizes a monoclonal antibody against prostatic acid phosphatase (PAP) was compared with current radioimmunoassay (RIA) methodology which uses a polyclonal antibody. Both assays are double antibody immunoassays with the major difference being the method of antibody preparation. A study of intrarun precision using control material showed an average within run coefficient of variation (CV) percent as 7.0 percent and 6.9 percent for ELISA and RIA, respectively, while between run CV percent averaged 11.1 percent and 11.5 percent, respectively. Thus, precision results compare similarly between the two assays. The specificity showed significantly different results. Patterns of correlation between the two methods indicate differing specificities of the primary antibodies. The values for ELISA were greater than RIA for control sera and patient samples when values fell outside the reference range; however, RIA values exceeded ELISA values with patient samples which fell within the reference ranges as provided by each manufacturer. Therefore, there exists a question of specificity of antibody employed in each of the two assays. The PAP antigen is prepared from two different sources for each kit. The ELISA manufacturer prepares antigen from seminal fluid and RIA manufacturer prepares antigen from normal human prostate. The question of specificity may be influenced by: (1) source of antigen used in immunizing animals and (2) monoclonal versus polyclonal means of producing antibody.     

Preparation and characterization of anti-sulphamethoxazole (SMX) monoclonal antibody

Anti-sulphamethoxazole (SMX) monoclonal antibody (MAb) has a significant role in monitoring of SMX residue levels in edible animal products. In this study, SMX was diazotizated and coupled to human serum albumin to form an antigen for animal immunization. Anti-SMX hybridomas were prepared by cell fusion using an enzyme-linked immunosorbent assay (ELISA) method to screen for positive hybridomas. A monoclonal antibody (MAb) against SMX was secreted by the hybridomas and its specificity was characterized by ELISA, SDS-PAGE and Western-blot. The results showed that the SMX-MAb was of the IgG1 type and its molecular weight was 162 000 Daltons. The affinity constant of MAb for the conjugated antigen was 2.3×10⁸ l/mol. The detection limit of 50% inhibition rate in the range 10 pg/mL to 10 ng/mL was 0.82 ng/mL and the recoveries of SMX were above 90%. There was no cross-reaction between MAb and SMX analogues.