Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

Au sujet de la caractérisation des produits a base deBacillus thuringiensis Berliner par rapport au titrage biologique de la toxine soluble thermostable

Summary Industrial products based onB. thuringiensis have been found to contain several toxins, differing in their ranges of activity and occuring in varying quantities. The presence of the ?thermostable toxin activeper os? is in particular unnoticed in the assay of the active material in the products, in spite of the fact that many studies of the application of products based onB. thuringiensis, concern insects whose sensitivity is limited to or dominated by the thermostable toxin. Biological assay can be envisaged for the estimation of the thermostable toxin in industrial products, while awaiting the development of other assay methods In this manner, the products will be charasterized as to their active material, for the benefit of the understanding of experimental or practical results. It is suggested that it would be more rational, on the international scale, to use as reference a standard preparation based on an autoclaved filtrate ofB. thuringiensis var.thuringiensis, than to use a standard biological assay method which involves an test insect. The precision of assay for ?thermostable toxin activeper os? can be greater or smaller, according to the insects for which the product is recommanded or on which it is tested.

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Ce mémoire a été présenté à l' ?International Symposium on the identification and assay of viruses and Bacillus thuringiensisused for insect control?, Londres, 13 juillet 1964.

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I.N.R.A., Station de Recherches de Lutte biologique et de Biocœnotique, La minière.     

Monoclonal antibodies specific for Laelius pedatus (bethylidae) and Bracon hebetor (braconidae), two hymenopterous parasitoids of stored-product pests

Before parasitoids and predators are fully endorsed as biological control agents in storage facilities, a reliable technique must be developed to determine how much they contribute to the overall insect contamination of stored commodities. Because determining the origin of insect fragments by visual examination is difficult, labor-intensive, and requires special skills, we propose using immunological techniques to differentiate among insect species biochemically. In the example presented here, we generated monoclonal antibodies (MAbs) against the parasitic wasps Laelius pedatus (Say) and Bracon hebetor Say. The MAbs reacted with all life stages and both sexes of the parasitoids. In Western blots of acrylamide and agarose gels, the MAbs recognized high molecular weight proteins (>500 kDa) composed of multiple subunits, with mildly acidic to neutral isoelectric focusing points. The MAbs did not cross-react with the additional 22 insect species we tested in an indirect enzyme-linked immunosorbent assay. These data suggest that MAb-based immunoassays could be used to differentiate among beneficial and destructive insects found in stored products.     

Assessing insect biodiversity with automatic light traps in Brazil: Pearls and pitfalls of metabarcoding samples in preservative ethanol

Automated species identification based on data produced with metabarcoding offers an alternative for assessing biodiversity of bulk insect samples obtained with traps. We used a standard two‐step PCR approach to amplify a 313 bp fragment of the barcoding region of the mitochondrial COI gene. The PCR products were sequenced on an Illumina MiSeq platform, and the OTUs production and taxonomic identifications were performed with a customized pipeline and database. The DNA used in the PCR procedures was extracted directly from the preservative ethanol of bulk insect samples obtained with automatic light traps in 12 sampling areas located in different biomes of Brazil, during wet and dry seasons. Agricultural field and forest edge habitats were collected for all sampling areas. A total of 119 insect OTUs and nine additional OTUs assigned to other arthropod taxa were obtained at a ≥97% sequence similarity level. The alpha and beta diversity analyses comparing biomes, habitats, and seasons were mostly inconclusive, except for a significant difference in beta diversity between biomes. In this study, we were able to metabarcode and HTS adult insects from their preservative medium. Notwithstanding, our results underrepresent the true magnitude of insect diversity expected from samples obtained with automatic light traps in Brazil. Although biological and technical factors might have impacted our results, measures to optimize and standardize eDNA HTS should be in place to improve taxonomic coverage of samples of unknown diversity and stored in suboptimal conditions, which is the case of most eDNA samples.     

Quality analysis of commercial <Emphasis Type="Italic">Chlorella</Emphasis> products used as dietary supplement in human nutrition

Chlorella vulgaris is one of the best-studied phototrophic eukaryotes. From the 1950s on, C. vulgaris and some other algal species were cultivated in huge quantities to meet the growing demand for alternative protein sources. After drying, algal biomass can be merchandised as tablets, capsules, extract or powder with specific biochemical qualities. However, the products quality, e.g. the containing species, microbial contamination or content and quality of pigments varies enormously. In this study, commercial Chlorella products, unprocessed Chlorella powders and several production strains were investigated. Molecular analysis of the 18S rDNA confirmed either the existence of more than one species per product or only other green algae species in about half of the samples tested. Many of the examined samples contained critical amounts of bacterial contaminations. Furthermore, cyanobacteria were detected in some of the samples. The content of chlorophyll a varied greatly between the samples and pheophytin, a degradation product of chlorophyll, was detected in some samples in large concentrations. These data indicate that quality control of microalgal products is an important issue that should be addressed by the manufactures.     

A Multiplex Immunoassay Method for Simultaneous Quantification of Iron,Vitamin A and Inflammation Status Markers

Deficiencies of vitamin A and iron affect a significant portion of the world''s population, and efforts to characterize patterns of these deficiencies are hampered by a lack of measurement tools appropriate for large-scale population-based surveys. Vitamin A and iron are not easily measured directly, so reliable proxy markers for deficiency status have been identified and adopted. Measurement of inflammatory markers is necessary to interpret vitamin A and iron status markers, because circulating levels are altered by inflammation. We developed a multiplex immunoassay method for simultaneous measurement of five markers relevant to assessing inflammation, vitamin A and iron status: α-1-acid glycoprotein, C-reactive protein, retinol binding protein 4, ferritin and soluble transferrin receptor. Serum and plasma specimens were used to optimize the assay protocol. To evaluate assay performance, plasma from 72 volunteers was assayed using the multiplex technique and compared to conventional immunoassay methods for each of the five markers. Results of the new and conventional assay methods were highly correlated (Pearson Correlations of 0.606 to 0.991, p<.0001). Inter-assay imprecision for the multiplex panel varied from 1% to 8%, and all samples fell within the limits of quantification for all assays at a single dilution. Absolute values given by the multiplex and conventional assays differed, indicating a need for further work to devise a new standard curve. This multiplexed micronutrient immunoassay technique has excellent potential as a cost effective tool for use in large-scale deficiency assessment efforts.     

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.     

赭曲霉毒素A和玉米赤霉烯酮-二联胶体金免疫层析试纸条的制备及应用

【背景】真菌毒素为真菌的有毒次级代谢产物,混合污染时毒性显著增强,可对人类和动物健康造成严重伤害。制备二联胶体金免疫层析试纸条,实现对常见真菌毒素混合污染的快速监测,具有重要意义。【目的】制备赭曲霉毒素A (Ochratoxin A,OTA)和玉米赤霉烯酮(Zeralenone,ZEN)金标单克隆抗体,基于免疫层析原理,采用竞争反应模式,建立二联胶体金免疫层析检测法用于污染样品中OTA和ZEN的同时快速检测。【方法】采用柠檬酸钠还原法制备胶体金颗粒,并标记获得两种真菌毒素金标单克隆抗体,通过优化相关条件,建立稳定的二联胶体金免疫层析检测方法,用于同时检测谷物和饲料样品中的OTA和ZEN。【结果】制备的OTA和ZEN二联胶体金试纸条对OTA和ZEN的检测限分别为0.625 ng/mL和1.25 ng/mL,且与谷物和饲料中其它真菌毒素(黄曲霉毒素B1、伏马毒素B1、桔青霉毒素、展青霉毒素和呕吐毒素)均无交叉反应,人工添加试验结果准确。对天然样本检测结果表明该方法与LC-MS/MS一致性良好。【结论】本研究制备的二联胶体金试纸条可用于实际样品中OTA和ZEN的同时快速筛查。     

Microbiological Evaluation of Suture Items Before Radiation Sterilization

Microbiological contamination levels of suture samples taken at various stages of the manufacturing process in a new hygienically controlled plant were determined by employing a membrane filturation technique. Both raw material and materials handled manually in the production process were tested to assess the effect of manual handling on the product contamination level. Evaluation of the efficacy of contamination control, however, was directed primarily to the finished, packaged products, just prior to the processing with cobalt 60. The suture material for testing was divided into two groups, namely, wet and dry products, the wet being packaged in a special "tubing" fluid consisting mainly of isopropyl alcohol. Initial contamination results are reported as the average of values obtained on the test day and the preceding 9 consecutive production days. A total of 1,787 suture samples tested in the dry group showed daily averages varying between 2.1 and 14.8 contaminants per suture. The 2,980 wet-packaged suture samples tested gave daily averages varying from 0.7 to 4.2 contaminants per suture. The highest values obtained for an individual suture were 400 for the dry and 89 for the wet. Identification studies of the contaminants revealed that fungi predominated. Most of the bacterial contaminants proved to be spore-forming rods.     

Infestation of Chocolate-based Products: Insects Responsible and Origins of Contamination

In response to on-going consumer complaints regarding insect infestation of chocolate-based products manufactured at a factory in southern Australia, research was undertaken to determine the insects responsible for infestation and locate the points along the manufacture/distribution network where insect pests were most likely to be entering the produce. Phycitine moths were responsible for almost all cases of product infestation, with most infestation occurring after goods had been packaged. Methods of identifying storage environments suspected of unknowingly harbouring phycitine populations or of regularly handling infested goods are discussed. the detrimental consequences for the manufacturer, and for the processed food industry in general, of the presence of stored product insects in wholesale and retail outlets are also considered.     

ADP inhibition of myosin V ATPase activity

The kinetic mechanism of myosin V is of great interest because recent evidence indicates that the two-headed myosin V molecule functions as a processive motor, i.e., myosin V is capable of moving along an actin filament for many catalytic cycles of the motor without dissociating. Three recent publications assessing the kinetics of single-headed myosin V provide different conclusions regarding the mechanism, particularly the rate-limiting step of the cycle. One study (, Proc. Natl. Acad. Sci. USA. 96:13726-13731) identifies ADP release as the rate-limiting step and provides a kinetic explanation for myosin V processivity. The others (, J. Biol. Chem. 274:27448-27456;, J. Biol. Chem. 275:4329-4335) do not identify the rate-limiting step but conclude that it is not ADP release. We show experimental and simulated data demonstrating that the inconsistencies in the reports may be due to difficulties in the measurement of the steady-state ATPase rate. Under standard assay conditions, ADP competes with ATP, resulting in product inhibition of the ATPase rate. This presents technical problems in analyzing and interpreting the kinetics of myosin V and likely of other members of the myosin family with high ADP affinities.     

Sensitive enzyme immunoassay for Manduca allatotropin and the existence of an allatotropin-immunoreactive peptide in Periplaneta americana

Antisera to Manduca sexta allatoropin were raised in rabbits and were used to develop a competitive enzyme immunoassay for this neuropeptide. The detection limit of the assay is less than 2 fmol/well. A useful quantification can be obtained from 2 to 30 fmol/well. No cross-reactivity was observed with several insect peptides, but the enzyme-linked immunosorbent assay does recognize [Ala⁶, Leu⁷, Ser⁸]-allatotropin, a myotropin recently isolated from Locusta migratoria. The assay was used to study the distribution of allatotropin within the nervous system of Manduca sexta. The peptide is present in the retrocerebral complex, the brain, and the ventral nerve cord of this species, in quantities of respectively 0.01, 1.2, and 1.7 pmol per insect. An allatotropin-immunoreactive peptide was found in the nervous system of Periplaneta americana. It is present in the ventral nerve cord (3.3 pmol/insect), brain (1.9 pmol/insect), and retrocerebral complex (0.09 pmol/insect). These data suggest that peptides of this family are generally present in insects. © 1993 Wiley-Liss, Inc.     

Rapid method for the detection of storage mites in cereals: feasibility of an ELISA based approach

This paper describes the development of rapid immunodiagnostic tests for the detection of storage mite infestations in cereals and cereal products. The study's first phase (proof of concept) involved the production of a species-specific enzyme-linked immunoassay (ELISA) for the flour mite, Acarus siro (L.), a major pest of stored commodities. The specificity of this new assay was assessed against key stored product contaminants (13 species of mites of which three were predatory, five species of insects and five species of fungi) in the presence and absence of grain. The assay was species-specific (no cross-reactivity to other storage contaminants) and was unaffected by the presence of cereal antigens in the extract. In the study's second phase, species- and genera-specific ELISAs were developed for a range of key storage mite pests: the cosmopolitan food mite (Lepidoglyphus destructor), the grocers' itch mite (Glycyphagus domesticus), the grainstack mite (Tyrophagus longior), mites of the Tyrophagus and Glycyphagus generas, and all storage mites. All tests were demonstrably specific to target species or genera, with no cross-reactions observed to other storage pest contaminants or cereals. The final, validation phase, involved a comparative assessment of the species-specific A. siro and the genus-specific Tyrophagus ELISAs with the flotation technique using laboratory and field samples. Both ELISAs were quantitative (0-30 mites per 10 g wheat) and produced good comparative data with the flotation technique (A. siro r(2)=0.91, Tyrophagus spp. r(2)=0.99).     

Arrivals of Hitchhiking Insect Pests on International Cargo Aircraft at Miami International Airport

In a study of hitchhiking or contaminating insect pests on international cargo aircraft at Miami International Airport from 1998 to 1999, it was found that contamination rates were greatest, 23%, on cargo flights from Central America and much lower, near 5%, on flights from all other regions. We reanalyzed the study data to test for associations between contaminated flights and factors such as season, cargo type, and time of departure (night or day), and developed probabilistic models for predicting insect pest arrivals by region and pest risk levels. Significant (P < 0.05) associations were detected between contaminated flights and (1) wet season flights from Central America, (2) flights carrying plant products and clothing or fabrics, and (3) flights departing at night from the country of origin. In Monte Carlo simulations, numbers of arriving mated insect pests were greatest for cargo flights from Central America, because of great contamination rates, and South America, because of the large volume of flights from there. Few insects arrived on flights from the Caribbean, and few high-risk insects arrived from anywhere. Although the likelihood of establishment in South Florida via this pathway could not be estimated, based upon arrivals the greatest threats were posed by moderate-risk insect pests on flights from Central and South America. Simulations indicated that switching to daytime departures only reduced pest arrivals by one-third. The simplest mechanism for pathway entry that explains the associations found is that insects entered aircraft randomly but sometimes remained because of the presence of certain cargo types. Hence, contamination rates were greater during the wet season because of greater abundance locally, and on nighttime flights because of greater abundance around lighted loading operations. Empty planes probably had no pests because pests had no access to holds. Thus, the best mitigation strategies for this pathway will likely be those that exclude insects from holds or reduce the attractiveness of night loading operations. Optimizing inspections based on associations is also possible but will be less effective for regions such as South America, with high flight volumes and low contamination rates. Comparisons to other pathways indicates the potential importance of hitchhikers on cargo aircraft at MIA.     

An affinity-amplified immunoassay for juvenile hormone esterase.

A method is described for increasing the specificity of an immunoassay for catalytically active enzymes and is specifically illustrated with a sensitive assay for an important regulatory enzyme from insects. Trifluoromethyl ketone haptens, potent inhibitors of insect juvenile hormone esterase, were bound to proteins such as hemocyanin (keyhole limpet) and conalbumin (chicken embryo). Haptens containing a thiol group were conjugated using heterobifunctional coupling reagents, and haptens with a carboxylic acid moiety were conjugated by the mixed anhydride method. The trifluoromethyl ketone-protein conjugates, shown to retain their inhibitory activity against juvenile hormone esterase, were used as coating antigens in several solid-phase enzyme-linked immunosorbent assay formats along with specific antibodies raised in rabbits against purified juvenile hormone esterase. The previously unreported format, termed affinity-amplified immunoassay (AAIA), was successfully used for quantitative monitoring of low levels of the esterase in dilute hemolymph and egg homogenates from various lepidopteran insect species, as well as for detection of the native and mutant forms of the enzyme obtained in a recombinant baculovirus expression system. The AAIA format was more sensitive for the target esterase and detected only the catalytically active form of the enzyme.     

Citrus limonoid by-products as insect control agents

Limonoids are a group of chemically related bitter tetranortriterpene derivatives found predominantly in Rutaceae and Meliaceae plants (Ourison et al., 1964). Interest in the Rutaceae limonoids has centered around limonoid removal from consumable citrus products. For example, bitterness in citrus juices (as well as in other citrus products) due to limonoids has become an increasingly serious economic problem (Wilson & Crutchfield, 1968; Sinclair, 1972). Interest in the Meliaceae limonoids, on the other hand, has centered on their efficacy as pest control and/or antitumor agents (Kubo & Klocke, 1981, 1982; Nakanishi, 1977, 1980). For example, azadirachtin, isolated from several Meliaceae trees, has proven to be a potent natural product against a myriad of insect and nematode pests (Warthen, 1979). In fact, we have isolated azadirachtin from the fresh fruit of Azadirachta indica as a potent insect ecdysis inhibitor against four agricultural pest insects with artificial diet feeding assay (Kubo & Klocke, in litt).     

Impact of environmental manipulation for Anopheles pseudopunctipennis Theobald control on aquatic insect communities in southern Mexico.

Extraction of filamentous algae from river pools is highly effective for the control of Anophelespseudopunctipennis in southern Mexico. We determined the magnitude of changes to the aquatic insect community following single annual perturbations performed over two years. In 2001, algae were manually removed from all the pools in a 3 km long section of the River Coatán, Mexico, while an adjacent section was left as an untreated control. In 2002, the treatments of both zones were switched and algal extraction was repeated. The abundance of An. pseudopunctipennis larvae + pupae was dramatically reduced by this treatment and remained depressed for two to three months. A total of 11,922 aquatic insects from ten orders, 40 families, and 95 genera were collected in monthly samples taken over five months of each year. Algal extraction did not reduce the overall abundance of aquatic insects in river pools, but a greater abundance and a greater richness of taxa were observed in 2002 compared to the previous year. This was associated with reduced precipitation and river discharge in 2002 compared to 2001. Shannon diversity index values were significantly depressed following algal extraction for a period of three months, in both years, before returning to values similar to those of the control zone. However, differences between years were greater than differences between treatments within a particular year. When insects were classified by functional feeding group (FFG), no significant differences were detected in FFG densities between extraction and control zones over time in either year of the study. Similarly, percent model affinity index values were classified as "not impacted" by the extraction process. Discriminant function analysis identified two orders of insects (Diptera and Odonata), water temperature, dissolved oxygen and conductivity, and river volume (depth, width, and discharge) as being of significant value in defining control and treatment groups in both years. We conclude that habitat manipulation represents an effective and environmentally benign strategy for control of An. pseduopunctipennis. Variation in precipitation and river discharge between years was much more important in determining aquatic insect community composition than variation generated by the filamentous algal extraction treatment.     

Ex vivo cytotoxicity of the Bacillus thuringiensis Cry4B delta-endotoxin to isolated midguts of Aedes aegypti larvae

The pathological effect of the Bacillus thuringiensis Cry delta- endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.     

Friction force reduction triggers feet grooming behaviour in beetles

In insects, cleaning (grooming) of tarsal attachment devices is essential for maintaining their adhesive ability, necessary for walking on a complex terrain of plant surfaces. How insects obtain information on the degree of contamination of their feet has remained, until recently, unclear. We carried out friction force measurements on walking beetles Gastrophysa viridula (Coleoptera, Chrysomelidae) and counted grooming occurrence on stiff polymer substrata with different degrees of nanoroughness (root mean square: 28-288 nm). Since nanoscopically, rough surfaces strongly reduced friction and adhesion without contaminating feet, we were able to demonstrate, for the first time to our knowledge, that friction force between tarsal attachment pads and the substrate provides an insect with information on the degree of contamination of its attachment structures. We have shown that foot grooming occurrence correlates not only with the degree of contamination but also with the decrease of friction force. This result indicates that insects obtain information about the degree of contamination, not statically but rather dynamically and, presumably, use mechanoreceptors monitoring either tensile/compressive forces in the cuticle or tensile forces between leg segments.     

Natural occurrence of aflatoxin B<Subscript>1</Subscript> in some Indonesian food and feed products in Yogyakarta in year 1998–1999

A survey was conducted between 1998–1999 to evaluate the level of aflatoxin B₁ (AfB₁) contamination in some selected Indonesian food products, mainly peanuts and peanut products for sale in supermarkets or traditional markets in Yogyakarta, Indonesia. Quantitative analysis was carried out on 118 samples using the ELISA (Enzyme-Linked Immunosorbent Assay) technique. The results indicate that (61.1%) samples were contaminated with AfB₁ at range 2.0 to 249.0 μg/kg. Approximately 50% of the baby food products analysed were contaminated with AfB₁ and the maximum level found was 7.0 μg/kg. In corn products and fermented products, AfB₁ was detected in 66.7 and 50.0% of samples, respectively. A level as high as 5.6 μg/kg of AfB₁ was found in the corn and 6.0 μg/kg in fermented product. AfB₁ was also detected in all rice products, feed products, and other processed products at levels of up to 7.0, 27.0, and 26.0 μg/kg, respectively.