Monoclonal Antibody-based Sandwich ELISA Research Articles

Detection of Mycoplasma mycoides sub-species mycoides small colony by a specific capture/enrichment monoclonal antibody-based sandwich ELISA

The present study describes the development of a specific Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) monoclonal antibody (MAb), 6E3, and its application in a sandwich ELISA (sELISA) format. Mab 6E3 reacted only to the 12 MmmSC within the 32 M. mycoides cluster strains and 12 representative strains of other bovine, ovine and caprine associated mycoplasmas examined. A capture/enrichment format of the sELISA that combined MAb 6E3 with a previously developed MAb 3H12 that cross reacted with Mmm Large Colony [Rodriguez, F., Ball, H.J., Finlay, D., Campbell, D., Mackie, D.P., 1996. Detection of Mycoplasma mycoides sub-species mycoides by monoclonal antibody-based sandwich ELISA. Veterinary Microbiology 51, 69–76], retained MmmSC specificity and improved the sensitivity from the 1.2×107cfu/ml for a standard 2h capture stage sELISA down to as low as 2cfu/ml for a 72h capture. A low level of false positives (1%) was observed when this assay was applied to 200 bovine respiratory and milk samples submitted for diagnostic investigation. This simple and specific sELISA provides a suitable assay for screening large numbers of samples for CBPP.

Rapid quality control of a live attenuated Peste des petits ruminants (PPR) vaccine by monoclonal antibody based sandwich ELISA

Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of goats and sheep. A homologous Vero cell-based attenuated PPR vaccine developed in our laboratory and used extensively throughout the country, is available for control of PPR. The presently used quality control test, titration in Vero cells for PPR virus titre in vaccine batches, takes at least 6–8 days to determine the quality and dose of vaccine. In this study, 74 freeze-dried PPR vaccine batches were tested simultaneously by both virus titration and PPR sandwich ELISA (S-ELISA) to correlate the titre of the vaccine virus with reactivity in S-ELISA. It was found that the vaccine batches with titre more than 10 3 TCID 50/ml gave positive results in S-ELISA and correlated well with the virus titre of the freeze-dried vaccines. The correlation coefficient between the virus titration and S-ELISA reactivity was estimated as 0.96, indicating a high correlation between the two parameters based on 74 batches of freeze-dried PPR vaccine. The vaccine batches with titres of 3.0, 4.3, 4.5, 5.0, 6.5 and 7.0 had shown a positive reaction when tested in two-fold dilutions in S-ELISA at 1, 5, 6, 7, 8 and 9 log 2 titres, respectively. The test vaccine batches were found to be negative in S-ELISA when the titre of the vaccine was less than 10 3 TCID 50/ml, suggesting that the vaccine could not be passed for field use. It is concluded that S-ELISA could be a preliminary tool useful for the quality control of PPR vaccine as it is rapid and easy to perform when compared to virus titration.

Novel Polyclonal−Monoclonal-Based ELISA Utilized To Examine Lupine (Lupinus Species) Content in Food Products

Sweet lupines are increasingly used in food production. Cause for concern has been expressed due to the increase in reported lupine-induced allergic incidents and the association between lupine and peanut allergies. In the current study, a polyclonal-monoclonal antibody-based sandwich ELISA for the detection of lupine proteins in foods was developed. The assay was sensitive to both native and processed proteins from Lupinus angustifolius and Lupinus albus and had a detection limit of 1 mug/g. Intra- and interassay coefficients of variation were <5 and <17%, respectively. A selection of 112 food samples, both with and without lupine declaration, was evaluated for their content of lupine. The data showed that the majority were in agreement with the respective labeling. However, some inconsistency was seen, typically in bread/rolls and soy flours.

Detection of Mycoplasma agalactiae Antigen in Sheep and Goats by Monoclonal Antibody-Based Sandwich ELISA

Zendulkova D., H. J. Ball, A. Madanat, P. Lany, Z. Pospi‰il: Detection of Mycoplasma agalactiae Antigen in Sheep and Goats by a Monoclonal Antibody- Based Sandwich ELISA, Acta Vet. Brno 2004, 73: 461-464. A preliminary epidemiological survey was carried out to find out whether contagious agalactia of sheep and goats was present in herds in the Czech Republic or Jordan. A total of 99 animals were examined for the presence of Mycoplasma agalactiaeantigen; there were 64 animals from the Czech Republic and 35 from Jordan. All Jordanian animals showed signs of clinical disease. Biological materials for examination included 353 swabs (133 from sheep and 220 from goats) collected from conjunctival, nasal and vaginal mucosae and the external ear canal. A monoclonal antibody-based sandwich ELISA was used to detect the antigen. The results were positive in 11 animals, ambiguous in 10 animals and negative in the rest of them (78). They confirmed our assumption that the Czech Republic is still free from contagious agalactia of sheep and goats but demonstrated that Jordanian herds of small ruminants are infected with Mycoplasma agalactiae. Sheep, goats, contagious agalactia, antigen, Mycoplasma agalactiae, ELISA

Taeniosis–cysticercosis in man and animals in the Sierra of Northern Ecuador

Taenia solium is endemic in the Andean region of Ecuador. The recent rediscovery of Taenia saginata in humans urges to reconsider some assumptions in relation to the epidemiology of the taeniosis/cysticercosis complex in this country. Therefore, data were compiled on the infection of both tapeworms in man and animals in Pichincha and Imbabura provinces in the Andean region, north of Quito. On post mortem inspection 3 out of 806 (0.37%) carcasses had T. saginata metacestodes, however, 35 sera out of 869 (4.03%) showed circulating antigen in a monoclonal antibody-based sandwich ELISA (Ag-ELISA). Porcine cysticercosis was detected in 15 out of 2896 (0.52%) carcasses and 93 out of 1032 serum samples (9.01%) were positive in Ag-ELISA. In humans, 4.99% (215 out of 4306) cases of antigen positives were found, whereas coprological examination of 1935 stools resulted in 30 positive cases (1.55%). The limited number of adult tapeworms (29) that were collected does not allow firm conclusions on the proportion of each species, but in total 21 specimen were identified as T. saginata and 8 as T. solium. These data have been discussed in view of the epidemiology of human cysticercosis.

Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA.

Grass pollen extracts currently used for allergy diagnosis and immunotherapy are a complex mixture of proteins of which only a few have allergenic activity. Lol p 1 is one of the most important allergens in grass pollen extracts. To develop a two-site enzyme-linked immunosorbent assay for the quantification of Lol p 1 and other group 1 allergens from grass species, and to assess its suitability for quantifying this group of allergens. Balb/c mice immunized with recombinant Lol p 1 were used for the production of monoclonal antibodies. Screening of hybridomas was performed by direct ELISA, and selected monoclonal antibodies were immobilized on ELISA plates and incubated with samples containing group 1 allergens. Bound allergens were detected by a combination of biotinylated Lol p 1-specific monoclonal antibody and peroxidase-streptavidin conjugate. The assay is based on three Lol p 1-specific monoclonal antibodies with different epitope specificities. The optimized ELISA measured Lol p 1 concentrations ranging from 125 to 1000 ng/mL and could quantify group 1 allergen from grass species belonging to the Pooidea subfamily. The assay does not depend on anti-sera production or availability of human sera and thus reactives can be produced in unlimited amounts. This sensitive and specific Lol p 1 assay will be helpful both for quantifying the group 1 allergen content of Pooideae pollen extracts intended for clinical use and for studying cross-reactivities among pollen extracts.

Development of a Monoclonal Antibody-Based Sandwich ELISA for Detection of the Latex Allergen Hev b 1

Background: Natural rubber latex (NRL) products are complex mixtures consisting of different allergenic components. Among them, Hev b 1 belongs to the important and well-characterized ones. To quantify the relevant allergen Hev b 1 in NRL products, a two-site monoclonal antibody (mAb)-based assay was developed. Methods: Two Hev b 1-specific mAbs with different epitope recognition and ability to bind simultaneously to an Hev b 1 molecule were used in the study. Both mAbs (II4F9 and II4G9) were enriched by in vitro production in a modular minifermenter and affinity purified. Wells of micro-ELISA plates coated with captured mAb II4G9 were incubated with samples containing Hev b 1. Bound Hev b 1 was detected by a combination of biotinylated mAb II4F9 as detection antibody and peroxidase-labeled avidin. Results: The optimized sandwich ELISA was highly reproducible in the linear range of the standard curve and Hev b 1 concentrations ranging from 12.5 to 400 ng/100 μl could be detected. The assay was suitable for the detection of Hev b 1 concentrations in latex sap and latex products, e.g. gloves, with a detection limit of 1.25 μg of Hev b 1/g of rubber. In a preliminary study with five different brands of latex gloves, Hev b 1 concentrations were found to be in the range of 18–40 μg per gram of rubber material, corresponding to 2–4% of the total extractable protein content in latex glove extracts. Conclusions: A sensitive sandwich assay was developed to quantify the latex allergen Hev b 1. This assay can be used to standardize latex extracts with regard to the content of the major allergen Hev b 1.

Quantification of profilins by a monoclonal antibody-based sandwich ELISA

Profilins are plant allergens responsible for cross-reactivities in pollen and fruit-allergic patients. A two-site enzyme-linked immunosorbent assay has been developed for the quantification of profilins and its suitability for quantifying profilin in different plant extracts has been evaluated. The assay is based on two profilin-specific monoclonal antibodies (mAbs) with different epitope specificities. These antibodies were immobilized on ELISA plates and incubated with samples containing profilin. Bound profilin was detected by a combination of biotinylated profilin-specific antiserum and peroxidase–streptavidin conjugate. The optimized ELISA measured profilin concentrations ranging from 4 to 250 ng/ml and could quantify profilins from plant species of a variety of different botanical families. No reactivity to mites, molds, or crustaceans was detected, suggesting that the immunoassay is plant-specific. The results indicate that this sensitive profilin-assay will be helpful both for quantifying the profilin content of allergenic extracts intended for clinical use and for studying cross-reactivities between pollen extracts.

Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA

Background: Among 13 allergens found in extracts of cooked brown shrimp ( Penaeus aztecus) the 36 kd muscle protein tropomyosin has been identified as the only major shrimp allergen (Pen a 1). Cross-reacting molecules with similar molecular weights were detected in other crustacea species such as crab, lobster, and crawfish. Because Pen a 1 and Pen a 1-like allergens are important in crustacea allergy, the aim of this study was to develop a monoclonal antibody (mAb)–based sandwich ELISA to quantify Pen a 1 and to evaluate Pen a 1 levels in four commercial shrimp, crab, and lobster extracts. Methods: Two Pen a 1-specific mAbs with different epitope specificities were selected. ELISA plates coated with captured mAb 3.2 were incubated with samples containing Pen a 1. Bound Pen a 1 was detected by a combination of biotinylated mAb 4.9.5 and alkaline phosphatase–labeled streptavidin. Results: The optimized sandwich ELISA could detect Pen a 1 concentrations ranging from 4 to 125 ng/ml. Four commercial shrimp extracts demonstrated a 40-fold difference in Pen a 1 levels (24 to 920 μg/ml). Crab and lobster extracts contained detectable levels of Pen a 1-like proteins. No reactivity to cockroach, house dust mite, oyster, codfish, or peanut extracts was detected, which indicates that the developed assay is crustacea-specific. Conclusion: A sensitive sandwich assay was developed to quantify Pen a 1. This assay will be helpful to standardize shrimp extracts in regard to the content of the major allergen, Pen a 1, and to study cross-reactivities among and evaluate occupational exposure to different crustacea species. (J Allergy Clin Immunol 1997;100:229-34.)

604 A monoclonal antibody-based sandwich ELISA to quantify the major shrimp allergen Pen a 1

604 A monoclonal antibody-based sandwich ELISA to quantify the major shrimp allergen Pen a 1

Development of Monoclonal Antibody-Based Sandwich ELISA for the Detection of Nuclear Polyhedra of Nuclear Polyhedrosis Virus Infection in Bombyx mori L.

Murine monoclonal antibodies (MCAs) were produced against Borrelina bombycis nuclear polyhedra. In ELISA all five MCAs reacted with nuclear polyhedra of B. bombycis and to a variable extent with other strains of nuclear polyhedra, namely Amsaeta olbistriga, Heliothis armigera, and Spodoptera litura . These were devoid of reactivity with normal hemolymph proteins, Serratia marcescens, Nosema bombycis, group A streptococci, Staphylococcus aureus, and Escherichia coli, except Bacillus thuringiensis. However, MA-321 showed a low degree of reaction with B. thuringiensis. In Western blots, these recognized antigens of 31 (polyhedrin protein) and 67 kDa of nuclear polyhedra (MA-321 showed reduced binding). The MCAs specifically recognized nuclear polyhedra in either infected Bran cells or mid gut of Bombyx mori larvae. In sandwich enzyme immunoassay employing purified rabbit polyclonal antibodies and MA-321 as low as 100 nuclear polyhedra could be detected. The results obtained by sandwich ELISA on hemolymph samples of infected as well as normal larvae collected from field revealed 100% correlation with results by microscopic examination. Hemolymph samples collected on various days from II instar larvae infected in the laboratory revealed that the sandwich ELISA could detect infection as early as 96 hr after infection. These antibodies could be useful for detection of nuclear polyhedra infection at early stages, i.e., II to III instar of larvae.

Monoclonal antibody ELISA to quantitate wheat gliadin contamination of gluten-free foods

A sensitive reproducible monoclonal antibody-based sandwich ELISA has been developed to measure the gliadin content of foods. Using the assay we have confirmed that nominally gluten-free products based on wheat starch contain trace amounts of gliadin. The level of gliadin contamination in these foods is sufficient to cause relapse of coeliac disease in specific patients. We have also examined the cross-reactivity of different cereal proteins in the assay and found good correlation with their known toxicity to patients with coeliac disease.