Indirect Enzyme-linked Immunosorbent Assay Procedure Research Articles

Identification of animal glue and hen-egg yolk in paintings by use of enzyme-linked immunosorbent assay (ELISA)

We report the development of an indirect ELISA procedure for specific identification of chicken-egg yolk and animal glues in painting micro-samples. The results presented integrate previously published work on ELISA recognition of bovine β-casein and chicken ovalbumin in painting materials. The integrated final ELISA procedure-optimised for protein extraction, immuno-reagent concentrations, blocking solution, incubation time, and temperature-enables multiplex identification, in single samples, of proteinaceous materials, i.e. chicken-egg yolk and albumen, animal glues, and bovine milk and/or casein, mainly used by painters in the past. The procedure has been systematically tested on laboratory models of mural and easel paintings, both naturally and artificially aged, to assess possible inhibitory effects on the immuno-reaction caused by inorganic painting materials (pigments and substrates) and by protein degradation resulting from aging processes. Real samples from case studies, which had previously been investigated and characterised by spectroscopy and chromatography, were successfully studied by use of the developed ELISA procedure. The commercial availability of all the immuno-reagents used, the affordable analytical equipment, and the specificity, sensitivity, and rapidity of ELISA make this method very attractive to diagnostic laboratories in the field of cultural heritage science. Possible further developments to the analytical potential of this technique include improvement of antibody performance and inclusion of other classes of bio-molecules as analytical targets.

Development and validation of an indirect Enzyme-linked Immunosorbent Assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3–100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

Indirect ELISA‐based approach for comparative measurement of high‐affinity cohesin–dockerin interactions

The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) to non-catalytic scaffoldin units (carrying multiple cohesins) in cellulosome, a multienzyme plant cell-wall degrading complex. This interaction is species-specific, for example, the enzyme-borne dockerin from Clostridium thermocellum bacteria binds to scaffoldin cohesins from the same bacteria but not to cohesins from Clostridium cellulolyticum and vice versa. We studied the role of interface residues, contributing either to affinity or specificity, by mutating these residues on the cohesin counterpart from C. thermocellum. The high affinity of the cognate interactions makes it difficult to evaluate the effect of these mutations by common methods used for measuring protein-protein interactions, especially when subtle discrimination between the mutants is needed. We described in this article an approach based on indirect enzyme-linked immunosorbent assay (ELISA) that is able to detect differences in binding between the various cohesin mutants, whereas surface plasmon resonance and standard ELISA failed to distinguish between high-affinity interactions. To be able to calculate changes in energy of binding (ΔΔG) and dissociation constants (K(d)) of mutants relative to wild type, a pre-equilibrium step was added to the standard indirect ELISA procedure. Thus, the cohesin-dockerin interaction under investigation occurs in solution rather than between soluble and immobilized proteins. Unbound dockerins are then detected through their interaction with immobilized cohesins. Because our method allows us to assess the effect of mutations on particularly tenacious protein-protein interactions much more accurately than do other prevalent methods used to measure binding affinity, we therefore suggest this approach as a method of choice for comparing relative binding in high-affinity interactions.

Development of Anti-Isoproturon Polyclonal Antibody

Abstract A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropy1)-3-(4-isopropylphenyl)-1-methylurea (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 x 10 5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L -1 and 1.0 x 10 5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC 50) was 0.07 mg mL -1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.

Avances de las pruebas únicas para detectar la brucelosis

This paper describes an indirect enzyme-linked immunosorbent assay (I-ELISA) and a fluorescence polarisation assay (FPA), each capable of detecting antibody in several species of hosts to smooth and rough members of the genus Brucella. The I-ELISA uses a mixture of smooth lipopolysaccharide (SLPS) and rough lipopolysaccharide (RLPS) as the antigen, and a recombinant protein A/G conjugated with horseradish peroxidase as the detection reagent. When using individually determined cutoff values, the SLPS/RLPS combined-antigen I-ELISA detected antibody in slightly more animals exposed to SLPS or to RLPS than did I-ELISA procedures using each individual antigen separately. Similarly, the assay using combined antigens detected antibody in slightly fewer animals not exposed to Brucella sp. When a universal cutoff of 10% positivity was used (relative to strongly positive control sera of each species), the overall performance index (percentage sensitivity plus percentage specificity) value decreased by 1.0 (from 199.4 to 198.4). In the FPA, it was not possible to use a universal cutoff without significant loss of performance. The overall sensitivity value for the FPA using the combined FPA antigen was 1.0% lower than using the O-polysaccharide (OPS) from SLPS and 9.1% higher than using the core antigen (CORE) from RLPS. When the combined antigen was used, the FPA specificity was slightly higher (1.2%) than from only the OPS, and considerably higher (12.6%) than the CORE. Overall, both the I-ELISA and the FPA with combined antigens were suitable as screening tests for all species of Brucella in the animal species tested.

Recognition of multiple Mycoplasma bovis antigens by monoclonal antibodies.

To produce monoclonal antibodies (MAbs), Balb/c AnN Crl BR mice were inoculated with the cell suspension of a Hungarian Mycoplasma bovis strain designated 26034. Three days after the last immunization the spleen of the immunized mouse was removed aseptically. The fusion of spleen cells with Sp2/0-Ag14 murine myeloma cells was performed in the presence of polyethylene glycol. The obtained hybrid cells were selected with hipoxantine, aminopterine and thymidine (HAT) medium. Two weeks after the fusion, the supernatants of the grown cells were tested by a self-developed indirect enzyme-linked immunosorbent assay (ELISA). The results showed that 63 antibody-producing hybridomas had been obtained. For accurate determination of the molecular weight of antigen determinants, the supernatants giving positive reaction in the ELISA were tested by Western blotting. According to the results, the obtained MAbs recognize the antigen determinants of the following molecular weights: 1B11: 63 kDa, 1C7: 63 kDa, 2C5: 22, 25 and 27 kDa, 2C9: 69 kDa, 3G12: 67, 69 and 72 kDa, 4H9: 63 kDa, 5B8: 22, 25 and 27 kDa, 5D3: 22, 25 and 27 kDa, 5C11: 69 kDa, 5E5: 22, 25 and 27 kDa, 6F11: 63 kDa, and 6H10: 22, 25 and 27 kDa. The 12 cell groups selected on the basis of the Western blotting were cloned twice by end-point dilution method. The cloned cells were propagated, and with 5 cell lines antibodies were produced in the CELLine bioreactor (Integra Biosciences, Zurich, Switzerland). Cell line 3G12 showed the highest productivity with an average daily output of 1.5 mg immunoglobulin. Cell line 5E5 produced 1.1 mg, 6H10 0.8 mg, 2C9 0.47 mg and 6F11 0.4 mg antibody per day. The isotype of the antibodies was determined by ELISA. The antibodies produced by the 12 cell lines tested were assigned to the IgG(1) subclass according to the heavy chain. Ten cell lines produced kappa and two produced lambda light-chain antibody. Possible cross-reactions of the produced monoclonal anti-M. bovis antibodies with certain Mycoplasma, Ureaplasma and Acholeplasma species were tested by an indirect ELISA procedure. All of the 12 antibodies tested gave a reaction with the antigen of M. bovis strain designated 26034. MAbs 3G12 (67, 69, and 72 kDa) and 5B8 (22, 25, and 27 kDa) gave no cross-reaction with antigens other than strains of the homologous Mycoplasma species. The other antibodies reacted with the M. bovigenitalium F7, M. anseris 8389, M. oculi, and M. gallisepticum s6 Holland antigens. Owing to its high specificity and affinity, the antibody produced by the cell line 3G12 is primarily considered suitable for use in immunodiagnostic tests of M. bovis infections.

Petri Dish-ELISA, a Simple and Economic Technique for Detecting Plant Viruses

A large number of microtiter plates are needed for mass testing of planting material for viruses in seed certification and plant quarantine. In the case of poorly equipped laboratories, problems with availability of microtiter plates have economic implications for the broad acceptance of enzyme-linked immunosorbent assay (ELISA) for e.g. seed health testing in developing countries. In this experiment the potential of an alternative, cheaper technique was investigated. A conventional indirect ELISA procedure was followed for comparison between the polystyrene solid phases of plastic Petri dishes and microtiter plates for detection of three viruses belonging to the Tobamovirus, Comovirus and Potyvirus genera. A wax pen was used to divide the inner surface of a polystyrene Petri dish into many circles or squares.

Detection of cucumber mosaic cucumovirus in weed species: a cautionary report on nonspecific reactions in ELISA

The reliability of enzyme-linked immunosorbent assay (ELISA) to detect cucumber mosaic cucumovirus (CMV) coat protein (CP) in three weed species (Taraxacum officinale, Gnaphalium obtusifolium, and Conyza canadensis) was compared with other detection methods, including Western blot analysis and a bioassay. Analysis of extracts prepared from these species by double antibody sandwich (DAS) ELISA yielded high absorbance values (A4O5nm) that were indicative of CMV concentrations of purified CMV standards of greater than 50 mg/mL. However, results from an ELISA procedure designed such that no viral antigen should be detected suggested that the strong ELISA absorbance values were nonspecific reactions with material(s) in the weed extracts. These findings were substantiated when no CMV CP was detected by Western blot analysis or infectious CMV was detected in bioassays involving inoculation of extracts onto an indicator host (Cucurbita pepo ‘Crookneck’ (squash)). In both DAS ELISA and indirect ELISA procedures, the nonspecific reaction was shown to result from the interaction of weed extract and alkaline phosphatase-conjugated immunoglobulin (including anti-CMV and goat anti-rabbit). Addition of bovine serum albumin to the extraction buffer for DAS ELISA or to the extraction buffer and at each antibody step for indirect ELISA dramatically reduced the nonspecific reactions.

The use of enzyme-linked immunosorbent assay for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom and venom antibodies

The specificity and sensitivity of an indirect and two (an ‘ordinary’ and a ‘rapid’) double sandwich enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom were examined. The three assays were equally sensitive and the accuracy of the assays was not substantially affected by individual variation in the venom composition. The specificity of the assays was examined against 26 venoms from snakes of the families Viperidae and Elapidae. While the double sandwich ELISA procedures were sufficiently specific to be used in the clinical immunodiagnosis of C. rhodostoma bite in Malaysia, the indirect ELISA procedure exhibited extensive cross-reactivity with other Malaysian pit viper venoms. Attempts were made to improve the specificity of the indirect ELISA procedure for the quantitation of C. rhodostoma venom. A ‘low ELISA cross-reactivity’ venom fraction (termed VF52) was isolated from C. rhodostoma venom by repeated Sephadex G-100 gel filtration chromatography. The indirect ELISA procedure using antibodies to VF52 as immunoreagent showed an improvement in specificity. The use of the indirect ELISA procedure for the detection of C. rhodostoma antibodies was also examined and the results show that the assay was sufficiently specific to be used for retrospective diagnosis of C. rhodostoma bite in Malaysia, in particular when VF52 was used as the coating antigen.

The effects of curing and cooking on the detection of species origin of meat products by competitive and indirect ELISA techniques

The purposes of this study were: (1) to develop and compare competitive and indirect enzyme linked immunosorbent assay (ELISA) methods for detecting species specific albumins in meat samples and (2) to examine the effect of curing and cooking on the identification of species origin of meats. Commercially obtained rabbit anti-pig serum albumin (anti-PSA) and rabbit anti-sheep serum albumin (anti-SSA) were affinity purified, and used to develop competitive and indirect ELISA procedures for PSA and SSA. The competitive ELISA procedures showed the lowest cross-reactivity with related serum albumins. Both ELISA procedures were capable of detecting as little as 5% pork or sheep in beef. Curing resulted in little or no inhibition in the ability of ELISA procedures to detect pork or sheep in beef. Cooking completely eliminated the ability of the competitive PSA ELISA to detect pork in beef, and of both SSA ELISA procedures to detect sheep in beef. Cooking also greatly reduced, but did not eliminate, the ability of the indirect PSA ELISA to detect pork in beef. Curing and cooking essentially eliminated the ability of the PSA ELISA procedure to detect pork in beef. Curing and cooking resulted in a 70 to 74% decrease in the signal of sheep meat in SSA ELISA procedures. These results demonstrated that competitive and indirect ELISA procedures are capable of determining the species origin of raw and cured meat. Heating raw or cured meats greatly reduced, but did not always eliminate, the ability of ELISA procedures to detect species origin of meats.

A modified indirect ELISA procedure for raw meat speciation using crude anti‐species antisera and stabilised immunoreagents

AbstractIndirect enzyme‐linked immunosorbent assay (ELISA) for species identification of unheated meat materials has been modified to facilitate the use of non‐specific (unpurified) anti‐species antisera by integral assay inhibition of heterologous cross‐reactivity. Effective ELISA treatments of three antisera (identifying beef, horse and pig) and four commercial products (identifying beef, horse, pig and sheep/goat) were determined using chequer‐board microtitration assays against pure species antigens. Subsequent tests with aqueous meat extracts gave consistent absorbance differences between the homologous (high colour) and heterologous (low colour) responses and provided reliable, accurate species identification without the extensive prior purification of antisera (i.e. by affinity chromatography) previously required. This modified ELISA enabled efficient use of stabilised anti‐species sera discs, prepared initially for application in a simplified agar‐gel immunodiffusion (AGID) test; i.e. one disc (20 μl freeze‐dried antiserum) for testing two meat samples by the prescribed AGID made sufficient treated solution to test 50 meat samples on microtitre plates. Authentic species meat extracts are also stabilised on discs for convenience (reference responses and routine controls). ELISA in this form is a practical alternative to AGID screening tests or can be used in parallel back‐up tests providing sensitive results in less than 3 h on a large or small scale.

Comparison of two indirect enzyme-linked immunosorbent assay procedures for detecting zucchini yellow mosaic virus.

Two indirect enzyme-linked immunosorbent assay (I-ELISA) procedures employing intermediate antibodies of Japanese quail (Coturnix coturnix japonica Temminck et Schlegel) and hen immunoglobulins were compared on their sensitivity for detecting zucchini yellow mosaic virus (ZYMV). The I-ELISA employing quail immunoglobulin on plates coated with gamma-globulin from rabbit antiserum against ZYMV was sensitive as that on non-coated plates, capable of detecting ZYMV in purified preparation at detection limitation of 10-50μg/ml and in crude extracts of infected pumpkin leaves at dilution end point of 104-105. In the assay with hen immunoglobulin, both procedures permitted to detect purified ZYMV at minimum concentration of 5-10ng/ml. However, when antigen in crude extracts of infected pumpkin leaves was assayed, the dilution end point obtained by non-precoated I-ELISA was 106-107, compared with 105-106 of precoated I-ELISA procedure. Of the procedures tested, non-precoated I-ELISA employing hen immunoglobulin showed the highest non-specific absorbance values. Despite of the relatively low sensitivity of non-precoated I-ELISA employing quail immunoglobulin, this procedure was relatively simple and useful tool for disease diagnosis of field samples. Quail appears to have potential as a source of anti-viral immunoglobulins in non-precoated and precoated I-ELISA for detecting ZYMV and perhaps for other plant viruses.

Variants of elisa in plant virus diagnosis

Variations of enzyme-linked immunosorbent assay (ELISA) were compared with respect to their ability to detect and to differentiate serologically related plant viruses. The broadest range of serologically related viruses was detected by an indirect ELISA on unprecoated plates. Coating the plates with F(ab') 2 fragments led to narrowing of the specificity in heterologous reactions of tymo-, tombusand tobamoviruses in indirect ELISA. With Andean potato latent virus (APLV) heterologous reactions were weaker on plates precoated with F(ab') 2 fragments than on those precoated with intact antibodies. Even on plates precoated with F(ab') 2 fragments the indirect ELISA detected a broader range of serologically related viruses than the direct double antibody sandwich method. Heterologous reactions in indirect ELISA procedures on plates precoated with either intact antibodies or F(ab') 2 fragments were always weaker than homologous reactions independent of the concentration of coating reactants and detecting antibodies. Attempts to differentiate closely related strains of APLV or radish mosaic virus by direct ELISA using F(ab') 2ments either for coating the plates or after labelling with alkaline phosphatase for detecting the trapped antigens failed. Under suitable conditions, the additional working step usually necessary for indirect ELISA could be avoided by using a short procedure which at low concentrations of detecting antibodies was more sensitive than the conventional procedure.