Plate-trapped Antigen Research Articles

A monoclonal antibody for the detection of Serpula lacrymans

An indirect, enzyme-linked immunoabsorbent assay (ELISA) using plate-trapped antigen, has been used to demonstrate the specificity of an IgM monoclonal antibody obtained against Serpula lacrymans mycelium. The antibody was employed in tests against a range of wood-rotting fungi including several isolates of S. lacrymans obtained from different sources. All S. lacrymans isolates gave significantly higher ELISA values (at A 450nm ) than any of the other species. The differences were maintained when the fungi were present in naturally or artificially infected timber, or in culture medium. The efficacy of the method was further assessed in a blind test using samples of material provided by Cuprinol Ltd. Samples containing S. lacrymans were positively identified.

The interactionin plantaof polygalacturonases fromBotrytis cinereawith a cell wall-bound polygalacturonase-inhibiting protein (PGIP) in raspberry fruits

The activities of four fungal polygalacturonases (endoPGI, endo-PGII, exo-PGI, exo-PGII), detected when Botrytis cinerea was grown on immature fruits of red raspberry (Rubus idaeus), were fractionated into soluble and wall-bound fractions. Western blots and plate-trapped antigen ELISA showed that endo-PGI and endo-PGII were most abundant in the cell wallbound fractions of the host. Immuno-inhibition studies using a polyclonal antiserum against polygalacturonase-inhibiting protein (PGIP), purified from immature raspberry fruits, showed that the low level of fungal PG activity detected in fractions containing endo-PGI was due to the presence of PGIP. When a purified preparation of endo-PGI and endo-PGII from B. cinerea was allowed to react in vitro with either a crude host cell wall preparation, or one which had previously been treated to remove cell wall-bound proteins, both endo-PG isozymes had a greater binding capacity towards the former wall preparation. Endo-PGI and endo-PGII also had an affinity for fungal cell walls. Exo-PGI and exo-PGII bound to both fungal and host cell walls. Greater quantities of fungal endo-PGs were detected by ELISA in fruits previously frozen and thawed ('freeze-thawed') and inoculated, than in fresh inoculated fruit. This result paralleled the extent of fungal growth in these tissues as assessed by chitin assay and suggests that the resistance shown by raspberries is dependent on continual replacement of inhibitory substances or induced resistance mechanisms

Comparison of penicillinase-based and alkaline phosphatase-based enzyme-linked immunosorbent assay for the detection of six potato viruses

A penicillinase (PNC)-based enzyme-linked immunosorbent assay (ELISA) was compared with one based on alkaline phosphatase (ALP) to detect potato viruses A, S, V, X and Y, and potato leafroll virus. Tests were made with different ratios (w/w) of γ-globulin∶enzyme in the conjugation reaction. The γ-globulin fraction of antisera to the viruses was conjugated to ALP or to PNC by the single step glutaraldehyde bridge method using the ratio of 1∶1 (γ-globulin∶enzyme). The ALP conjugates worked well but PNC conjugates gave substantial non-specific reactions and in general were not suitable for ELISA. However, PNC conjgates made with a ratio of 1∶0.05 (γ-globulin∶enzyme) gave little or no non-specific reaction and were at least as sensitive as ALP conjugates. Both ALP and PNC conjugates were found to be useful in three variants of ELISA (double antibody sandwich; plate-trapped antigen and a simplified plate-trapped antigen form of ELISA) to detect the six potato viruses. Although PNC-based ELISA often required longer incubation with substrate to achieve the same sensitivity of detection as with ALP-based ELISA, this is only a minor disadvantage and PNC should prove to be more economical and safer to use than ALP.

Production of Monoclonal Antibodies Against Banana Bunchy Top Virus and Their Use in Enzyme‐Linked Immunosorbent Assay

AbstractWhen purified banana bunchy top virus (BBTV) was used to immunize Balbc mice, all 480 wells from one fusion contained hybridomas and culture fluid from 92% of them showed a positive reaction to BBTV using plate trapped antigen (PTA) ELISA (also known as indirect ELISA). After a third screening, 84 hybridoma cell lines retained their ability to produce BBTV‐specific antibodies. Fitry‐eight monoclonal cell lines with the ability to produce BBTV‐specific antibodies. were obtained when these hybridomas were cloned by limiting dilution to single cells. The antibodies produced by these monoclonal hybridomas were mostly isotype IgG2a. The titres of antibody in culture supernatant and ascitie fluid of one clone were 104 and 104, respectively, while those of another clone were as high as 104 and 104. Hybridomas produced 40 to 62 times more anti‐BBTV specific antibodies in mouse ascites than in culture. The minimum active concentration of purified immunoglobulin produced by three hybridomas ranged from 0.03 to 0.06 μg/ml, while the minimum detectable concentration of BBTV antigen was 0.26 μg/ml with PTA ELISA. Antibody trapped antigen (ATA) ELISA (also known as direct ELISA) was 16 times more sensitive than PTA ELISA in detecting BBTV. Although PTA ELISA could not detect BBTV in crude extract of diseased tissues, ATA ELISA was able to detect the virus even when the extract was diluted 1/512.

A Monoclonal Antibody That Discriminates Strains of Citrus Tristeza Virus

(...) Discrimination of CTV isolates by CTV-MCA13 was similar in indirect ELISA with plate-trapped antigen and in double antibody sandwich indirect ELISA with antigen trapped on polyclonal antibody-coated plates. Results of immunoelectron microscopy suggest that CTV-MCA13 may react to a cryptic epitope on the viral coat protein. The monoclonal antibody is an IgG2a immunoglobin and did not react to extracts of healthy citrus or citrus infected with other viruses