Triple Antibody Sandwich ELISA Research Articles

SEROLOGICAL COMPARISON OF TOSPOVIRUS ISOLATES USING POLYCLONAL AND MONOCLONAL ANTIBODIES

A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four serogroups, 96 were chosen because they could be recovered after inoculation to Nicotiana benthamiana and N. rustica. Antigen extracts were tested at 1:100 and 1:1000 in parallel in two different enzyme-linked immunosorbent assay (ELISA) protocols: double antibody sandwich ELISA (DAS-ELISA) with 10 polyclonal antisera and triple antibody sandwich ELISA (TAS-ELISA) with 33 monoclonal antibodies (MAbs). According to both tests, 47 isolates belonged to serogroup 1 (TSWV); 8 belonged to serogroup II (2 TCSV, 2 GRSV, and 4 of uncertain classification); 8 belonged to serogroup III (INSV); and 6 belonged to serogroup IV. Among the remaining 27 isolates, 16 did not react with any of the different antibodies; whereas, 11 were detected by only a few antibodies that related them either to serogroup I or II. It can be assumed that they include hitherto unknown tospovirus isolates. DAS-ELISA revealed that polyclonal antisera, prepared against complete virions, had a much broader reactivity than nucleocapsid (N)-specific antisera; however, in this comparison they, were only available for TSWV. TAS-ELISA included both N-and glycoprotein (G)-specific MAbs against TSWV and revealed a high degree of variability, reflecting the presence of several different epitopes on both proteins. In addition, a polymerase chain reaction (PCR) detection method was tested with three different primer sets that were designed to detect tospoviruses in general as well as specifically. Two different primer sets were able to detect tospoviruses from all four serogroups.

Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

Summary.Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study.Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate‐trapped antigen ELISA (PTA‐ELISA) and dot‐blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS‐ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA‐ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse‐grown plants.

A comparison of four serological tests for the detection of banana bunchy top virus in banana

Four different serological tests for detection of banana bunchy top virus (BBTV) in banana sap are compared: (i) a triple-antibody sandwich ELISA for BBTV, utilising anti-BBTV polyclonal antibodies for virus capture, and anti-BBTV monoclonal antibodies, alkaline phosphatase-labelled sheep anti-mouse antibodies, and p-nitrophenyl phosphate for detection (ELISA-NPP); (ii) an alternative enzyme-substrate system for ELISA involving an amplification step (AmpakTM enzyme amplification kit) (ELISA-A); (iii) a colorimetric dot immunobinding assay (DIBA-C), in which the enzyme-substrate system was alkaline phosphatase and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate; (iv) an enhanced chemiluminescent form (DIBA-ECL), in which the enzyme-substrate system was horseradish peroxidase and luminol. For both DIBA-C and DIBA-ECL, maximum sensitivity was obtained by pre-coating the nitrocellulose membrane with anti-BBTV polyclonal antibodies, by using 0.05 M sodium carbonate (pH 9.6) as the coating buffer, and by clarifying the sap by centrifugation and extraction with chloroform or dichloroniethane. Treatment of the sap before centrifugation by snap-freezing at -70�C, or heating at either 30 or 50�C for 10 min, had no effect on sensitivity; heating at 70�C for 10 min eliminated antigenicity. ELISA-NPP, ELISA-A, and DIBA-ECL had equivalent sensitivity, but DIBA-C was up to 8-fold less sensitive than the former 3 assays. ELISA-NPP was adjudged to be the best compromise between sensitivity, cost and completion time.

Setting confidence limits for the detection of prune dwarf virus in Prunus avium with a monoclonal antibody‐based triple antibody‐sandwich ELISA†

SUMMARYA triple antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf virus (PDV) in sweet cherry trees (Prunus avium). An independent reverse transcribed polymerase chain reaction test was also developed to establish, in conjunction with a bioassay, the incidence of PDV in 40 sweet cherry trees and to confirm the absence of virus in 15 control trees. Trees with two‐thirds of their leaves positive for PDV would be identified with 99% probability by testing four leaves per tree with TAS‐ELISA. The monoclonal antibody did not cross‐react with Prunus necrotic ringspot virus in the TAS‐ELISA.

Studies on Variability of Peanut Clump Virus: Symptomatology and Serology

AbstractIntensive survcys of groundnut virus diseases were carried out in Senegal from 1986 to 1990. Peanut clump virus (PCV; furovirus group) was detected in several regions in groundnuts (Arachis hypogaea), showing typical symptoms namely, small dark green leaves, short petioles and internodes, and reduced shoot size resulting in a dwarfed and bushy appearance (clumping) of the infected plants. PCV was also detected in groundnuts exhibiting variable symptoms like chlorotic leaf spots, specking, chlorotic rings or ringspots, line patterns, vein yellowing, mottle or light mosaic etc. with or without clumping. Symptoms induced by these different isolates on the test plant Chenopodium amaranticolor also showed considerable variability. Serological studies of 41 isolates of PCV (collected from Senegal, Burkina Faso, Niger and India), using seven monoclonal antibodies in Triple Antibody Sandwich ELISA (TAS‐ELISA), permitted us to distinguish five different serogroups based on their reaction profiles. However, these did not correspond to the five groups formed in an arbitrary classification based on the symptomatology of C. amaranticolor. Serogroups do not correlate with the geographic origin.

Inconsistent transmission of banana bunchy top virus in micropropagated bananas and its implication for germplasm screening

Banana bunchy top virus (BBTV) was readily transmitted through tissue culture in banana (Musa sp.) cv. Lady finger (AAB) and Cavendish cv. Williams (AAA). Lines derived from infected and healthy field plants had similar in vitro multiplication rates. BBTV-infected in vitro cultures displayed symptoms of stunting, leaf curling, chlorotic and green flecks, and poor root growth. Symptoms became milder with time, and were often difficult to discern in older, rapidly multiplying cultures. A triple antibody sandwich ELISA using polyclonal and monoclonal antibodies was very efficient for detecting BBTV in vitro. Symptomless, ELISA-negative plants arose in 10 out of 11 lines derived from BBTV-infected field plants and first appeared after 9 months continuous in vitro culture at a constant 28�C. Meristem tip culture or heat therapy was not used. These plants remained symptomless and ELISA-negative after planting out in the glasshouse (individual plants checked for up to 16 months). The implications of this inconsistent transmission of BBTV for germplasm indexing and exchange are discussed.

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein

D. S. Smith and D. D. Kitts. Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein. Toxicon 32, 317–323, 1994.—A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

Consecutive elisa screening with monoclonal antibodies to detect potato virus YN

In a 1992 survey of potato fields in Canada for the tobacco veinal necrosis strain of potato virus Y (PVYN), initial enzyme-linked immunosorbent assay (ELISA) screening with one monoclonal antibody (MAb 295.5) followed by a second ELISA screening with a panel of several MAbs (4E7, Bioreba, 1F5 and 295.5) was assessed as a means of rapidly identifying positive samples prior to confirmation by bioassay. MAbs 4E7 and Bioreba were used in double antibody sandwich (DAS) ELISA, while MAbs 1F5 and 295.5 were used in triple antibody sandwich (TAS) ELISA. In the second ELISA, MAbs 4E7, 1F5 and Bioreba all enhanced test specificity relative to the retest with MAb 295.5. All the MAbs were found to react, to some degree, with isolates of the common strain of PVY (PVYO), but MAb 4E7 was the most specific widi no cross-reaction being observed at, or above, the signal to noise (S:N) threshold of 4:1. However, this MAb was also the least sensitive. At the S:N threshold of 4:1, its sensitivity was 68%, compared to a sensitivity of close to 100% for the other MAbs

Beet Western Yellows Virus Is Not an Important Component of Potato Leafroll Disease in Canada and the United States

We tested 801 samples of potato leaves with leafroll-like symptoms for potato leafroll virus (PLRV) and beet western yellows virus (BWYV) using virus-specific monoclonal antibodies in triple-antibody sandwich enzyme-linked immunosorbent assay (TAS ELISA). The samples represented 32 cultivars and originated in eight Canadian provinces and 12 states in the United States. None of the samples tested positive for BWYV, whereas 774 (96.6%) tested positive for PLRV. Neither virus could be recovered via aphid (Myzus persicae) transfers to indicator hosts from 18 samples that tested negative for both viruses. Tubers collected from 134 of the samples were sprouted and retested by both TAS ELISA and a polyclonal antiserum-based double-antibody sandwich (DAS) ELISA

The Relationship Between Aphid-Transmissibility of Potato Leafroll Virus and Surface Epitopes of the Viral Capsid

A panel of nine monoclonal antibodies (MAbs) was used to investigate the relationship between aphid-transmissibility and surface epitopes of the capsid of potato leafroll virus (PLRV). In immunoblocking experiments, mixtures of purified PLRV and MAbs were fed to 1-day-old Myzus persicae nymphs, and their transmission efficiency was then studied. The MAbs were applied in triple-antibody sandwich enzyme-linked immunosorbent assay (TAS ELISA) to characterize antigenically two phenotypic variants of the Wageningen isolate of PLRV (PLRV-Wag) that differ in transmissibility and a highly aphid-transmissible isolate of PLRV. It was previously established that these MAbs re-acted with epitopes exposed on the surface of the virion. However, the MAbs can be separated into two groups on the basis of their reactivity with intact and disrupted particles of PLRV [...]

Reliable Detection of Barley Yellow Dwarf Viruses in Field Samples by Monoclonal Antibodies

Monoclonal antibodies in triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and polyclonal antibodies in double-antibody sandwich ELISA (DAS-ELISA) were used for detection of three serotypes of barley yellow dwarf viruses (BYDVs) in field collected oat and wheat leaf samples. Agreement between the two ELISA systems was >99% for 900 and 1,050 samples collected in 1989 and 1990, respectively, indicating that the monoclonal antibodies were detecting highly conserved viral epitopes (...)

Comparison and Differentiation of Potyvirus Isolates and Identification of Strain-, Virus-, Subgroup-specific and Potyvirus Group-common Epitopes Using Monoclonal Antibodies

A panel of monoclonal antibodies (MAbs) generated against an admixture of 12 potyvirus isolates was used to compare and differentiate diverse potyviruses. Both native and denatured virions of strains of bean yellow mosaic (BYMV), potato virus Y, tobacco etch, pea seed-borne mosaic, iris severe mosaic, iris mild mosaic and asparagus virus-1 potyviruses were used as immunogen and as antigen for screening of the hybridoma cell lines. Thirty cell lines secreting potyvirus-specific antibodies reactive in indirect antigen-coated plate (ACP-) ELISA were selected for detailed analysis. All 30 MAbs reacted with at least one strain of BYMV; 11 MAbs reacted with between one and eight of the nine BYMV strains and an additional three MAbs reacted only with isolates within the BYMV subgroup (BYMV, pea mosaic virus and clover yellow vein virus). The remaining 16 MAbs reacted with a BYMV isolate and with at least one of the other 43 potyvirus isolates tested. MAb PTY 1 reacted with all 55 potyvirus isolates tested (representing at least 33 different and distinct aphid-transmissible potyviruses). The potyvirus cross-reactive MAbs generally gave higher reactivity values in ACP-ELISA with dissociated virus than with polyclonal antibody-trapped intact virions in triple antibody sandwich ELISA (i.e. were cryptotope-specific). The BYMV strain- and virus-specific MAbs reacted strongly with both types of antigens (i.e. were metatope-specific). At least 25 distinct epitopes (12 cryptotopes and 13 metatopes) could be identified from the MAb-antigen reactivity patterns. The distribution of these epitopes between virus isolates can be used to detect and differentiate potyviruses in infected plant extracts and to examine virus architectures. Some of these epitopes are shared by potyvirus isolates not previously shown to be serologically related. The broad spectrum-reacting MAb PTY 1 recognizes a cryptotope conserved on all of the aphid-transmissible potyviruses examined and should be a valuable tool for the detection and assay of these potyviruses.

Survey of Spring Oats for Barley Yellow Dwarf Viruses in Illinois

During the spring of 1987, 1,215 samples of spring oats (Avena sativa L.) were collected in Madison, Champaign, Woodford, Warren, and DeKalb counties, Illinois. At each site on each of three sampling dates, 45 samples were collected (regardless of symptoms) in a W pattern in I ha and tested for the PAY, MAV, RPV, and RMV serotypes of barley yellow dwarf virus (BYDV) by direct doubleantibody sandwich enzyme-linked immunosorbent assay (ELISA). RMV was not detected at any location. PAY and RPV were detected at all locations, as early as 17 April in Champaign County. The incidences of P A V and RPV from all plants sampled ranged from 2 to 64% and from 2 to 88%, respectively. Highest incidences of both strains were in May samples [rom Woodford County. MAV was detected in lower incidences (2-16%) only in samples from the central region of the state (Champaign, Woodford, and Warren counties). The presence of MA V serotypes was confirmed in triple-antibody sandwich ELISA with the MA V -specific MAFF2 monoclonal antibody from L. Torrance. In the last previous survey for BYDV in Illinois during 1967-1968 (1), about 75% of the isolates were PAY and about 20% were RPV; single isolates of RMV and MAV were found. Twenty years later, 55% were PAY, 39% were RPV, and 6% were MAV.

Use of monoclonal antibody to potato leafroll virus for detecting groundnut rosette assistor virus by ELISA*

SUMMARYThree of 10 monoclonal antibodies (MAbs) produced to potato leafroll luteovirus (PLRV) were found to react in triple antibody sandwich ELISA (TAS‐ELISA) with groundnut rosette assistor luteovirus (GRAV), though none reacted with four other luteoviruses (barley yellow dwarf, bean leaf roll, beet western yellows or carrot red leaf)‐ The most effective PLRV MAb, SCR 6, was used in TAS‐ELISA to detect isolates of GRAV from groundnut plants with chlorotic, green and mosaic forms of rosette from Nigeria and Malawi. The test also detected GRAV in extracts of single Aphis craccivora.