Indirect Double Antibody Sandwich ELISA Research Articles

Development and evaluation of indirect double-antibody sandwich ELISA for rapid detection of Salmonid Alphavirus using Baculoviridae expressed E1 Protein

Salmon alphavirus (SAV) is a widespread virus that infects Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Europe. It has a fatality rate of ≤48% and can cause pancreatic and sleeping disease. With the frequent exchange of salmon farming trade, it may become a global pathogen; therefore, a sensitive, effective, and high-throughput detection method is necessary. The serological diagnoses for SAV are indirect immunofluorescence assays (IFA) and indirect enzyme-linked immunosorbent assays (ELISA), which can only detect one of the six subtypes of SAV. In this study, an insect cell/baculovirus expression system was used for simultaneous detection of multiple genotypes (SAV1, SAV2, and SAV5) and an indirect double-antibody sandwich-enzyme linked immunosorbent assay (IDAS-ELISA) method was adopted, based on the highly conserved region (E1) of SAV1-6. The results showed that the IDAS-ELISA had no cross-reaction with other common RNA viruses and high sensitivity with 103.4TCID50/0.1 mL detection line. SAV 1 (98.33%) and SAV 2 (91.67%) samples were positively identified by IDAS-ELISA and real-time polymerase chain reaction (PCR), respectively. This assay can simultaneously detect multiple subtypes of SAV and provide technical support for clinical diagnosis and the epidemiology of SAV.

Behavior of profilins in the atmosphere and in vitro, and their relationship with the performance of airborne pollen

Most pollen allergens in the air are carried by pollen grains, but the presence of airborne smaller respirable particles containing pollen allergens has also been demonstrated. Meteorological factors drastically affect the occurrence of pollen, allergen release in the air and diffusion of the latest. In order to shed light on this phenomenon, the dynamics of pollen and the pollen panallergen profilin in the air of two European cities (León, Spain and Bologna, Italy) having different weather conditions, were analyzed. Pollen sampling was performed continuously from March to June 2015 using two seven-day recording volumetric trap of Hirst-type, while the particles for aeroallergen quantification were sampled with a Burkard Cyclone sampler and the profilin content in aerosol samples was quantified using an indirect double-antibody sandwich ELISA. In both cities, pollen and profilin concentrations followed a similar trend and showed a significant correlation; however, peaks were often misaligned, with the profilin peaks following those of pollen. Several meteorological parameters, such as relative humidity, significantly influenced pollen and allergen dispersion. In vitro pollen tests were thus performed in order to mimic pollen rehydration, occurring in natural conditions and a massive protein release from allergenic pollen was detected during the early stages of pollen rehydration when profilin was also extruded from the grains. The different timing and protein amounts released from different pollen during hydration might explain, at least in part, the non-synchronous pollen and profilin peaks detected in the atmosphere.

Estimation of the accuracy of two diagnostic methods for the detection of Plum pox virus in nursery blocks by latent class models

The control of Plum pox virus (PPV), the most important viral disease that affects stone fruit trees, requires the use of reliable detection methods. The effectiveness of spot real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) for the detection of PPV in samples collected from nursery blocks was compared with a validated PPV detection technique, the double antibody sandwich indirect enzyme‐linked immunosorbent assay (DASI‐ELISA) using the PPV‐specific monoclonal antibody 5B‐IVIA/AMR. In total, 5047 nursery plants were analysed by both techniques. The agreement between the techniques was almost perfect (Cohen’s kappa index of 0·88 ± 0·01). The diagnostic parameters (sensitivity, specificity and likelihood ratios) of both techniques were simultaneously evaluated in 2473 nursery plants by latent class models using maximum likelihood functions and a Bayesian approach. The sensitivity and specificity of both techniques did not vary according to the latent model applied. Spot real‐time RT‐PCR was more sensitive while DASI‐ELISA was more specific for PPV detection. In addition, the findings demonstrate that latent class models are a flexible and potent statistical method to estimate the accuracy of diagnostic tests for plant pathology.

Identification and detectability of broad bean stain virus in broad bean seeds and effects on nodulation

During a survey for broad bean viruses in Sohag Governorate, a virus was identified as Broad bean stain virus (BBSV), a member of the genus Comovirus, based on host reactions, symptomatology, electron microscopy and serologically by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) technique. The virus was mechanically transmitted to some members of Leguminosae and also through seeds. The ultraviolet absorption spectrum of the purified virus showed a typical curve of nucleoprotein with an A260/280 ratio of about 1.25–1.34. The yield of purified virus was 0.61–0.63 mg/kg infected tissue. Electron microscopy of purified preparations revealed the presence of isometric virus particles, 27 nm in diameter. The polyclonal antibodies against BBSV were produced and the antiserum titre of three bleedings was determined by indirect DAS-ELISA technique. Gross reduction of nodulation was achieved by virus inoculation on broad bean plants cv. Giza 402. It produced smaller, fewer nodules and reduced its leghaemoglobin content. As well as seed yield quality and quantity was strongly affected due to infection. When cells of root nodules in BBSV-infected broad bean plants were investigated by transmission electron microscopy, a decrease of number, volume of bacteroids in nodule cells and the space between the bacteroid and its membrane envelope (ME) were observed when compared with healthy cells. This difference was accompanied with the presence of BBSV particles in the root nodule cells. Seeds taken from these plants were tested for the presence of the virus by DAS-ELISA and symptoms development on the seedlings produced. There was a good correlation between ELISA detection of BBSV in tissue taken from single broad bean seeds and subsequent development of infected plants grown from the same seeds. The ELISA detected BBSV in the cotyledons and developing axis of the embryo, but not in seed coat tissues. When mixtures of infected and healthy seeds in different ratios were tested, BBSV was detected in mixtures up to 1:100 (infected: healthy). The ELISA technique is reliable for selecting BBSV-free stocks of broad bean seeds.

Susceptibility of Prunus rootstocks to natural infection of Plum pox virus and effect of mineral oil treatments

The use of rootstocks that are less susceptible or resistant to natural Plum pox virus (PPV) infection and/or the application of mineral oil treatments are two possible strategies to reduce viral incidence in nursery plots. We evaluated the susceptibility of Prunus rootstocks used in the Spanish stone fruit industry and the effect of mineral oil treatment (Sunspray Ultrafine at 1%) on the spread of the virus at two different localities in Valencia, Spain, under different natural PPV inoculum pressures (high inoculum pressure, 2006‐08; low inoculum pressure, 2006‐07). Samples from both plots were analysed by double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) and spot real-time RT-PCR. Under high inoculum pressure, the assayed rootstocks exhibited significant differences in their susceptibility to natural infection. The most susceptible rootstocks at the end of the experiment were Adesoto 101 and Mariana GF8-1. Cadaman and Garnem rootstocks presented the fewest PPV-infected plants; these infections could be detected only by spot real-time RT-PCR. No differences among the assayed rootstocks were found under low PPV inoculum pressure. Aphid species were monitored using Moericke yellow water traps and sticky-plant methods at both localities in May 2006 and 2007. Aphis spiraecola was the most abundant aphid species monitored by both methods at both localities. The average percentage of A. spiraecola carrying PPV PCR-amplifiable targets was 30.37% in the plot with high PPV inoculum pressure and only 7.98% in the plot with low inoculum pressure. We found significant differences in PPV incidence between Mariana GF8-1 plots that were treated with mineral oil and those that were not treated after one year under natural conditions and high PPV inoculum pressure.

Serum midkine depends on lymph node involvement and correlates with circulating VEGF-C in oesophageal squamous cell carcinoma

Lymph node metastasis (LNM) is a key factor for selection of treatment method and patients’ prognosis in oesophageal squamous cell carcinoma (ESCC). However, no biomarkers able to support the clinical detection of LNM have been reported. Recently, vascular endothelial growth factor C (VEGF-C) was found to be a more accurate marker of LNM in lung cancer than computed tomography. Midkine is a multifunctional cytokine involved in cancer development. We investigated circulating midkine levels in ESCC patients (n=73) compared with those in healthy subjects (n=42) with double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). We found that midkine was elevated in ESCC and involved in metastatic disease. Serum midkine (sMK) was a good marker of LNM, evaluated both clinically and pathologically, as revealed by ROC analysis. It also correlated with serum levels of VEGF-C. The increase of sMK was related to cancer cells, although a weak correlation was observed between sMK and platelet and leucocyte counts.

Seasonal Variation of Ralstonia solanacearum Biovar 2 Populations in a Spanish River: Recovery of Stressed Cells at Low Temperatures

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14 degrees C or higher, but its isolation was usually unsuccessful at temperatures below 9 degrees C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35 degrees C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum. Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35 degrees C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.

Interleukin-2 potentiates foot-and-mouth disease vaccinal immune responses in mice

The present study describes the role of recombinant human interleukin-2 (rh IL-2) as immunomodulatory molecule in foot-and-mouth disease (FMD) vaccinal immune response in a murine model. The humoral immune response was evaluated by examining the antibody titre against FMD virus type O, A 22 and Asia 1 in serum samples obtained from different groups of mice inoculated with PBS, FMD vaccine alone; vaccine along with rh IL-2 on 0, 7, 14, 21, and 30 days post vaccination (DPV) by indirect double antibody Sandwich ELISA. The cellular immune response was also examined on different DPV by an MTT based lymphoproliferation assay in splenic mononuclear cells (SMNC) obtained from different groups. IL-2 was able to enhance the specific immune response against FMD virus type O, A 22 and Asia 1 as evident by significantly higher ELISA antibody titres ( P < 0.05) in serum obtained from mice receiving IL-2 along with vaccine as compared to mice immunized with vaccine alone. Similarly, the same group of mice showed significantly higher lymphoproliferative responses in SMNC against mitogen PHA and FMD virus types O, A 22 and Asia 1 on all DPVs as compared to the group inoculated with vaccine alone.

Current situation of sharka disease in Ankara, Turkey

Sharka disease has a limited distribution in Turkey and does not present a problem for stone fruit production. However, sharka is the most common virus disease of apricots, plums and peaches in Ankara, although it is not a common disease in other cities in Turkey. In different parts of Ankara, 212 private gardens in 21 locations were surveyed forPlum pox virus (PPV) incidence. All together 935 trees of apricot, plum, peach, sweet cherry and sour cherry were sampled and tested for the presence ofPPV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA).PPV was identified in numerous trees,viz. 286 apricots, 172 plums and 65 peaches. Strain differentiation ofPPV was accomplished using double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). These assays confirmed the presence of isolates belonging toPPV-M, PPV-D and mixed infections ofPPV-M+D. This is the first report of the presence ofPPV-D and mixed infections ofPPV-M+D in Turkey.

Natural Spread of Plum Pox Virus in Ankara, Turkey

AbstractPlum pox virus (PPV) has a limited distribution in Turkey, so is not a serious problem for stone fruit production. However, it is widespread in apricot, plum and peach trees in the Ankara region where its natural spread was studied between 1995 and 2002 in 10 home gardens planted with apricot, plum and peach trees. The presence of the virus was determined by visual observation of symptoms in leaves and fruits and by double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA); strains were identified by double antibody sandwich indirect enzyme‐linked immunosorbent assay (DASI‐ELISA) and immunocapture reverse transcription‐polymerase chain reaction (IC‐RT‐PCR) tests. Within 8 years, PPV incidence in the gardens near to, and 0.5 km from an infection source was 85.7–100 and 25–29.4%, respectively. This difference in incidence was attributed to the distance from the infection source. All PPV isolates were identified as PPV‐M. It was found that sharka disease was introduced to the healthy gardens by mealy plum aphids (Hyalopterus pruni), the only vector of PPV found in Ankara. This is the first report of PPV spread in Turkey.

Elimination of Citrus psorosis virus by somatic embryogenesis from stigma and style cultures

Somatic embryogenesis was used to eliminate Citrus psorosis virus (CPsV) from three citrus species (common mandarin, sweet orange and Dweet tangor), all of which regenerated somatic embryos with different embryogenic potential from stigma and style explants. CPsV was detected by double antibody sandwich‐indirect‐enzyme‐linked immunosorbent assay (DASI‐ELISA) in explants and embryogenic callus, but was not detected in any of the plants obtained from somatic embryos, even 24 months after regeneration. Loss of juvenile characters (disappearance of thorns) was observed in the first year of growth and was retained in plants propagated by grafting from thornless stems. Somatic embryogenesis appears to be a very promising technique for the production of healthy citrus stocks.

Characterization and expression of the coat protein-coding region of banana bract mosaic potyvirus, development of diagnostic assays and detection of the virus in banana plants from five countries in southeast Asia.

We have sequenced the entire coat protein (CP)-coding region and 5' 162 nucleotides of the 3' untranslated region (UTR) of nine different isolates of banana bract mosaic virus (BBrMV) from five different countries. Further, we have sequenced the 3' 621 nucleotides of the NIb-coding region of a Philippines isolate. This is the first report of BBrMV in Thailand, Vietnam and Western Samoa. When the sequences of the CP-coding region and 3' UTR were compared to each other, variability of between 0.3% and 5.6%, and 0.3% and 4. 3%, was observed at the nucleotide and amino acid levels, respectively. Phylogenetic analysis of the BBrMV isolates did not reveal any relationship between the geographic location of the isolates. The BBrMV CP was expressed in Escherichia coli as a fusion protein and the purified recombinant protein was used to produce a high titre BBrMV-specific polyclonal antiserum. This antiserum was used to develop a F(ab')(2) indirect double antibody sandwich ELISA and compared with immuno-capture PCR (IC-PCR) and reverse transcription PCR (RT-PCR) assays for BBrMV detection. RT-PCR was shown to be the most sensitive test followed by ELISA and IC-PCR. http://link.springer. de/link/service/journals/00705/bibs/9144009/91441725.htm</++ +HEA

Modulation of the antigenic reactivity of the citrus tristeza virus coat protein

Trapping properties of a panel of monoclonal antibodies (Mabs) raised against citrus tristeza virus (CTV) were analyzed in an indirect double-antibody sandwich ELISA (I-DAS-ELISA). These antibodies had been previously assigned by serological specificity into five groups (I to V). Mabs from group V, which are directed to conformational epitopes, trapped significant amounts of virus antigen from CTV-infected plant tissue at IgG concentration above 10 ng/ml. Mabs from groups I to IV, which are directed to linear, continuous epitopes, performed poorly as coating antibodies, even at a 1 μg/ml concentration of the IgG's, indicating that the respective linear epitopes were inaccessible. However, when Mabs from groups I to IV were combined with a small amount of Mabs from group V, a substantial increase in trapping of the CTV antigen was recorded. In this `two antibody-binding assay' previously cryptic, linear epitopes of the CTV CP apparently became accessible to the Mabs from groups I to IV. Modulation of the antigenic reactivity of the CTV CP was also recorded upon binding of the Mabs directed to the conformational epitopes in solution. Induced exposure of the linear epitopes of the CTV CP was revealed in `two antibody-binding assays' with pairwise combinations of different mouse Mabs and several rabbit and chicken polyclonal antisera with different serological specificities, including antisera to bacterially expressed CP fragments. This mixed coating in I-DAS-ELISA resulted in substantially increased efficiency of the virus antigen trapping by antisera produced against bacterially expressed protein fragments and an increased sensitivity of the CTV detection after optimization of the ratio between conformational and linear antibodies.

Quantitation of monoclonal antibodies by ELISA: The use of purified mouse IgG and mouse IgM monoclonal antibodies as standards in a quantitative ELISA measuring monoclonal antibodies produced by cell culture

Murine monoclonal antibodies (MAbs) of IgG1, IgG2a and IgG2b subclasses and IgM class in hybridoma culture supernatants were quantified using a sensitive, reliable, optimized indirect double antibody sandwich ELISA. In the ELISA, the MAb in the culture supernatants was sandwiched between affinity isolated heavy chain specific polyclonal antibodies used for capture and detection. Quantitation was achieved by comparison with a standard curve produced by a purified MAb of the same class, subclass or ideally the same clone as the MAb to be quantified. These quantitative results were compared with those obtained using purified IgG and IgM polyclonal serum samples as standards and those obtained by total protein estimation using measurement at OD 280 nm. The IgG subclass MAbs used as standards were purified using protein G and the IgM class MAb was purified by ion exchange followed by gel filtration chromatography. Bovine IgG contamination of the MAb supernatants and the purified MAbs was also measured by a double antibody sandwich ELISA.

Detection of maize streak virus antigens over time in different parts of maize plants of a sensitive and a so-called tolerant cultivar by ELISA

Maize streak virus (MSV) capsid antigens were detected over time in different parts of maize plants of a sensitive (INRA508) and a so-called tolerant (?tolerant?) cultivar (IRAT297) using a direct or an indirect double antibody sandwich ELISA. Based on three types of experiments, it was shown that the antigens were distributed in the plant according to the age of the tissues. When the virus was inoculated on a particular leaf of 18-day old plants with infective Cicadulina mbila, only the young leaves above the inoculated one were positive by ELISA but not the older ones below. The antigens could not be detected in the inoculated leaf. At day 3 after inoculation, the antigens were detected in the sheath and/or in the whorl of the third leaf above the inoculated one but not in the oldest part of the leaf, the unfolded lamina. Plants of the sensitive cultivar were inoculated at 9 days with C. mbila deposited in the whorl. At 23 h after inoculation, the antigens were detected in the sheath but not in the whorl which was found to be positive only at 32 h. On the basis of these results, a hypothesis of the mode of virus infection is proposed. Our results contribute to a better understanding of the relationship between the age of the plant at inoculation and yield loss as well as secondary infection. By transmission tests with C. mbila, it was shown that virus could only be acquired from leaves exhibiting symptoms. Virus concentrations were measured in plant samples by ELISA using a range of dilutions of purified virus. The virus concentrations were higher in the sensitive than in the ?tolerant? cultivar, but no difference in antigen distribution was observed between the two cultivars. The ?tolerant? cultivar appeared to be resistant to virus multiplication.(resme d'auteurs)

Monoclonal antibodies for the detection ofRhizomonas Suberifacienscausal agent of corky root of lettuce, with enzyme immunoassays

Monoclonal antibodies (MAbs) were produced to Rhizomonas suberifaciens strain CA1. Initial screening of MAbs was performed with strains CA1 and FL1 of R. suberifaciens and 82 strains of other genera in antibody capture indirect enzyme‐linked immunosorbent assay (ELISA) tests. One MAb (MAb‐Rs 1) reacted with both strains of R. suberifaciens and another (MAb‐Rs 2) only with R. suberifaciens strain CA1. Both MAbs were further screened for cross‐reactivity with 28 strains of R. suberifaciens, four strains of Rhizomonas sp., and 56 strains of unknown bacteria isolated from lettuce, prickly sowthistle and melon roots with symptoms of corky root. All these strains were tested for pathogenicity on lettuce, DNA homology with DNA of R. suberifaciens strain CA1, and cross‐reactivity with both MAbs in indirect antibody capture and indirect double antibody sandwich (DAS) ELISA tests. All strains of R. suberifaciens reacted with MAb‐Rs1 in both indirect ELISA tests, while none of the other strains reacted. In antibody capture indirect ELISA tests, MAb‐Rs2 had affinity to strains of R. suberifaciens from California and New York but not to those from Florida, Wisconsin and one from New York. In indirect DAS ELISA tests, MAb‐Rs2 reacted with all strains of R. suberifaciens. The indirect DAS ELISA tests were more sensitive than the indirect antibody capture tests.

Indirect double antibody sandwich ELISA for detecting alfalfa mosaic virus in aphids after short probes on infected plan

An indirect double antibody sandwich enzyme-linked immunosorbent assay (IDAS-ELISA) system using antibodies elicited in rabbits and chickens is described for the detection of alfalfa mosaic virus (AMV). The method is capable of detecting 40 pg of homologous purified AMV and was shown to be suitable for detecting the virus in aphids. AMV in the order of 150 pg was detected in single aphids. It was shown that a significant proportion of insects could acquire this or higher amounts of virus during 1 min probes on the leaves of infected plants. The IDAS-ELISA should find applicability in research on the acquisition of viruses by aphids and in epidemiological studies for detecting viruses in insects from traps.

In vitro culture of sugarcane infected with maize streak virus (MSV)

Young buds and leaf tissues of sugarcane cv. R 574 (a Saccharum hybrid), healthy or infected with Maize Streak Virus (MSV) were cultured in vitro. The success rate of the cultures from infected material is lower than that from healthy material. The symptoms of the disease were displayed on most of the plantlets produced from buds but only on 6% of the plantlets regenerated from calli. The indirect double-antibody sandwich ELISA technique using a monoclonal antibody makes it possible to identify virus-infected plantlets. In vitro, the infection results in the reduced growth of shoots.

Production and Characterization of Monoclonal Antibodies to Potato Virus A

Abstract Purified potato virus A (PVA) was used for immunization to produce monoclonal antibodies (MAb). The type of ELISA with purified PVA or non–purified PVA, played an essential role in selecting MAb with different specificity.Two MAb's (MAb–1 and MAb–2) were selected, using indirect ELISA (I–ELISA) with purified PVA. Competition experiments suggested that MAb–1 and MAb–2 reacted with the same epitope on purified PVA (epitope 1). ELISA, IEM and SDS–PAGE–immunoblotting experiments showed that epitope–1 was only present on purified PVA but not on non–purified PVA, suggesting that this epitope was introduced during the purification. Assays at different steps during purification indicated that epitope–1 was only exposed after plant components and reducing agents were removed from the PVA extract.Three MAb's (MAb–3, MAb–4 and MAb–5) were selected by indirect double antibody sandwich ELISA (IDAS–ELISA) with non–purified PVA. These MAb's reacted in I–ELISA or IDASELISA with purified PVA as well as with non–purified PVA and might be useful for routine diagnosis. MAb–3, 4 and 5 cross–reacted with some other potyviruses in I–ELISA and in IDAS–ELISA. MAb–1 cross–reacted with 5 out of 7 other potyviruses in I–ELISA, but not in IDAS–ELISA.

On the screening procedures of ELISA for monoclonal antibodies against three luteoviruses.

Total of one hundred and thirty-five antibody-secreting hybridomas were fused from luteovirus-immunized spleen cells. Hybridomas secreting monoclonal antibodies (MoAbs) for potato leafroll virus (PLRV), beet western yellows virus (BWYV) and tobacco necrotic dwarf virus (TNDV), were screened by four different procedures of enzyme-linked immunosorbent assay (ELISA). There are procedure 1; antigen adsorption ELISA (AAI-ELISA), in which purified virus in phosphate buffered saline (PBS) at pH 7.4 was adsorbed onto the microplate wells, procedure 2; AAI-ELISA in which purified virus in sodium carbonate-bicarbonate buffer (SCB) at pH 9.6 was adsorbed onto the microplate wells, procedure 3; indirect double antibody sandwich ELISA (IDAS-ELISA) in which polyclonal antibody was used as trapping antibody and purified preparations diluted in PBS-T (containing Tween-20) as antigens were used, procedure 4; IDAS-ELISA in which polyclonal antibody was used for trapping antibody and crude saps of virus infected plants in PBS-T as antigens were used. Based on the results, all of the antibody-secreting hybridomas can be screened by AAI-ELISA of procedure 1. On the other hand IDAS-ELISA is indispensable for screening virus-specific MoAbs. We recommend that combination of two ELISA procedures, such as AAI-ELISA of procedure 1 and IDAS-ELISA of procedure 4, will be used for the screening of luteovirus antibody-secreting hybridomas.