Stable recombinant alpaca antibodies for detection of Tulip virus X

Beekwilder, Jules; Houwelingen, Adèle van; Beckhoven, José van; Speksnijder, A.G.C.L.

doi:10.1007/s10658-007-9265-y

Cited by 18 publications

(28 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recombinant expression of these heavy chain variable domains yields single domain antibodies (VHHs). The single domain nature of VHHs provides many advantages over the other recombinant antibody fragments; in particular, ease of expression in various expression systems, high thermal stability, excellent solubility, resistance to proteolysis, and ease of genetic manipulation 15–17 . Antigen-binding patterns of VHHs are very unique compared to conventional antibodies.…”

Section: Introductionmentioning

confidence: 99%

Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive Immunoassay

et al. 2011

View full text Add to dashboard Cite

Some unique subclasses of Camelidae antibodies are devoid of light chain and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA (150% cross reactivity) with negligible cross reactivity with other tested structural analogs and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.

show abstract

Section: Introductionmentioning

confidence: 99%

Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive Immunoassay

et al. 2011

View full text Add to dashboard Cite

show abstract

“…This would give rise to false-negative results because they are not detected by ELISA. Specific antisera are also available in ELISA to detect tulip virus X (TVX) and cucumber mosaic virus (CMV) in tulips (Beekwilder et al 2008;Niimi et al 2003).…”

Section: Overall Resultsmentioning

confidence: 99%

Detection of the tulip breaking virus (TBV) in tulips using optical sensors

Polder

Heijden

Doorn³

et al. 2010

Precision Agric

View full text Add to dashboard Cite

The tulip breaking virus (TBV) causes severe economic losses for countries that export tulips such as the Netherlands. Infected plants have to be removed from the field as soon as possible. There is an urgent need for a rapid and objective method of screening. In this study, four proximal optical sensing techniques for the detection of TBV in tulip plants were evaluated and compared with a visual assessment by crop experts as well as with an ELISA (enzyme immunoassay) analysis of the same plants. The optical sensor techniques used were an RGB color camera, a spectrophotometer measuring from 350 to 2500 nm, a spectral imaging camera covering a spectral range from 400 to 900 nm and a chlorophyll fluorescence imaging system that measures the photosynthetic activity. Linear discriminant classification was used to compare the results of these optical techniques and the visual assessment with the ELISA score. The spectral imaging system was the best optical technique and its error was only slightly larger than the visual assessment error. The experimental results appear to be promising, and they have led to further research to develop an autonomous robot for the detection and removal of diseased tulip plants in the open field. The application of this robot system will reduce the amount of insecticides and the considerable pressure on labor for selecting diseased plants by the crop expert.

show abstract

“…In the present work, VHH was preferred over scFv, because scFvs are larger, and often suffer from poor stability, unless genetically engineered [25], [26], while VHHs often display high stability over long periods of exposure to ambient temperature [10], which allowed for extensive re-use of the SPR chip.…”

Section: Discussionmentioning

confidence: 99%

“…Bacteria were induced to express the VHH and urea-lysed as described previously [10] using PBS as buffer. VHHs were purified using Ni-NTA Superflow resin (QIAGEN, Germany) as reported before [10].…”

Section: Methodsmentioning

confidence: 99%

A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

Trilling

Ronde

Noteboom³

et al. 2011

PLoS ONE

Self Cite

View full text Add to dashboard Cite

BackgroundRecombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.Methodology/Principal FindingsAntibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10−10 M.Conclusions/SignificanceA broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

show abstract

Stable recombinant alpaca antibodies for detection of Tulip virus X

Cited by 18 publications

References 22 publications

Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive Immunoassay

Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive Immunoassay

Detection of the tulip breaking virus (TBV) in tulips using optical sensors

A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

Contact Info

Product

Resources

About