Protein Bait Research Articles

Single-Molecule Protein Detection in a Biofluid Using a Quantitative Nanopore Sensor.

Protein detection in complex biological fluids has wide-ranging significance across proteomics and molecular medicine. Existing detectors cannot readily distinguish between specific and nonspecific interactions in a heterogeneous solution. Here, we show that this daunting shortcoming can be overcome by using a protein bait-containing biological nanopore in mammalian serum. The capture and release events of a protein analyte by the tethered protein bait occur outside the nanopore and are accompanied by uniform current openings. Conversely, nonspecific pore penetrations by nontarget components of serum, which take place inside the nanopore, are featured by irregular current blockades. As a result of this unique peculiarity of the readout between specific protein captures and nonspecific pore penetration events, our selective sensor can quantitatively sample proteins at single-molecule precision in a manner distinctive from those employed by prevailing methods. Because our sensor can be integrated into nanofluidic devices and coupled with high-throughput technologies, our approach will have a transformative impact in protein identification and quantification in clinical isolates for disease prognostics and diagnostics.

Proximity-dependent biotinylation mediated by TurboID to identify protein-protein interaction networks in yeast.

The use of proximity-dependent biotinylation assays coupled to mass spectrometry (PDB-MS) has changed the field of protein-protein interaction studies. However, despite the recurrent and successful use of BioID-based protein-protein interactions screening in mammalian cells, the implementation of PDB-MS in yeast has not been effective. Here, we report a simple and rapid approach in yeast to effectively screen for proximal and interacting proteins in their natural cellular environment by using TurboID, a recently described version of the BirA biotin ligase. Using the protein arginine methyltransferase Rmt3 and the RNA exosome subunits, Rrp6 and Dis3, the application of PDB-MS in yeast by using TurboID was able to recover protein-protein interactions previously identified using other biochemical approaches and provided new complementary information for a given protein bait. The development of a rapid and effective PDB assay that can systematically analyze protein-protein interactions in living yeast cells opens the way for large-scale proteomics studies in this powerful model organism.

A Method of Lysate Preparation to Improve the Isolation Efficiency of Protein Partners for Target Proteins Encoded by the Genes of Human Chromosome 18

The aim of this work was to test modifications of the standard protocol for the sample preparation of cell/tissue lysate before performing the affinity isolation of lysate protein partners for the target protein (bait protein) which is covalently immobilized on an inert sorbent (e.g. BrCN-, SH-Sepharose 4B) or a carrier (e.g. paramagnetic nanoparticles). The series of our previous works on applying the approach to direct molecular fishing procedure with combination of affinity chromatography and LC-MS/MS analysis using a number of proteins, encoded by the genes of human chromosome 18, have shown that there are at least two problems affecting the specificity and the effectiveness of this procedure. These include: (i) redundancy of the background proteins in the eluates from an affinity sorbent (carrier) due to isolation of multiprotein complexes “labeled” with a direct protein partner which binds with a bait protein immobilized on the sorbent; (ii) low enrichment of the eluates with appropriate protein partners due to the fact that some direct protein partners in the lysate exist in stable “wild type” complexes with the bait protein itself. This means that latter group of protein partners will not be sufficiently isolated from lysate. Therefore, in order to increase the specificity and efficiency of affinity isolation of protein partners for the bait protein, we modified the standard protocol of lysate preparation and the preliminary step on dissociation of lysate protein complexes was added. Several model experiments for the choice of regeneration solution, assessment of their efficiency in the dissociation of lysate protein complexes as well as the stability and binding capacity of proteins were performed under the control of surface plasmon resonance (SPR) biosensor Biacore 3000 using HepG2 cell lysate. It was shown that acid treatment and incubation of the cell lysate for one min on ice (final lysate dilution 20 times) and subsequent neutralization (pH shift from 2.0 to 7.4) resulted in maximal dissociation of the lysate protein complexes without significant negative effects on the protein-protein interactions tested.

Evaluation of traps and attractants for mass trapping of African Invader Fly, <i>Bactrocera invadens</i> on mango in south west Nigeria

Mass trapping has demonstrated to be a powerful tool in the control of fruit flies, its use in African countries has currently increased remarkably as a control method. This study evaluated the efficacy of two traps; Lynfield Trap (LT) and Modified Lynfield Trap(MLT) and two lures (Methyl Eugenol (ME) and Protein Bait from Brewery Waste (PBBW)) for mass trapping of Bactrocera invadens Drew, Tsuruta, & White (Diptera: Tephritidae) on mango during 2014 and 2015 fruiting seasons. Traps containing attractants were randomly set on mango homestead trees in three replicates at three selected locations in Ibadan Southwest Nigeria. Results showed no significant difference (p > 0.01) between the trap types baited with ME attractant, although the MLT recorded higher catches of B. invadens during the two seasons. Methyl eugenol was significantly (p < 0.01) more effective than hydrolyzed protein bait from brewery waste in mass trapping B. invadens in the three locations of study. However, ME trapped only male B invadens while PBBW trapped both male and female. B. invadens population was highest at the peak of mango ripening (June) and the density of flies trapped in 2015 was significantly (p < 0.05) higher than 2014 in all the locations. Suitable choice of traps, lures and season enhances the mass trapping results. The use of Protein bait from brewery waste and modified lynfield trap for monitoring B. invadens and population suppression is promising and should be encouraged since they attract both sexes and less cost effective.Keywords: trapping, mango, lures, fruit fly, control

Bait visitation by Formica lemani (Hymenoptera: Fomicidae) indicates shortage of carbohydrates in alpine grasslands

Insights can be gained by analysing the feeding decisions of animals in terms of nutrient demands at a species or community level. Using carbohydrate and protein food baits, resource use and food preferences of Formica (Serviformica) lemani were determined at nine locations situated at different altitudes (1875 to 2400 m a.s.l.) in the alpine grassland belt above the tree line in Austria and northern Italy. F. lemani is the most common species of ant in this habitat. Sucrose baits placed around ant colonies were visited by significantly (3.9 times) more workers than protein baits. This indicates that sources of sugar (carbohydrate) are in short supply in the alpine zone, whereas availability of prey items appears to be less constraining. Overall, we recorded a decrease in the incidence of visits to baits from low (31.9% baits attracting ants at least once) to high altitudes (16.7%). Foraging ants never visited 51.5% of the baits exposed for periods of 75 min. This indicates that with increasing altitude competition for food among ant colonies becomes less intense in alpine grassland ant communities.

Effect of Physiological State on Female Melon Fly (Diptera: Tephritidae) Attraction to Host and Food Odor in the Field.

Foraging behavior of wild female melon fly, Bactrocera (Zeugodacus) cucurbitae Coquillett, a worldwide pest of economically important cucurbit crops, was examined through mark and recapture studies in both wild (Kona: dominated by the invasive weed ivy gourd, Coccinea grandis [L.] Voigt [Cucurbitaceae]), and cultivated (Kapoho: dominated by papaya, Carica papaya L. [Caricaceae] orchards) habitats on Hawaii Island. In particular, the extent to which wild melon flies and color-marked F2 females responded to cucumber odor and Solulys yeast hydrolysate laced with ammonium acetate (1%, wt/vol) according to sexual maturity stage and degree of protein hunger was documented. Kona results indicated that more wild and color-marked F2 females responded to cucumber (Cucumis sativus L. [Cucurbitaceae]) odor than to protein odor with the exception of captured wild flies without eggs, which responded similarly to protein bait and cucumber odor. Results with captured wild females and color-marked F2 females in Kapoho suggested a significant preference for cucumber odor over protein odor regardless of whether or not they had eggs in their ovaries with the exception of protein-deprived color-marked F2 females, which responded to both odors in equal numbers. Implications of these new findings based on wild melon flies in natural habitats are discussed with respect to integrated pest management control strategies with protein bait sprays used in Hawaii. The possibility of adding cucurbit volatiles to protein-based baits is discussed.

Soil surface complexity has a larger effect on food exploitation by ants than a change from grassland to shrubland

1. Changes in vegetation community composition, such as a transition from grassland to shrubland (woody encroachment), are associated with reductions in plant cover and increases in bare ground. Encroachment‐driven changes in surface cover at small spatial scales can alter ant community assemblages by changing their foraging behaviour and their ability to locate and monopolise resources.2. Artificial arenas with three levels of complexity were used to examine changes in ant foraging efficiency, body size and ability to monopolise food. The three levels of complexity included a control (no substrate), low‐complexity treatment (woody debris) and high‐complexity treatment (leaf litter).3. No difference was found in ant species composition within the complexity arenas between grassland and shrubland, but ant functional groups ‘generalised Myrmicinae’ and ‘subordinate Camponotini’ were more abundant in grassland arenas, whereas ‘opportunists’ were more abundant in shrubland arenas. Ants took twice as long to find baits in high‐complexity treatments, and 1.5 times as long in low‐complexity treatments, than in control treatments, which were bare arenas with no substrate. Ant body size declined with increasing surface complexity, suggesting that larger ants are discouraged from foraging in complex habitats.4. There was also significantly greater monopolisation of the protein bait (tuna) in low‐ and high‐complexity treatments, but there were no differences between tuna and carbohydrate (honey) in the control treatment. Consistently, no differences were found in ant behaviour between grasslands and shrublands.5. The present study shows that ants are more responsive to small‐scale alterations in soil surface complexity than to changes in vegetation community composition. Changes in soil surface complexity select for ants based on body size, which in turn influences their foraging success. Changes in vegetation complexity at small spatial scales are therefore likely to influence ant behaviour and abundance of some functional groups, potentially having an effect on the many ecosystem functions carried out by ants.

Suppression of female melon fly, Zeugodacus cucurbitae, with cue‐lure and fipronil bait stations through horizontal insecticide transfer

AbstractMelon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), is an important quarantine tephritid fruit fly with resident populations established in Hawai'i, USA. In the male‐annihilation approach, male flies are targeted using dispensers with cue‐lure (C‐L) and insecticides, typically organophosphates. The efficacy of the male annihilation approach is thought to be limited to individual male flies, contacting the lure and the pesticide, after which they die. Alternative classes of insecticides, such as fipronil, have been investigated for use in male‐annihilation. We hypothesized that ingestion of fipronil by male flies could lead to horizontal transfer and mortality in female flies. Horizontal insecticide transfer extends pesticide control beyond the individual contacting the toxicant through indirect contact via food sharing or other mechanisms. We tested the possibility for horizontal transfer of fipronil from male to female Z. cucurbitae through field and laboratory studies. Two repeated field trials were conducted to compare the numbers of female flies collected in fields treated with Amulet C‐L (0.34% fipronil active ingredient) bait stations, sanitation, and spot treatments of GF‐120 Fruit Fly Bait to numbers collected in fields where sanitation and spot‐treatments were used without Amulet C‐L. In fields with Amulet C‐L bait stations in conjunction with sanitation and weekly protein bait spot treatments of GF‐120 Fruit Fly Bait, female captures were significantly lower than those in field plots treated with weekly protein bait spot treatments and sanitation. In subsequent laboratory studies, all females died within 6 h after direct exposure to male flies that had access to Amulet C‐L for 1–4 min. The possibility that male regurgitant could be a mechanism for horizontal transfer and subsequent female mortality was determined by collecting regurgitated droplets from fipronil‐fed male flies and feeding them to males and females. Both male and female flies exposed to regurgitant from fipronil‐fed male flies or droplets containing fipronil had higher mortality than the male and female flies that were exposed to regurgitant or droplets with only the C‐L compound or sugar solution. Thus, female flies do experience mortality from exposure to regurgitant from males that have fed on fipronil laced solutions. This provides evidence of at least one mechanism of horizontal transfer of insecticide in tephritid fruit flies. These findings are discussed in the context of Z. cucurbitae integrated pest management programs in Hawai'i.

Survey of Formicidae Attracted to Protein Baits on Georgia's Barrier Island Dunes

Although insects have been identified as valuable bioindicator species, insect diversity in coastal sand dunes is understudied. Our study presents the first survey focused on Georgia's barrier island ant assemblage. We surveyed the primary and secondary dunes of Cumberland, Little St. Simons, and Sapelo islands in the summers of 2016 and 2017 using protein baits to recruit scavenging ants that forage on dunes and beaches. We placed 4863 baits over the 2 sampling seasons; 2458 recruited ants. We documented 29 ant species, including 3 new records for the state: Dorymyrmex reginicula, Pheidole navigans, and Solenopsis globularia. Our survey provides a baseline for future projects to evaluate disturbance and ecosystem health on Georgia's barrier islands.

Screening Arrayed Libraries with DNA and Protein Baits to Identify Interacting Proteins.

Molecular interactions are an integral part of the regulatory mechanisms controlling gene expression. The yeast one- and two-hybrid systems (Y1H/Y2H) have been widely used by many laboratories to detect DNA-protein (Y1H) and protein-protein interactions (Y2H). The development of efficient cloning systems have promoted the generation of large open reading frame (ORF) clone collections (libraries) for several organisms. Functional analyses of such large collections require the establishment of adequate protocols. Here, we describe a simple straightforward procedure for high-throughput screenings of arrayed libraries with DNA or protein baits that can be carried out by one person with minimal labor and not requiring robotics. The protocol can also be scaled up or down and is compatible with several library formats. Procedures to make yeast stocks for long-term storage (tube and microplate formats) are also provided.

Microbial Carriage and Contamination of Mangoes by the Oriental Fruit Fly

Background: Fruit flies, especially of the Family Terphritidae, are economically important pests for the horticulture industry because many species cause serious mechanical damage to a number of crops of different plant families. Studies have shown that some species of fruit flies have the potential to contaminate fruits and vegetables with enteric bacterial pathogens. However, this has not been conclusively demonstrated. Methods: In this study, we investigated enteric bacteria carriage by Bactrocera dorsalis and its possible role in transmission of microbes into internal tissues of fruits. Fruit flies trapped using liquid protein bait, ripe mango fruits exposed to the fruit flies and controls, as well as mangoes obtained from farms with and without fly-control traps, were analyzed for microbes, such as total aerobic bacteria, total coliforms, yeast and molds, Escherichia coli and Salmonella/Shigella spp. using direct culture methods. Results and Discussion: The results revealed that a high percentage of these insects carries pathogenic bacteria. This finding shows that, like B. cacuminata and B. tryoni, B. dorsalis also carries pathogenic microbes. It was also observed that mangoes sampled from fly-control farms had significantly lower microbial loads and proportions of fruits contaminated compared to those from farms without fly-control. Additionally, all microbial counts of internal tissues were significantly higher for exposed mangoes compared to unexposed mangoes. These data indicate that B. dorsalis contaminates not only the external but also internal tissues of mangoes. Conclusion: These findings show that B. dorsalis carries pathogenic bacteria and plays a direct role in internalization of microbes in mangoes.

Heptyl butyrate, a putative pheromone involved in social communication of Vespula germanica wasps

Greater knowledge on the social communication of invasive Vespula germanica wasps is needed to fully understand their foraging behavior. This is particularly valuable considering that poison baiting is currently the only effective way of reducing wasp density. Heptyl butyrate is an attractant for many wasp species, but attempts to determine effects on V. germanica have yielded mixed results. We studied the behavior elicited by heptyl butyrate on V. germanica wasps in field experiments with foraging wasps. We also analyzed headspace volatiles of live V. germanica workers, to determine if heptyl butyrate is emitted. Heptyl butyrate was present in the headspace of live workers. Amounts of heptyl butyrate averaged 1.4 ± 0.2 ng per sample per hour, where each sample included six workers. Wasps approaching filter papers treated with different concentrations of heptyl butyrate hovered over it, but only a small percentage landed on it. Pure heptyl butyrate elicited the greatest response although all the concentrations tested were attractive. When heptyl butyrate was applied to protein baits, a greater number landed on the treated baits than on untreated ones, demonstrating this compound enhances attractiveness of baits. Results from our study suggest that heptyl butyrate is a pheromonal compound involved in attracting conspecifics to food resources, but other cues are needed to trigger landing.

Management and eradication options for Queensland fruit fly

AbstractSeveral tephritid fruit flies have explosive population growth and a wide host range, resulting in some of the largest impacts on horticultural crops, reducing marketable produce, and limiting market access. For these pests, early detection and eradication are routinely implemented in vulnerable areas. However, social and consumer concerns can limit the types of population management tools available for fruit fly incursion responses. Deterministic population models were used to compare eradication tools used at typical densities alone and in combination against the Queensland fruit fly (‘Qfly’), Bactrocera tryoni. The models suggested that tools that prevent egg laying are likely to be most effective at reducing populations. Tools that induced mortality once Qfly was sexually mature only slowed population growth, as successful mating still occurred. Release of sterile Qfly when using the sterile insect technique (SIT) interferes with the successful mating of wild flies, and of the tools investigated here, SIT caused the greatest reduction in the population at the prescribed release rate. Used in tandem with SIT, protein baits slightly improved the rate of population reduction, but the male annihilation technique (MAT) almost nullified control by SIT due to the mortality induced on sterile flies. The model suggested that the most rapid decrease in population size would be achieved by SIT plus protein baits. However, the model predicted both the SIT and protein baits when used alone would result in population reduction. The MAT can be used prior to SIT release to increase overflooding ratios.

Evaluation of Lures for Monitoring Silk Flies (Diptera: Ulidiidae) in Sweet Corn

Abstract Several morphologically similar species of picture-winged flies (the silk fly complex, Diptera: Ulidiidae) are severe primary pests of sweet corn (Zea mays L.; Poaceae) in Florida. Monitoring traps for these pests may aid threshold development and species complex determination in the field. This study evaluated floral lures, some previously used to monitor pest Lepidoptera, and liquid protein baits, used for other pest Diptera, for efficacy in attraction of silk flies. Baited universal moth traps were deployed for several weeks and placed in a summer fallow field (field trial 1), a fall sweet corn field (field trial 2), and a spring sweet corn field (field trial 3). Flies were removed weekly during each experiment. In field trial 1, traps baited with 1,4-dimethoxybenzene captured the most flies. The majority of flies captured were Chaetopsis massyla Walker. In field trial 2, aged torula yeast—baited traps captured more flies than other treatments, (1,4-dimethoxybenzene, geraniol, phenylacetaldehy...

Capture ofZeugodacus cucurbitae(Diptera: Tephritidae) in Traps Baited with Torula Yeast Solution Versus Cucumber Volatile Plugs

Several species of invasive fruit flies (Diptera: Tephritidae) are among the most damaging pests of agricultural crops in the world. Detection of these pests relies primarily on traps baited with male-specific lures. Traps with protein food baits are also employed to attract females, but these baits are fairly weak, and there is considerable interest in developing more powerful lures for female tephritids. Recently, a plug that emits cucumber volatiles was developed as a lure for cucurbit-infesting species, and a recent field study showed that traps baited with these plugs attracted more females of the Australian cucurbit pest Bactrocera cucumis (French) than traps containing a protein bait. The objective of the present study was to gather additional data regarding captures of a closely related species, the melon fly, Zeugodacus cucurbitae (Coquillett), in traps baited with a torula yeast and borax (TYB) solution (a standard food lure) compared with traps baited with the cucumber volatile (CV) plug. Data collected from 5 locations on Oahu, Hawaii, showed that traps baited with the TYB solution captured significantly more melon flies than traps baited with the CV plugs. At 4 sites, where hosts were scattered, melon fly captures were relatively low, and males were trapped in significantly greater numbers than females in both TYB- and CV-baited traps. At the remaining site, hosts were very abundant, melon fly captures were high, and females were trapped in significantly greater numbers than males in both TYB- and CV-baited traps. Possible explanations for the superior performance of TYB-baited traps are discussed.

Attraction ofBactrocera cucurbitaeandBactrocera dorsalis(Diptera: Tephritidae) to Beer Waste and Other Protein Sources Laced with Ammonium Acetate

Adult tephritid fruit fly females require protein sources for adequate egg production, and ammonia and its derivatives serve as volatile cues to locate protein-rich food. The attractiveness of beer waste and the commercially available baits Nu-Lure® Insect Bait, Buminal®, and Bugs for Bugs® Fruit Fly Bait with and without ammonium acetate or ammonium carbonate to males and females of Bactrocera dorsalis (Hendel) and B. cucurbitae (Coquillett) (Diptera: Tephritidae) was quantified in semi-field cage studies in Hawaii. Evaluations also compared the relative attractiveness of the baits to that of the standard bait GF-120® NF Naturalyte® Fruit Fly Bait. Our findings indicate that ammonium carbonate did not exert any effect on the response of B. cucurbitae or B. dorsalis to the protein baits evaluated and that the addition of ammonium acetate to beer waste and to the commercially available bait Bugs for Bugs® can improve bait attractiveness in particular to females of B. cucurbitae. Beer waste laced with ammonium acetate performed as well as the standard fruit fly bait GF-120®. There were variations in the level and type of response between B. cucurbitae and B. dorsalis, and such variability was dependent upon the type of bait being evaluated. For example, there were several instances where baits lacking ammonium acetate (e.g., beer waste and Nu-Lure® for B. cucurbitae; Buminal® for B. dorsalis) were as attractive as GF-120®. Results are discussed in light of potential applications associated with the use of beer waste as a low-cost, readily available material for fruit fly monitoring and suppression.

System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants

Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems.

Yeast One- and Two-Hybrid High-Throughput Screenings Using Arrayed Libraries.

Since their original description more than 25 years ago, the yeast one- and two-hybrid systems (Y1H/Y2H) have been used by many laboratories to detect DNA-protein (Y1H) and protein-protein interactions (Y2H). These systems use yeast cells (Saccharomyces cerevisiae) as a eukaryotic "test tube" and are amenable for most labs in the world. The development of highly efficient cloning methods has fostered the generation of large collections of open reading frames (ORFs) for several organisms that have been used for yeast screenings. Here, we describe a simple mating based method for high-throughput screenings of arrayed ORF libraries with DNA (Y1H) or protein (Y2H) baits not requiring robotics. One person can easily carry out this protocol in approximately 10 h of labor spread over 5 days. It can also be scaled down to test one-to-one (few) interactions, scaled up (i.e., robotization) and is compatible with several library formats (i.e., 96, 384-well microtiter plates).

Effects of Northern Bobwhite Habitat Management Practices on Red Imported Fire Ants

Management practices that create early successional plant communities through disturbance (discing and prescribed fire) often are prescribed for restoration of declining northern bobwhite (Colinus virginianus) populations. Because disturbance may facilitate invasion of exotic flora and fauna such as red imported fire ants (RIFA, Solenopsis invicta), we hypothesized that habitat management practices commonly used to enhance bobwhite habitat might have the unintended consequence of increasing local abundance of RIFA. During 1999, we tested effects of 4 treatments (spring discing, spring prescribed burning, spring mowing, and no management), in a randomized complete block design (n = 10) on RIFA abundance in Conservation Reserve Program (CRP) fields in central Mississippi. We surveyed RIFA abundance using 3 measures: 1) mound density, 2) a population index based on worker ant and brood estimates, and 3) foraging activity as indexed by attraction to protein bait cups. During May 1999, mound density (P = 0.0136) and population index (P 0.0078) differed among treatments, with abundance values greatest in plots treated with fire, and lowest in disced plots. The index of foraging activity did not differ among treatments (P = 0.6637). During October 1999, mound density (P = 0.0334) and population index (P = 0.0451) differed among treatments with abundance values greatest in plots receiving fire and disc treatments, and lowest abundance in control plots. The index of foraging activity did not differ among treatments (P = 0.9079). Disturbance tools such as prescribed fire and discing are essential to maintain plant communities to which bobwhite are adapted; however, they may have the unintended consequence of facilitating invasion of RIFA and increasing local RIFA populations.

Identification and specificity of broadly neutralizing antibodies against HIV.

SummaryBeginning in 2009, studies of the humoral responses of HIV‐positive individuals have led to the identification of scores, if not hundreds, of antibodies that are both broadly reactive and potently neutralizing. This development has provided renewed impetus toward an HIV vaccine and led directly to the development of novel immunogens. Advances in identification of donors with the most potent and broad anti‐HIV serum neutralizing responses were crucial in this effort. Equally, development of methods for the rapid generation of human antibodies from these donors was pivotal. Primarily these methods comprise single B‐cell culture coupled to high‐throughput neutralization screening and flow cytometry‐based sorting of single B cells using HIV envelope protein baits. In this review, the advantages and disadvantages of these methodologies are discussed in the context of the specificities targeted by individual antibodies and the need for further improvements to evaluate HIV vaccine candidates.