Analytical applications of aptamers

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets.

Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.

Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.     

Advances in SELEX and application of aptamers in the central nervous system

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a screening technique that involves the progressive selection of highly specific ligands by repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of the selection are called aptamers, which are short single stranded DNA or RNA molecules, binding with high affinity, attributed to their specific three-dimensional shapes, to a large variety of targets, ranging from small molecules to complex mixtures. Various improvement of the original SELEX method described in 1990 have been obtained recently, such as capillary electrophoresis SELEX, Toggle-SELEX, Tailored-SELEX, Photo-SELEX, and others. These new variants greatly shorten time of selection and improve aptamer affinity and specificity. Such aptamers have great potential as detecting and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, and are considered as potent therapeutic lead structures evaluated in preclinical disease models. Recently, one aptamer has been approved by Food and Drug Administration of US for treating age-related macular degeneration. This review presents recent advances in the field of SELEX with special emphasis on applications of aptamers as analytical, diagnostic and therapeutic tools in the central nervous system.     

Probing the limits of aptamer affinity with a microfluidic SELEX platform

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K_d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K_d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K_d.     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Aptamers against extracellular targets for in vivo applications

Oligonucleotides are multifunctional molecules which can interfere with gene expression by different mechanism such as antisense, RNA interference, ribozymes, etc. For most in vivo diagnostic and therapeutic applications, oligonucleotides need to be delivered to the intracellular compartment of a specific organ, a difficult task which limits considerably their use. However, aptamer oligonucleotides which target extracellular markers obviate this problem. Aptamers are short oligonucleotides (<100 bases) selected from large combinatorial pools of sequences for their capacity to bind to many types of different targets, ranging from small molecules (amino acids, antibiotics...) to proteins or nucleic acid structures. Aptamers present the same high specificity and affinity for their targets as antibodies. In addition to efficient binding, aptamers have been shown in many cases to display an inhibitory activity on their targets. Moreover, they seem to lack immunogenicity and can be chemically modified in order to improve their stability against nucleases or extend their blood circulation time, two properties which are particularly useful for in vivo applications. Recently, aptamers have been selected against whole living cells, opening a new avenue which presents three major advantages 1) direct selection without prior purification of the targets; 2) conservation of membrane proteins in their native conformation similar to the in vivo conditions and 3) identification of (new) targets for a specific phenotype. Many aptamers are now being developed against biomedical relevant extracellular targets: membrane receptor proteins, hormones, neuropeptides, coagulation factors... Among them, one aptamer that inhibits the human VEGF165 has recently been approved by FDA for the treatment of age-related macular degeneration. Here we discuss the recent developments of aptamers against extracellular targets for in vivo therapy and as tools for diagnosis using molecular imaging.     

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

Background

Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.

Principal Findings

Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.

Conclusion

We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.     

Aptamer directly evolved from live cells recognizes membrane bound immunoglobin heavy mu chain in Burkitt's lymphoma cells

The identification of tumor related cell membrane protein targets is important in understanding tumor progression, the development of new diagnostic tools, and potentially for identifying new therapeutic targets. Here we present a novel strategy for identifying proteins that are altered in their expression levels in a diseased cell using cell specific aptamers. Using an intact viable B-cell Burkitt's lymphoma cell line (Ramos cells) as the target, we have selected aptamers that recognize cell membrane proteins with high affinity. Among the selected aptamers that showed different recognition patterns with different cell lines of leukemia, the aptamer TD05 showed binding with Ramos cells. By chemically modifying TD05 to covalently cross-link with its target on Ramos cells to capture and to enrich the target receptors using streptavidin coated magnetic beads followed by mass spectrometry, we were able to identify membrane bound immunoglobin heavy mu chain as the target for TD05 aptamer. Immunoglobin heavy mu chain is a major component of the B-cell antigen receptor, which is expressed in Burkitt's lymphoma cells. This study demonstrates that this two step strategy, the development of high quality aptamer probes and then the identification of their target proteins, can be used to discover new disease related potential markers and thus enhance tumor diagnosis and therapy. The aptamer based strategy will enable effective molecular elucidation of disease related biomarkers and other interesting molecules.     

An RNA aptamer that selectively recognizes symmetric dimethylation of arginine 8 in the histone H3 N-terminal peptide

Epigenetic modifications of N-terminal histone tails, especially histone H3, are important for the regulation of the target genes in chromatin. Specific methods for detection of these modifications in histone H3?N-terminal peptides are valuable tools for diagnostic and therapeutic purposes. As an alternative to antibodies, RNA aptamers display compatible binding affinities and selectivites against various biologically relevant targets. Systematic evolution of ligands by exponential enrichment (SELEX) was performed against histone H3R8Me2sym. A 14-amino acid peptide that mimics this modified histone tail was prepared in a biotinylated form and 10 selection cycles of SELEX were carried out. This produced 4 aptamers, one of which (clone 1) was observed to have low nanomolar binding affinity (K(d)=12 nM) against the cognate peptide. The affinity of this aptamer is comparable to 2 commercially available antibodies against differently modified histone H3 peptides and it displays a greater selectivity than the antibodies.     

RNAapt3D: RNA aptamer 3D-structural modeling database

RNA aptamers are oligonucleotides with high binding affinity and specificity for target molecules and are expected to be a new generation of therapeutic molecules and targeted delivery materials. The tertiary structure of RNA molecules and RNA-protein interaction sites are increasingly important as potential targets for new drugs. The pathological mechanisms of diseases must be understood in detail to guide drug design. In developing RNA aptamers as drugs, information about the interaction mechanisms and structures of RNA aptamer-target protein complexes are useful. We constructed a database, RNA aptamer 3D-structural modeling (RNAapt3D), consisting of RNA aptamer data that are potential drug candidates. The database includes RNA sequences and computationally predicted RNA tertiary structures based on secondary structures and implements methods that can be used to predict unknown structures of RNA aptamer-target molecule complexes. RNAapt3D should enable the design of RNA aptamers for target molecules and improve the efficiency and productivity of candidate drug selection. RNAapt3D can be accessed at https://rnaapt3d.medals.jp.     

CS-SELEX Generates High-Affinity ssDNA Aptamers as Molecular Probes for Hepatitis C Virus Envelope Glycoprotein E2

Currently, the development of effective diagnostic reagents as well as treatments against Hepatitis C virus (HCV) remains a high priority. In this study, we have described the development of an alive cell surface -Systematic Evolution of Ligands by Exponential Enrichment (CS-SELEX) technique and screened the functional ssDNA aptamers that specifically bound to HCV envelope surface glycoprotein E2. Through 13 rounds of selection, the CS-SELEX generated high-affinity ssDNA aptamers, and the selected ssDNA aptamer ZE2 demonstrated the highest specificity and affinity to E2-positive cells. HCV particles could be specifically captured and diagnosed using the aptamer ZE2. A good correlation was observed in HCV patients between HCV E2 antigen-aptamer assay and assays for HCV RNA quantities or HCV antibody detection. Moreover, the selected aptamers, especially ZE2, could competitively inhibit E2 protein binding to CD81, an important HCV receptor, and significantly block HCV cell culture (HCVcc) infection of human hepatocytes (Huh7.5.1) in vitro. Our data demonstrate that the newly selected ssDNA aptamers, especially aptamer ZE2, hold great promise for developing new molecular probes, as an early diagnostic reagent for HCV surface antigen, or a therapeutic drug specifically for HCV.     

综述与专论: 核酸适配体筛选与亲和力评价技术主要研究方向、进展与挑战

核酸适配体是一类具有特异性分子识别能力的单链DNA或者RNA分子,通过指数富集的配体系统进化技术（SELEX）筛选得到。核酸适配体相比抗体具有热稳定性高、便于化学合成与修饰、免疫原性低等优点,在生物分析、生物医学、生物技术等众多领域引起广泛关注。高质量的核酸适配体是应用的基础,然而目前能够满足实际应用的核酸适配体数量还非常有限。如何获得高亲和力、高特异性、高体内稳定性的核酸适配体是核酸适配体领域的技术瓶颈。本文首先简单介绍了SELEX技术的基本原理和核酸库的设计、筛选过程监控、次级文库制备、测序和候选适配体筛选等关键步骤。接着归纳总结了30多年来核酸适配体筛选技术的6个主要研究方向、研究进展和局限性。这6个主要研究方向分别是提高适配体特异性的筛选方法、提高适配体稳定性（抗核酸酶降解能力）的筛选方法、快速筛选方法、复杂靶标适配体筛选方法、小分子靶标适配体筛选方法、提高适配体亲和力的筛选方法。其中快速筛选技术是长期以来持续关注的研究方向,几乎所有物理分离手段都已用于提高SELEX的筛选效率。最近,高效化学反应与SELEX技术的结合为核酸适配体的快速筛选提供了新的策略。本文随后对适合小分子靶标核酸适配体筛选的3类方法进展和存在的问题进行了重点评述。这3类方法分别是基于靶标固定的筛选技术、基于文库固定的筛选技术（捕获-SELEX,Capture-SELEX）和均相筛选技术（氧化石墨烯-SELEX,GO-SELEX）。基于靶标固定的筛选技术尽管存在空间位阻等众多问题,由于其操作的简单性,目前依然应用广泛。近年来Capture-SELEX应用广泛。结合36种靶标适配体的筛选实验条件（文库设计、正筛靶标浓度、负筛靶标的选择和浓度）和所获得的适配体的亲和力（K_D,解离常数,dissociation constant）和特异性,对Capture-SELEX的实验条件与适配体性能的关系进行了讨论。统计数据表明,降低正筛靶标浓度有利于提高适配体的亲和力,但不是必要条件。负筛选是目前提高适配体特异性的主要技术手段,但适配体的特异性还不能满足实际需求。负筛选靶标及其浓度的选择差异很大,而且36种靶标中有20种靶标的适配体筛选没有进行负筛选。如何提高核酸适配体的特异性是目前小分子靶标核酸适配体所面临的难题,急需寻找新的策略。本文还列表归纳了近三年利用GO-SELEX进行的13种小分子靶标的实验条件和所获得的适配体的K_D和特异性。统计数据表明,GO-SELEX比Capture-SELEX所需要的筛选轮数少,两种方法所获得的适配体的亲和力多在纳摩尔每升水平。Capture-SELEX相对较低的筛选效率应该主要由于文库的自解离问题。核酸适配体的亲和力评价是候选核酸适配体结构与性能评价的重要组成部分。常用的核酸适配体亲和力评价技术包括基于分离、基于固定、均相体系三大类十多种方法。假阳性和假阴性是各种评价技术都有可能存在的问题。本文以纳米金比色法和等温热滴定技术为例评述技术进展,讨论导致不同亲和力评价技术结果不一致性问题的根本原因。本文最后对核酸适配体筛选技术、亲和力评价技术和技术的标准化的未来发展趋势进行了展望。     

Aptamer selection by high-throughput sequencing and informatic analysis

Traditional methods for selecting aptamers require multiple rounds of selection and optimization in order to identify aptamers that bind with high affinity to their targets. Here we describe an assay that requires only one round of positive selection followed by high-throughput DNA sequencing and informatic analysis in order to select high-affinity aptamers. The assay is flexible, requires less hands on time, and can be used by laboratories with minimal expertise in aptamer biology to quickly select high-affinity aptamers to a target of interest. This assay has been utilized to successfully identify aptamers that bind to thrombin with dissociation constants in the nanomolar range.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

鳗弧菌(Vibrio anguillarum)核酸适配体的筛选及其结合蛋白的分离鉴定

目的 鳗弧菌(Vibrio anguillarum)是水产养殖中的重要条件致病菌,每年给水产养殖业造成巨大的经济损失,研究其致病机制、对其进行快速的检测鉴定是其病害防治的前提和基础.核酸适配体因其高亲和力、高特异性等多种优点,在微生物的靶标分析、检测鉴定以及致病机制等多个领域都呈现出较好的应用潜力.因此,筛选鳗弧菌的核...     

Aptamers: versatile probes for flow cytometry

Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed “systematic evolution by exponential enrichment” make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.     

Peptide aptamers: specific inhibitors of protein function

In recent years, peptide aptamers have emerged as novel molecular tools that are useful for both basic and applied aspects of molecular medicine. Due to their ability to specifically bind to and inactivate a given target protein at the intracellular level, they provide a new experimental strategy for functional protein analyses, both in vitro and in vivo. In addition, by using peptide aptamers as "pertubagens", they can be employed for genetic analyses, in order to identify biochemical pathways, and their components, that are associated with the induction of distinct cellular phenotypes. Furthermore, peptide aptamers may be developed into diagnostic tools for the detection of a given target protein or for the generation of high-throughput protein arrays. Finally, the peptide aptamer technology has direct therapeutic implications. Peptide aptamers can be used in order to validate therapeutic targets at the intracellular level. Moreover, the peptide aptamer molecules themselves should possess therapeutic potential, both as lead structures for drug design and as a basis for the development of protein drugs.     

Unraveling the binding mode of a methamphetamine aptamer: A spectroscopic and calorimetric study

Nucleic-acid aptamers are bio-molecular recognition agents that bind to their targets with high specificity and affinity and hold promise in a range of biosensor and therapeutic applications. In the case of small-molecule targets, their small size and limited number of functional groups constitute challenges for their detection by aptamer-based biosensors because bio-recognition events may both be weak and produce poorly transduced signals. The binding affinity is principally used to characterize aptamer-ligand interactions; however, a structural understanding of bio-recognition is arguably more valuable in order to design a strong response in biosensor applications. Using a combination of nuclear magnetic resonance, circular dichroism, and isothermal titration calorimetry, we propose a binding model for a new methamphetamine aptamer and determine the main interactions driving complex formation. These measurements reveal only modest structural changes to the aptamer upon binding and are consistent with a conformational-selection binding model. The aptamer-methamphetamine complex formation was observed to be entropically driven, apparently involving hydrophobic and electrostatic interactions. Taken together, our results exemplify a means of elucidating small molecule-aptamer binding interactions, which may be decisive in the development of aptasensors and therapeutics and may contribute to a deeper understanding of interactions driving aptamer selection.     

综述: 适配子介导的siRNA转运

小分子干扰RNA(small interfering RNA,siRNA)因能快速抑制哺乳动物特定基因的表达而用于各种疾病的治疗,然而选择合适的载体将siRNA安全有效地转运进入靶细胞仍是制约siRNA应用于临床治疗的重要因素．越来越多的转运载体被开发出来,其中包括针对细胞表面蛋白的适配子(aptamer)．Aptamer是一种能与靶分子高特异性和高亲和结合的寡核苷酸,已经越来越多地用于疾病的诊断和治疗．Aptamer作为载体介导siRNA转运可提高治疗的靶向性并减少副作用,这将为siRNA应用于临床靶向治疗提供一种特异有效的途径．目前,已经发现几种aptamers可以介导siRNA的转运,如anti-PSMA aptamer,anti-HIV gp120 aptamer,anti-CD4 aptamer等．本文将综述aptamer介导siRNA转运的最新研究进展．     

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr‐activated sepharose‐4B affinity chromatography

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA‐aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (k_d = 2.3 × 10^?11). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.