Sequence-based design and construction of synthetic nanobody library.

Nanobody (Nb), the smallest antibody fragments known to bind antigens, is now widely applied to various studies, including protein structure analysis, bioassay, diagnosis, and biomedicine. The traditional approach to generating specific nanobodies involves animal immunization which is time-consuming and expensive. As the understanding of the antibody repertoire accumulation, the synthetic library, which is devoid of animals, has attracted attention widely in recent years. Here, we describe a synthetic phage display library (S-Library), designed based on the systematic analysis of the next-generation sequencing (NGS) of nanobody repertoire. The library consists of a single highly conserved scaffold (IGHV3S65*01-IGHJ4*01) and complementary determining regions of constrained diversity. The S-Library containing 2.19 × 108 independent clones was constructed by the one-step assembly and rapid electro-transformation. The S-Library was screened against various targets (Nb G8, fusion protein of Nb G8 and green fluorescent protein, bovine serum albumin, ovalbumin, and acetylcholinesterase). In comparison, a naïve library (N-Library) from the source of 13 healthy animals was constructed and screened against the same targets as the S-Library. Binders were isolated from both S-Library and N-Library. The dynamic affinity was evaluated by the biolayer interferometry. The data confirms that the feature of the Nb repertoire is conducive to reducing the complexity of library design, thus allowing the S-Library to be built on conventional reagents and primers.

A potent neutralizing nanobody targeting a unique epitope on the receptor-binding domain of SARS-CoV-2 spike protein.

SARS-CoV-2 and its variants continue to threaten public health. Nanobodies that block the attachment of the RBD to host cell angiotensin-converting enzyme 2 (ACE2) represent promising drug candidates. In this study, we reported the identification and structural biological characterization of a nanobody from a RBD-immunized alpaca. The nanobody, termed as 2S-1-19, shows outstanding neutralizing activity against both pseudotyped and authentic SARS-CoV-2 viruses. The crystal structure of 2S-1-19 bound to SARS-CoV-2 RBD reveals an epitope that overlaps with the binding site for ACE2. We also showed that 2S-1-19 reserves promising, though compromised, neutralizing activity against the Delta variant and that the trivalent form of 2S-1-19 remarkably increases its neutralizing capacity. Despite this, neither the monomeric or trimeric 2S-1-19 could neutralize the Omicron BA.1.1 variant, possibility due to the E484A and Q493K mutations found within this virus variant. These data provide insights into immune evasion caused by SARS-CoV-2 variants.

Sensitivity Intensified Ninhydrin-Based Chromogenic System by Ethanol-Ethyl Acetate: Application to Relative Quantitation of GABA.

Gamma-aminobutyric acid (GABA) is a functional metabolite in various organisms. Herein, a sensitivity intensified ninhydrin-based chromogenic system (SINICS), achieved by ethanol and ethyl acetate, is described for the reliable relative quantitation of GABA. A 2.9 mL SINICS kit comprises 1% ninhydrin, 40% ethanol, 25% ethyl acetate, and 35 µL 0.2 M sodium acetate buffer (pH 5.0). In practice, following the addition of a 0.1 mL sample to the kit, the chromogenic reaction is completed by heating at 70 °C for 30 min. The kit increased the color development sensitivity of L-glutamic acid and GABA, with the detection limits being reduced from 20 mM and 200 mM to 5 mM and 20 mM, respectively. The chromophore was stable for at least 2 h at room temperature, which was sufficient for a routine colorimetric analysis. The absorbance at 570 nm with the deduction of background directly represents the content of amino acid. For a proof-of-concept, the SINICS was adopted to optimize the GABA fermentation process of Levilactobacillus brevis CD0817. The results demonstrated that SINICS is an attractive alternative to the available ninhydrin-based colorimetric methods.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Tandem nanobody: A feasible way to improve the capacity of affinity chromatography.

Nanobodies, referred to the binding domain of the heavy-chain-only antibodies, are the smallest antigen recognition unit. The molecular weight of monomeric nanobodies is about one-tenth of the conventional antibodies. The small size of nanobodies facilitates genetic manipulation and recombinant expression. This study aimed to investigate the effects of nanobody multivalency on the binding capacity of affinity resin. The nanobody (namely AFV), which binds to the fragment crystallizable (Fc) region of immunoglobulin G (IgG), was fused to the N-terminal of HaloTag in the form of monomeric (H-AFV), dimer (H-diAFV), trimer (H-triAFV), and tetramer (H-tetAFV). The fusion proteins were solubly expressed in Escherichia coli yielding at least 9.9 mg L-1. The biolayer interferometry confirmed an increment of avidity as the increase of AFV valences. The four recombinant proteins in crude cell lysate were site-specifically immobilized onto the Halo ligand resin via the self-labeling HaloTag, respectively. The generated affinity resins were able to isolate high purity IgG from mouse plasma. The highest improvement of the static binding capacity was achieved 73.7% by the H-diAFV resin other than the H-triAFV or H-tetAFV, as compared to the H-AFV resin.

Stability comparison of four lipases and catalytic mechanism during the synthesis of 1,3-di-oleic-2-medium chain triacylglycerols in a trace water-in-oil system: Experimental analyses and computational simulations.

In the present study, a kind of structured lipids, namely 1,3-di-oleic-2-medium chain (OMO) triacylglycerols, were synthesized through lipase-catalyzed reactions using coconut oil and rapeseed acid as materials in a trace water-in-oil system. Experimental analysis and computational simulations were undertaken to compare the stability of four lipases including Lipozyme RMIM, Lipozyme TLIM, Novozym 435, and Aspergillus oryzae immobilized lipase (AOIM), and illustrate catalytic mechanism of Novozym 435 during the synthesis of OMO. Fourier transform infrared and molecular dynamics simulation results demonstrated that a decrease in ordered structure (α-helix and ß-sheet) led to a reduction in enzyme activity. Compared with Lipozyme RMIM and Novozym 435, Lipozyme TLIM and AOIM exhibited better stability due to a short-chain lid in TLIM, which covers activity sites, and hydrogen bonds formed between activity center of AOIM and water. Among four lipases, AOIM exhibited best catalytic performance: a OMO yield of 30.7% at 3 hr and a good stability of long term (48 hr). Density functional theory results demonstrated that specifically, during the synthesis of OMO triacylglycerol, the addition of Novozym 435 (derived from Candida antarctica lipase B, CALB) substantially lowered reaction barriers (64.4 KJ/mol with CALB vs. 332.7 KJ/mol with no lipase), aiding in the generation of OMO because of the formations of transitional tetrahedral intermediates. A trace water-in-oil system was a green and efficient alternative for lipase-catalyzed production of OMO, and this study provided crucial insights into the stability/instability and catalytic mechanisms of lipase in the synthesis of structured lipids. PRACTICAL APPLICATIONS: We compared the stability of Lipozyme RMIM, Lipozyme 435, Lipozyme TLIM, and AOIM during the synthesis of the OMO triacylglycerols in a trace water-in-oil system, where exhibited a high catalytic activity of lipase in water-oil interface. AOIM had the highest stability and showed the best catalytic performance due to the formation of hydrogen bonds. Besides, for the first time, the transition tetrahedral structure was revealed in the enzymatic synthesis of medium- and long-chain triacylglycerols. This study provides a rational approach to compare lipase stability and a possible hint to choose appropriate enzyme in a specific catalytic condition.

Research on the Mechanism of Action of a Citrinin and Anti-Citrinin Antibody Based on Mimotope X27.

Immunoassays are developed based on antigen-antibody interactions. A mimotope is an effective recognition receptor used to study the mechanism of action of antigens and antibodies, and is used for improving the sensitivity of the antibody. In this study, we built a 3D structure of the citrinin (CIT) mimotope X27 and anti-CIT single-chain antibody fragment (ScFv) through a "homologous modeling" strategy. Then, CIT and X27 were respectively docked to anti-CIT ScFv by using the "molecular docking" program. Finally, T28, F29, N30, R31, and Y32 were confirmed as the key binding sites in X27. Furthermore, the result of the phage-ELISA showed that the mutational phage lost the binding activity to the anti-CIT ScFv when the five amino acids were mutated to "alanine", thereby proving the correctness of the molecular docking model. Lastly, a site-directed saturation strategy was adopted for the sites (T28, F29, N30, R31, and Y32). Eighteen different amino acids were introduced to each site on average. The activities of all mutants were identified by indirect competitive ELISA. The sensitivities of mutants T28F, T28I, F29I, F29V, N30T, and N30V were 1.83-, 1.37-, 1.70-, 2.96-, 1.31-, and 2.01-fold higher than that of the wild-type, respectively. In conclusion, the binding model between the CIT and antibody was elaborated for the first time based on the mimotope method, thereby presenting another strategy for improving the sensitivity of citrinin detection in immunoassays.

The ctnF gene is involved in citrinin and pigment synthesis in Monascus aurantiacus.

The application of Monascus is restricted by citrinin. So, it is important to explore the synthetic pathway of citrinin to completely inhibit the production of citrinin. In our previous study, we found that the protein encoded by the ctnF gene has a significant similarity to fructose-2,6-bisphosphatase (F26BPase). It is generally known that the bifunctional enzyme F26BPase regulates the glycolytic flux. So, we speculated that the CtnF protein strengthens carbon flux towards acetyl-CoA and malonyl-CoA which are precursor compounds in citrinin and pigment synthesis. In this study, the ctnF gene-targeting vector pctnF-HPH was constructed and transformed into Monascus aurantiacus. A ctnF-deficient strain was selected by four sets of primers and polymerase chain reaction amplification. Compared with the wild-type strain, citrinin content in the deficient strain was reduced by 34%, and the pigment production was decreased by 72%. These results indicate that the ctnF gene is involved in the common synthesis of citrinin and pigment, which is consistent with previous speculations.

Landscape of variable domain of heavy-chain-only antibody repertoire from alpaca.

Heavy-chain-only antibodies (HCAbs), which are devoid of light chains, have been found naturally occurring in various species including camelids and cartilaginous fish. Because of their high thermostability, refoldability and capacity for cell permeation, the variable regions of the heavy chain of HCAbs (VHHs) have been widely used in diagnosis, bio-imaging, food safety and therapeutics. Most immunogenetic and functional studies of HCAbs are based on case studies or a limited number of low-throughput sequencing data. A complete picture derived from more abundant high-throughput sequencing (HTS) data can help us gain deeper insights. We cloned and sequenced the full-length coding region of VHHs in Alpaca (Vicugna pacos) via HTS in this study. A new pipeline was developed to conduct an in-depth analysis of the HCAb repertoires. Various critical features, including the length distribution of complementarity-determining region 3 (CDR3), V(D)J usage, VJ pairing, germline-specific mutation rate and germline-specific scoring profiles (GSSPs), were systematically characterized. The quantitative data show that V(D)J usage and VHH recombination are highly biased. Interestingly, we found that the average CDR3 length of classical VHHs is longer than that of non-classical ones, whereas the mutation rates are similar in both kinds of VHHs. Finally, GSSPs were built to quantitatively describe and compare sequences that originate from each VJ pair. Overall, this study presents a comprehensive landscape of the HCAb repertoire, which can provide useful guidance for the modeling of somatic hypermutation and the design of novel functional VHHs or VHH repertoires via evolutionary profiles.

Single-chain variable fragment antibody-based immunochromatographic strip for rapid detection of fumonisin B1 in maize samples.

A rapid and sensitive immunochromatographic strip (ICS) based on a single-chain variable fragment (scFv) was developed for detecting fumonisin B1 (FB1). The ICS was based on a competitive reaction for colloidal gold-labeled scFv between FB1 and FB1-BSA, which was used along with sheep anti-mouse IgG as capture reagents immobilized at test and control lines, respectively, on a nitrocellulose membrane of the strip. The limit of detection of the ICS was 2.5 ng/mL (25 µg/kg) FB1 in buffer, and the sensitivity was eight times higher than that of monoclonal antibodies for the preparation of the scFv. The cross-reactivity of the scFv with common mycotoxins was determined by ICS, the results showed that the scFv were not against other mycotoxins. Eight naturally contaminated maize samples were analyzed with the scFv-based ICS and by LC-MS/MS. The results of analysis obtained with the strip assay showed good agreement with those obtained by LC-MS/MS.

Engineering a recombination neutral protease I from Aspergillus oryzae to improve enzyme activity at acidic pH.

Extracellular neutral proteases (NPs) in Aspergillus oryzae (A. oryzae) play a role in hydrolyzing soybean proteins into smaller peptides at pH about 7.5. The optimum pH of moromi fermentation (The second stage of soy sauce fermentation.) is 4.5-5.5. NPI is acid sensitive. To decrease the pH optimum of NPI, we got a mutant NPI-Y122FK246ID382V from the error-prone PCR library that showed optimal activity at pH 5.5. The specific activity at 40 °C of the NPI-Y122FK246ID382V mutant was 1383.50 U mg-1, which was 2.75-fold that of wild-type (503.09 U mg-1). The Michaelis constants of the mutant decreased from 22.13 mM (wild-type) to 19.98 mM (NPI-Y122FK246ID382V). The residues at positions 122 and 246 are important in influencing hydrolytic activity at pH 5.5 through site-directed mutagenesis. And the pH optimum of double amino acid mutants (Y122FK246I) shifted dramatically to an acidic pH compared to those of single amino acid substitution. Molecular models and structural comparisons of native and mutant provided further insight on the basis to improve catalytic efficiency at acidic pH. These results indicated that we modified the neutral protease I of Aspergillus oryzae, which can effectively improve the application of the neutral protease in industrial production, and finally lay the foundation for improving the utilization rate of raw protein.

One-step orientated immobilization of nanobodies and its application for immunoglobulin purification.

Affinity chromatography technologies play an important role in the purification of antibodies. To prepare affinity materials, prior isolation and purification of affinity ligands are required before coupling onto solid supports, which is quite expensive and laborious in large-scale applications. In this study, a one-step approach which circumvents the ligand purification procedures was developed to fabricate affinity gel for purifying immunoglobulin G (IgG). A self-labeling tag, haloalkane dehalogenase, was fused to the C-terminal of an anti-Fc variable domain of the heavy chain of the heavy-chain antibody (AFV) which was isolated in previous work. The AFV binds to various sources of IgG and is highly thermal stable. The fusion protein, namely HAFV, was expressed in Escherichia coli as a soluble protein. The binding affinity of HAFV to the Fc region of IgG was characterized and compared with the untagged anti-Fc nanobody. Next, the HAFV was immobilized directly from the crude cell lysate of isopropylthio-ß-D-galactoside (IPTG) induced E. coli. The effects of NaCl concentrations and pH on the capacity of the HAFV resin were investigated. In addition, the one-step coupled HAFV resin was compared with the AFV resin and commercial resins (Protein A and Protein G) by evaluating the static capacity and stability. Though the Protein A (8.34 ±â€¯0.37 mg/ml) and Protein G (9.19 ±â€¯0.28 mg/ml) showed higher static capacity, the static capacity of HAFV resin (8.21 ±â€¯0.30 mg/ml) was better than that of the untagged AFV gel (6.48 ±â€¯0.56 mg/ml). The recovery results calculated for the reusability and stability show that there is no significant difference between the results obtained for the HAFV gel with those of the untagged AFV gel and commercial Protein A and G. After stored at 37 ℃ for 7 days and recycled 10 times, the static capacity of HAFV gel remains above 78%. Our strategy is site-specific, cost-effective, reproducible, and has the potential to dramatically cut down the costs of affinity materials for IgG purification.

One-Step Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Fusion for Detection of Aflatoxin B1 in Cereal.

Nanoluciferase (Nluc), the smallest luciferase known, was used as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection of the aflatoxin B1 in cereal. Nanobody (clone G8) against aflatoxin B1 was fused with nanoluciferase and cloned into a pET22b expression vector, and then transformed into Escherichia coli. The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography, yielding a biologically active fusion protein. The fusion protein G8-Nluc retained binding properties of the original nanobody. Concentration of the coelenterazine substrate and buffer composition were also optimized to provide high intensity and long half-life of the luminescent signal. The G8-Nluc was used as a detection antibody to establish a competitive bioluminescent ELISA for the detection of aflatoxin B1 in cereals successfully. Compared to classical ELISA, this novel assay showed more than 20-fold improvement in detection sensitivity, with an IC50 value of 0.41 ng/mL and linear range from 0.10 to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can be completed in one step within 2 h, from sample preparation to data analysis. These results suggest that nanobody fragments fused with nanoluciferase might serve as useful and highly sensitive dual functional reagents for the development of rapid and highly sensitive immunoanalytical methods.

Panning anti-LPS nanobody as a capture target to enrich Vibrio fluvialis.

Vibrio fluvialis is considered as a human pathogen in developing countries. This bacterium is widely distributed in seawater and harbors that contains traces of salt. V. fluvialis can cause human enteritis and diarrhea, which has broken out at a global scale. Lipopolysaccharide (LPS) is a key bacterial antigen used to classify V. fluvialis serogroups. In this research, phage display technology was adopted to isolate nanobodies from a naïve phage library by using LPS as the target antigen. The isolated nanobody was tested in LPS ELISA and bacterial enzyme-linked immunosorbent assay Nanobody V23 had a high affinity toward the pathogen and was utilized to synthesize immunomagnetic beads for the enrichment of V. fluvialis. The capture efficiency of the immunomagnetic beads against V. fluvialis was 90.7 ±â€¯3.2% (N = 3) through the plate-counting method. We generated a high-affinity nanobody against LPS from V. fluvialis and developed a rapid method of enriching V. fluvialis by using immunomagnetic beads.

Substrate sustained release-based high efficacy biosynthesis of GABA by Lactobacillus brevis NCL912.

BACKGROUND: Gamma-aminobutyric acid (GABA) plays a significant role in the food and drug industries. Our previous study established an efficient fed-batch fermentation process for Lactobacillus brevis NCL912 production of GABA from monosodium L-glutamate; however, monosodium L-glutamate may not be an ideal substrate, as it can result in the rapid increase of pH due to decarboxylation. Thus, in this study, L-glutamic acid was proposed as a substrate. To evaluate its potential, key components of the fermentation medium affecting GABA synthesis were re-screened and re-optimized to enhance GABA production from L. brevis NCL912. RESULTS: The initial fermentation medium (pH 3.3) used for optimization was: 50 g/L glucose, 25 g/L yeast extract, 10 mg/L manganese sulfate (MnSO4·H2O), 2 g/L Tween-80, and 220 g/L L-glutamic acid. Glucose, a nitrogen source, magnesium, and Tween-80 had notable effects on GABA production from the L-glutamic acid-based process; other factors showed no or marginal effects. The optimized levels of the four key components in the fermentation medium were 25 g/L glucose, 25 g/L yeast extract FM408, 25 mg/L MnSO4·H2O, and 2 g/L Tween-80. A simple and efficient fermentation process for the bioconversion of GABA by L. brevis NCL912 was subsequently developed in a 10 L fermenter as follows: fermentation medium, 5 L; glutamic acid, 295 g/L; inoculum, 10% (v/v); incubation temperature, 32 °C; and agitation, 100 rpm. After 48 h of fermentation, the final GABA concentration increased up to 205.8 ± 8.0 g/L. CONCLUSIONS: L-Glutamic acid was superior to monosodium L-glutamate as a substrate in the bioproduction of GABA. Thus, a high efficacy bioprocess with 205 g/L GABA for L. brevis NCL912 was established. This strategy may provide an alternative for increasing the bioconversion of GABA.

Evaluation of truncated G protein delivered by live attenuated Salmonella as a vaccine against respiratory syncytial virus.

Human respiratory syncytial virus (RSV) can cause severe acute lower respiratory tract disease leading to numerous hospitalizations and deaths in the infant and elderly populations worldwide, while no vaccine or effective drug is available for RSV infections. In the present study, truncated G protein was successfully expressed both in prokaryotic and eukaryotic system, and high levels of serum IgG in response to truncated G protein were observed both in GD-protein group (intramuscularly with purified GD protein) and GD-VNP20009 group (challenged via the oral route with 1 × 109 CFU of pLIVE-RSV-GD-VNP20009 strains) since 21th day, and GD-VNP20009 significantly reduced the productions of IL-1ß, IL-6, and TNF-α, histamine and pathological features caused by the RSV Long strain (P < .01). Our data indicated that Salmonella typhimurium can be used to deliver truncated G DNA vaccine and represents a promising effect to protect host against RSV.

Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal.

A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB1 was developed. The anti-idiotypic nanobody-alkaline phosphatase (Ab2ß-Nb-AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB1 were 2.69 and 0.35 ng mL(-1), respectively, with a linear range of 0.93-7.73 ng mL(-1). The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL(-1), and the IC50 was 0.89 ± 0.09 ng mL(-1) with a linear range of 0.29-2.68 ng mL(-1). Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2ß-Nb-AP fusion protein based one-step competitive immunoassay for monitoring FB1 contamination in cereals. The Ab2ß-Nb-AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.

Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library.

Compared with peptide-based mimotope, anti-idiotypic antibodies (AIds) are considered as promising biosynthetic surrogate antigen because these antibodies display stable protein conformation. Nevertheless, conventional AIds are generated by immunizing animals with heterologous idiotypic antibody in vivo; isolated AIds commonly exhibit a higher affinity to primary antibodies than target analytes because AIds undergo an affinity-matured process during immune responses, resulting in low sensitivity in competitive immunoassay. In the present study, an anti-citrinin monoclonal antibody (anti-CIT McAb) was designed as primary antibody; one ß-type AI alpaca heavy chain single domain antibody (ß-AI VHH) was selected as a citrinin (CIT) surrogate from a naive phage-displayed VHH library. The affinity constant (K D) of obtained ß-AI VHH to anti-CIT McAb (160 nM) is 2.35 times lower than that of CIT and ovalbumin conjugates (CIT-OVA) to anti-CIT McAb (68 nM). The developed VHH-based enzyme-linked immunosorbent assay (V-ELISA) can be used to perform dynamic linear detection of CIT in 10% (v/v) methanol/PBS from 5.0 to 300.0 ng/mL, with a median inhibitory concentration (IC50) of 44.6 ng/mL (n = 3); this result was twice as good as that of indirect competitive ELISA (ic-ELISA, IC50 = 96.2 ng/mL) with CIT-OVA as a coating antigen. Moreover, the precision of V-ELISA was evaluated by analyzing average recoveries and coefficient of variations of CIT-spiked cereal sample; the reliability of V-ELISA was also validated with a conventional ic-ELISA. In summary, the proposed strategy has a great potential for panning other ß-AI VHH toward small organic molecules from a naive VHH library.