Novel Polystyrene-Binding Nanobody for Enhancing Immunoassays: Insights into Affinity, Immobilization, and Application Potential.

Nanobodies, which represent the next generation of antibodies due to their unique properties, face a significant limitation in their poor physical adsorption on solid supports. In this study, we successfully discovered polystyrene binding nanobodies from a synthetic nanobody library. Notably, bivalent nanobody B2 exhibited high affinity for polystyrene (0.7 nM for ELISA saturation binding analysis and 15.6 nM for isothermal titration calorimetry), displaying a pH-dependent behavior. Remarkably, hydrophobic and electrostatic interactions contribute minimally to the binding process. Molecular modeling provided insights into the interaction between B2 and polystyrene, revealing that the Trp51 residue within the CDR2 loop formed an aromatic H-bond with polystyrene at a distance of 2.74 Å, thus explaining the observed reduction in B2 affinity caused by Trp51 mutations. To explore B2's potential in protein immobilization, we constructed a bispecific nanobody by fusing B2 to an anticarcinoembryonic antigen nanobody 11C12, which cannot be immobilized on polystyrene through passive adsorption. Remarkably, the fusion construct achieved effective immobilization on polystyrene within 5 min by passing the need for periplasmic protein purification despite its low expression level. Moreover, the fusion construct demonstrated excellent linearity in the chemiluminescent enzyme immunoassay. For the first time, this study reports a simplified and seamless platform for the oriented immobilization of nanobody. Importantly, the entire process eliminated the need for protein purification, enabling efficient and rapid immobilization of fusion proteins directly from crude cell extracts, even when the expression level was low. Our developed process dramatically reduced the processing time from 2.5 days to just 5 min.

Toward evaluation of multiresolution cortical thickness estimation with FreeSurfer, MaCRUISE, and BrainSuite.

Advances in Magnetic Resonance Imaging hardware and methodologies allow for promoting the cortical morphometry with submillimeter spatial resolution. In this paper, we generated 3D self-enhanced high-resolution (HR) MRI imaging, by adapting 1 deep learning architecture, and 3 standard pipelines, FreeSurfer, MaCRUISE, and BrainSuite, have been collectively employed to evaluate the cortical thickness. We systematically investigated the differences in cortical thickness estimation for MRI sequences at multiresolution homologously originated from the native image. It has been revealed that there systematically exhibited the preferences in determining both inner and outer cortical surfaces at higher resolution, yielding most deeper cortical surface placements toward GM/WM or GM/CSF boundaries, which directs a consistent reduction tendency of mean cortical thickness estimation; on the contrary, the lower resolution data will most probably provide a more coarse and rough evaluation in cortical surface reconstruction, resulting in a relatively thicker estimation. Although the differences of cortical thickness estimation at the diverse spatial resolution varied with one another, almost all led to roughly one-sixth to one-fifth significant reduction across the entire brain at the HR, independent to the pipelines we applied, which emphasizes on generally coherent improved accuracy in a data-independent manner and endeavors to cost-efficiency with quantitative opportunities.

A novel silk fibroin protein-based fusion system for enhancing the expression of nanobodies in Escherichia coli.

Nanobodies show a great potential in biomedical and biotechnology applications. Bacterial expression is the most widely used expression system for nanobody production. However, the yield of nanobodies is relatively low compared to that of eukaryotic systems. In this study, the repetitive amino acid sequence motifs (GAGAGS) found in silk fibroin protein (SFP) were developed as a novel fusion tag (SF-tag) to enhance the expression of nanobodies in Escherichia coli. SF-tags of 1 to 5 hexapeptide units were fused to the C-terminus of 4G8, a nanobody against human epididymis protein 4 (HE4). The protein yield of 4G8 variants was increased by the extension of hexapeptide units and achieved a 2.5 ~ 7.1-fold increase compared with that of untagged 4G8 (protein yield of 4G8-5C = 0.307 mg/g vs that of untagged 4G8 = 0.043 mg/g). Moreover, the fusion of SF-tags not only had no significant effect on the affinity of 4G8, but also showed a slight increase in the thermal stability of SF-tag-fused 4G8 variants. The fusion of SF-tags increased the transcription of 4G8 by 2.3 ~ 7.0-fold, indicating SF-tags enhanced the protein expression at the transcriptional level. To verify the applicability of the SF-tags for other nanobody expression, we further investigated the protein expression of two other anti-HE4 nanobodies 1G8 and 3A3 upon fusion with the SF-tags. Results indicated that the SF-tags enhanced the protein expression up to 5.2-fold and 5.7-fold for 1G8 and 3A3, respectively. For the first time, this study reported a novel and versatile fusion tag system based on the SFP for improving nanobody expression in Escherichia coli, which may enhance its potential for wider applications.Key points• A silk fibroin protein-based fusion tag (SF-tag) was developed to enhance the expression of nanobodies in Escherichia coli.• The SF-tag enhanced the nanobody expression at the transcriptional level.• The fusion of SF-tag had no significant effect on the affinity of nanobodies and could slightly increase the thermal stability of nanobodies.

Towards Characterizing and Developing Formation and Migration Cues in Seafloor Sand Waves on Topology, Morphology, Evolution from High-Resolution Mapping via Side-Scan Sonar in Autonomous Underwater Vehicles.

Sand waves constitute ubiquitous geomorphology distribution in the ocean. In this paper, we quantitatively investigate the sand wave variation of topology, morphology, and evolution from the high-resolution mapping of a side scan sonar (SSS) in an Autonomous Underwater Vehicle (AUV), in favor of online sequential Extreme Learning Machine (OS-ELM). We utilize echo intensity directly derived from SSS to help accelerate detection and localization, denote a collection of Gaussian-type morphological templates, with one integrated matching criterion for similarity assessment, discuss the envelope demodulation, zero-crossing rate (ZCR), cross-correlation statistically, and estimate the specific morphological parameters. It is demonstrated that the sand wave detection rate could reach up to 95.61% averagely, comparable to deep learning such as MobileNet, but at a much higher speed, with the average test time of 0.0018 s, which is particularly superior for sand waves at smaller scales. The calculation of morphological parameters primarily infer a wave length range and composition ratio in all types of sand waves, implying the possible dominant direction of hydrodynamics. The proposed scheme permits to delicately and adaptively explore the submarine geomorphology of sand waves with online computation strategies and symmetrically integrate evidence of its spatio-temporal responses during formation and migration.

Development of a highly thermostable immunoassay based on a nanobody-alkaline phosphatase fusion protein for carcinoembryonic antigen detection.

Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) assays are employed in routine clinical settings to diagnose tumor. We selected two nanobodies with high-affinity to CEACAM-5, termed Nb11C12 and Nb2D5, using phage-display technology. The Nb2D5 fused with calf intestinal alkaline phosphatase (CAP), human placental alkaline phosphatase (HAP), or Pyrococcus abyssi alkaline phosphatase (PAP) were expressed in human embryonic kidney (HEK293) cells. The enzymatic activity of Nb2D5-HAP fusion protein was the best and remained stable at 60 °C for 7 days. The affinity of Nb2D5-HAP fusion protein to CEACAM-5 reached 42 pM. A chemiluminescent enzyme immunoassay (CLEIA) based on Nb2D5-HAP fusion protein was established for quantitative CEACAM-5 assay in clinical settings. The CLEIA exhibited a wide linear range of 0.31-640 ng/mL toward CEACAM-5, with a limit of detection (LOD) of 0.85 ng/mL. No cross-reactivity occurred with CEACAM-1, CEACAM-3, CEACAM-6, or CEACAM-8, and no interference was observed with rheumatoid factors. The CLEIA based on Nb2D5-HAP fusion protein was stable for 8 weeks at 37 °C and 50% relative humidity. The CLEIA developed from Nb2D5-HAP fusion protein had much better stability and linearity with similar reproducibility compared with the enzyme-linked immunosorbent assay developed from conventional monoclonal antibodies, which have been widely used in clinics over the past several decades. Graphical abstract.

Seminal plasma fractions can protect common carp (Cyprinus carpio) sperm during cryopreservation.

This study aimed to investigate the effect of fractionated seminal plasma on characteristics of common carp Cyprinus carpio cryopreserved sperm. Nanosep® centrifugal devices yielded four seminal plasma fractions with different total protein content ranging in molecular weight from less than 17 to almost 74 kDa. Each protein fraction was added to semen extender medium prior to freezing. Spermatozoon motility characteristics and DNA integrity were analyzed in supplemented and non-supplemented cryopreserved samples. The cryopreservation process strongly affected the swim-up sperm quality. Treatment with fractions 1, 2, 3, and 4 was associated with significantly higher spermatozoon motility rate and curvilinear velocity than seen in extender only, with highest values obtained with fraction 4 (78.21 ± 2.41% and 168.05 ± 4.46 µm/s, respectively). Significantly less DNA damage, expressed as percent tail DNA (12.23 ± 1.27) and olive tail moment (0.68 ± 0.12), was recorded in fraction 4. The findings indicated that addition of fractionated seminal plasma to cryopreservation medium can preserve the quality of common carp sperm. The protective effect of each fraction varied, suggesting the presence of distinct components exerting different effects on cryopreserved sperm function.

Production of functional human nerve growth factor from the submandibular glands of mice using a CRISPR/Cas9 genome editing system.

Nerve growth factor (NGF) is an essential trophic factor for the growth and survival of neurons in the central and peripheral nervous systems. For many years, mouse NGF (mNGF) has been used to treat various neuronal and non-neuronal disorders. However, the biological activity of human NGF (hNGF) is significantly higher than that of mNGF in human cells. Using the CRISPR/Cas9 system, we constructed the transgenic mice expressing hNGF specifically in their submandibular glands. As demonstrated by fluorescence immunohistochemical staining, these mice produced hNGF successfully, with 0.8 mg produced per gram of submandibular glands. hNGF with 99% purity was successfully extracted by two-step ion-exchange chromatography and one-step size-exclusion chromatography from the submandibular glands of these transgenic mice. Further, the purified hNGF was verified by LC-MS/MS. We analyzed the NH2-terminus of hNGF using both Edman degradation and LC-MS/MS-based methods. Both results showed that the obtained hNGF lost the NH2-terminal octapeptide (SSSHPIFH). Moreover, the produced hNGF demonstrated a strong promotion in the proliferation of TF1 cells.

An efficient constitutive expression system for Anti-CEACAM5 nanobody production in the yeast Pichia pastoris.

Nanobodies offer multiple advantages over conventional antibodies in terms of size, stability, solubility, immunogenicity, and production costs, with improved tumor uptake and blood clearance. Additionally, the recombinant expression of nanobodies is robust in various expression systems, such as Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. P. pastoris is the most widely used microorganism for nanobody production, but all or almost all expression vectors developed for this system are based on the regulated promoter of the alcohol oxidase 1 gene (AOX1) that requires methanol for full induction. In this study, a constitutive anti-CEACAM5 nanobody expression system was constructed under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) promoter. The effects of different carbon sources and pH on nanobody expression were evaluated in shaking flask cultures. After 96 h of constitutive expression in shaking flask, a yield of 51.71 mg/L was obtained. In addition, this constitutive expression system produced nanobodies at equivalent yield and affinity to that produced by methanol-induced expression. The results of this study indicated that the use of a constitutive expression system is a promising alternative for the production of nanobodies applied for cancer diagnosis and therapy.

Improvement of isoprene production in Escherichia coli by rational optimization of RBSs and key enzymes screening.

BACKGROUND: As an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum. As an alternative, bio-production of isoprene has attracted much attention in recent years. Previous researches were mostly focused on key enzymes to improve isoprene production. In this research, besides screening of key enzymes, we also paid attention to expression intensity of non-key enzymes. RESULTS: Firstly, screening of key enzymes, IDI, MK and IspS, from other organisms and then RBS optimization of the key enzymes were carried out. The strain utilized IDIsa was firstly detected to produce more isoprene than other IDIs. IDIsa expression was improved after RBS modification, leading to 1610-fold increase of isoprene production. Secondly, RBS sequence optimization was performed to reduce translation initiation rate value of non-key enzymes, ERG19 and MvaE. Decreased ERG19 and MvaE expression and increased isoprene production were detected. The final strain showed 2.6-fold increase in isoprene production relative to the original strain. Furthermore, for the first time, increased key enzyme expression and decreased non-key enzyme expression after RBS sequence optimization were obviously detected through SDS-PAGE analysis. CONCLUSIONS: This study prove that desired enzyme expression and increased isoprene production were obtained after RBS sequence optimization. RBS optimization of genes could be a powerful strategy for metabolic engineering of strain. Moreover, to increase the production of engineered strain, attention should not only be focused on the key enzymes, but also on the non-key enzymes.

Metabolic engineering for the production of isoprene and isopentenol by Escherichia coli.

The biotechnological production of isoprene and isopentenol has recently been studied. Isoprene, which is currently made mainly from petroleum, is an important platform chemical for synthesizing pesticides, medicines, oil additives, fragrances, and more and is especially important in the rubber production industry. Isopentenols, which have better combustion properties than well-known biofuels (ethanol), have recently received more attention. Supplies of petroleum, the conventional source of isoprene and isopentenols, are unsustainable, and chemical synthesis processes could cause serious environmental problems. As an alternative, the biosynthesis of isoprene and isopentenols in cell factories is more sustainable and environmentally friendly. With a number of advantages over other microorganisms, Escherichia coli is considered to be a powerful workhorse organism for producing these compounds. This review will highlight the recent advances in metabolic engineering for isoprene and isopentenol production, especially using E. coli cell factories.

Suppressor of cytokine signaling 3 (SOCS3) is related to pro-inflammatory cytokine production and triglyceride deposition in turbot (Scophthalmus maximus).

Turbot (Scophthalmus maximus) is an economically important fish that is farmed by aquaculture for human consumption. Aquacultured turbot are commonly fed a high-lipid diet; however, this diet causes excessive lipid deposition and the overexpression of pro-inflammatory cytokines. Studies in mammals have indicated that a relationship exists between pro-inflammatory cytokine overexpression and altered lipid metabolism through the activation of suppressor of cytokine signaling 3 (SOCS3). In this study, we investigated the relationship between SOCS3 and triglyceride (TG) deposition and mechanism of SOCS3 activation in farmed turbot fed high-lipid diet (HLD). TG content increased with SOCS3 production, mediated by toll-like receptor-nuclear transcription factor kappa-B (TLR-NFκB) signaling in the liver of turbot fed a HLD and in turbot primary liver cells incubated with oleic acid (OA). Overexpression of SOCS3 increased TG deposition via the increased production of mature sterol regulatory element binding protein 1 (m-SREBP-1). Knockdown of SOCS3 in turbot primary liver cells resulted in normalized TG deposition and decreased m-SREBP-1 production. These results suggest that the HLD and OA can induce cytokine expression by activating the TLR-NFκB signaling pathways, resulting in increased SOCS3 expression. It is proposed that SOCS3 enhances m-SREBP-1 production, leading to TG deposition. These findings provide important new insights into the relationship between cytokine expression and TG deposition and mechanism of HLD-induced pro-inflammatory response, which could help to improve the health of farmed turbot and a better understanding of fish immunity.

Pullulan production from synthetic medium by a new mutant of Aureobasidium pullulans.

Pullulan with different molecular-weight could be applied in various fields. A UV-induced mutagenesis Aureobasidium pullulans UVMU6-1 was obtained from the strain A. pullulans CGMCC3.933 for the production of low-molecular-weight pullulan. First, the obtained polysaccharide from A. pullulans UVMU6-1 was purified and identified to be pullulan with thin-layer chromatography, Fourier transform infrared, and nuclear magnetic resonance. Then, culture medium and conditions for this strain were optimized by flask fermentation. Based on the optimized medium and culture conditions (pH 4, addition of 4 g/L Tween 80 for 96 hr of cultivation), continuously fermentation was performed. The highest pullulan production and dry biomass was 109 and 125 g/L after fermentation for 114 hr, respectively. The average productivity was about 1 g/L/hr, which was intensively higher than the previous reported. This study would lay foundations for the industrial production of pullulan.

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3).

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39 kDa. The effects of induction temperature, concentration of isopropyl-ß-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23 U/mL (8.64 U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.

Histone-dependent IgG conservation in octanoic acid precipitation and its mechanism.

Octanoic acid (OA) precipitation has long been used in protein purification. Recently, we reported a new cell culture clarification method for immunoglobulin G (IgG) purification, employing an advance elimination of chromatin heteroaggregates with a hybrid OA-solid phase system. This treatment reduced DNA more than 3 logs, histone below the detection limit (LOD), and non-histone host cell proteins (nh-HCP) by 90 % while conserving more than 90 % of the IgG monomer. In this study, we further investigated the conservation of IgG monomer and antibody light chain (LC) to the addition of OA/OA-solid phase complex, with or without histone and DNA in different combinations. The results showed that highly basic histone protein was the prime target in OA/OA-solid phase precipitation system for IgG purification, and the selective conservation of IgG monomer in this system was histone dependent. Our findings partially support the idea that OA works by sticking to electropositive hydrophobic domains on proteins, reducing their solubility, and causing them to agglomerate into large particles that precipitate from solution. Our findings also provide a new perspective for IgG purification and emphasize the necessity to re-examine the roles of various host contaminants in IgG purification.

Simultaneous Localization and Mapping with Iterative Sparse Extended Information Filter for Autonomous Vehicles.

In this paper, a novel iterative sparse extended information filter (ISEIF) was proposed to solve the simultaneous localization and mapping problem (SLAM), which is very crucial for autonomous vehicles. The proposed algorithm solves the measurement update equations with iterative methods adaptively to reduce linearization errors. With the scalability advantage being kept, the consistency and accuracy of SEIF is improved. Simulations and practical experiments were carried out with both a land car benchmark and an autonomous underwater vehicle. Comparisons between iterative SEIF (ISEIF), standard EKF and SEIF are presented. All of the results convincingly show that ISEIF yields more consistent and accurate estimates compared to SEIF and preserves the scalability advantage over EKF, as well.

Effects of ultrasonic pretreatment on drying characteristics and water migration characteristics of freeze-dried strawberry.

The effects of ultrasonic pretreatment on the drying characteristics and microstructure of strawberry slices were investigated. The rehydration characteristics of freeze-dried products, which were pre-frozen at -20 °C and - 80 °C were explored, with a focus on water mobility and distribution. The ultrasonic pretreatment significantly increased the water mobility of the strawberry slices, resulting in a reduction in their water content. However, the application of ultrasound significantly decreased the rehydration speed, indicating a lower moisture absorption capacity in the pretreated sample. The micrographs revealed that the structure of the tissue was more uniform after ultrasonic treatment, and water loss was accelerated. In addition, the contact angle measurements showed that the samples were more hydrophobic after ultrasonic treatment, and the eutectic temperature and fold point of the samples increased. Therefore, this study found that ultrasonic-assisted freeze vacuum drying technology effectively reduces hygroscopicity, improves product storage, and represents a potential method for dried production.

A comprehensive review of engineered exosomes from the preparation strategy to therapeutic applications.

Exosomes exhibit high bioavailability, biological stability, targeted specificity, low toxicity, and low immunogenicity in shuttling various bioactive molecules such as proteins, lipids, RNA, and DNA. Natural exosomes, however, have limited production, targeting abilities, and therapeutic efficacy in clinical trials. On the other hand, engineered exosomes have demonstrated long-term circulation, high stability, targeted delivery, and efficient intracellular drug release, garnering significant attention. The engineered exosomes bring new insights into developing next-generation drug delivery systems and show enormous potential in therapeutic applications, such as tumor therapies, diabetes management, cardiovascular disease, and tissue regeneration and repair. In this review, we provide an overview of recent advancements associated with engineered exosomes by focusing on the state-of-the-art strategies for cell engineering and exosome engineering. Exosome isolation methods, including traditional and emerging approaches, are systematically compared along with advancements in characterization methods. Current challenges and future opportunities are further discussed in terms of the preparation and application of engineered exosomes.

The prognostic value of LAYN in HPV-related head and neck squamous cell carcinoma and its influence on immune cell infiltration.

BACKGROUND: HPV-positive head and neck squamous cell carcinoma (HNSCC) exhibits different characteristics from HPV-negative tumors in terms of tumor development, clinical features, treatment response, and prognosis. Layilin (LAYN), which contains homology with C-type lectins, plays a critical role in tumorigenesis and cancer progression. However, the prognostic value of LAYN and the relationship between LAYN and immune infiltration levels in HPV-related HNSCC patients still require a comprehensive understanding. Herein, we aimed to assess the prognostic value of LAYN and to investigate its underlying immunological function in HPV-related HNSCC. METHODS: Through various bioinformatics methods, we analyzed the data from The Cancer Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA) databases to explore the potential underlying oncogenic impression of LAYN, including the relevance of LAYN to survival outcomes, clinicopathological factors, immune cell infiltration, and immune marker sets in HPV-related HNSCC. The expression levels of LAYN and HPV were also verified in HNSCC patient tissues. RESULTS: LAYN was differentially expressed in a variety of tumors. The expression of LAYN in HNSCC was significantly higher than that in adjacent normal tissues (P < 0.0001), and high expression of LAYN was correlated with poor overall survival (OS) in HNSCC patients (Hazard Ratio (HR) = 1.3, P = 0.035). Moreover, LAYN expression level in HPV-positive HNSCC patients was significantly lower than that in HPV-negative patients, with HPV-positive HNSCC patients displaying a trend of favorable prognosis. In addition, the relationship between LAYN expression and immune infiltration levels in HPV-positive HNSCC group was less tightly correlated than that in HPV-negative HNSCC group, and there was a strong relationship between LAYN expression and markers of M2 macrophage (P < 0.001) and exhausted T cells (P < 0.05) in HPV-negative HNSCC. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis suggested that LAYN potentially influenced tumor progression through HPV infection and other cancer-related pathways. CONCLUSIONS: LAYN might contribute to tumorigenesis via its positive correlation with immune checkpoint molecules and tumor-associated macrophages (TAMs). Our study might provide a novel prognostic biomarker and latent therapeutic target for the treatment of HPV-related HNSCC.

An early underwater artificial vision model in ocean investigations via independent component analysis.

Underwater vision is one of the dominant senses and has shown great prospects in ocean investigations. In this paper, a hierarchical Independent Component Analysis (ICA) framework has been established to explore and understand the functional roles of the higher order statistical structures towards the visual stimulus in the underwater artificial vision system. The model is inspired by characteristics such as the modality, the redundancy reduction, the sparseness and the independence in the early human vision system, which seems to respectively capture the Gabor-like basis functions, the shape contours or the complicated textures in the multiple layer implementations. The simulation results have shown good performance in the effectiveness and the consistence of the approach proposed for the underwater images collected by autonomous underwater vehicles (AUVs).

Development of a highly sensitive immunoassay based on pentameric nanobodies for carcinoembryonic antigen detection.

BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) is a well-characterized biomarker for the clinical diagnosis of various cancers. Nanobodies, considered the smallest antibody fragments with intact antigen-binding capacity, have gained significant attention in disease diagnosis and therapy. Due to their peculiar properties, nanobodies have become promising alternative diagnostic reagents in immunoassay. However, nanobodies-based immunoassay is still hindered by small molecular size and low antigen capture efficacy. Therefore, there is a pressing need to develop novel nanobody-based immunoassays with superior performance. RESULTS: A novel pentameric nanobodies-based immunoassay (PNIA) was developed with enhanced sensitivity and specificity for CEACAM-5 detection. The binding epitopes of three anti-CEACAM-5 nanobodies (Nb1, Nb2 and Nb3) were analyzed. To enhance the capture and detection efficacy of CEACAM-5 in the immunoassay, we engineered bispecific nanobodies (Nb1-Nb2-rFc) as the capture antibody, and developed the FITC-labeled pentameric nanobodies (Nb3-VT1B) as the detection antibody. The binding affinities of Nb1-Nb2-rFc (1.746 × 10-10) and Nb3-VT1B (1.279 × 10-11) were significantly higher than those of unmodified nanobodies (Nb1-rFc, 4.063 × 10-9; Nb2-rFc, 2.136 × 10-8; Nb3, 3.357 × 10-9). The PNIA showed a linear range of 0.625-160 ng mL-1 with a correlation coefficient R2 of 0.9985, and a limit of detection of 0.52 ng mL-1, which was 24-fold lower than the immunoassay using monomeric nanobody. The PNIA was validated with the spiked human serum. The average recoveries ranged from 91.8% to 102% and the coefficients of variation ranged from 0.026% to 0.082%. SIGNIFICANCE AND NOVELTY: The advantages of nanobodies offer a promising alternative to conventional antibodies in disease diagnosis. The novel PNIA demonstrated superior sensitivity and high specificity for the detection of CEACAM-5 antigen. This bispecific or multivalent nanobody design will provide some new insights into the design of immunoassays for clinical diagnosis.