Search Results (69)

A key function of naturally occurring antibodies is to control pathologically altered cells, such as those with aberrant glycosylation. Age-related diminution in the pool of B cells producing these immunoglobulins is linked to impaired anti-tumor immunity. In this study, the levels of antibodies against tumor-associated carbohydrate antigens (TACAs)—common in gastric cancer (GC) and other malignancies—were analyzed in 235 treatment-naïve GC patients (stages I–IV) and 76 healthy donors using a printed glycan array (PGA). We found that anti-glycan IgM levels, but not IgG, reduced with age in both patients and donors. Crucially, IgM levels against most glycans were significantly lower in the GC cohort compared with healthy donors, a trend that remained after age adjustment. Furthermore, an immunohistochemical analysis revealed that human anti-GalNAcα (Tn) antibodies—a well-characterized TACA in gastrointestinal cancers—bound to tumor cells and exhibited perinuclear and membrane staining in non-tumor surface cells within the same organ. These data support the hypothesis that gastric cancer patients have reduced levels of anti-glycan IgMs, which are responsible for the early recognition of transformed cells. This specific immunodeficiency may contribute to a permissive environment for tumor development. Full article

Introduction. Over the past two decades, the αGal (Galα1–3Galβ1–4GlcNAc–R) epitope, a carbohydrate found in many non-primate mammals, has gained significant relevance in medicine due to its association with an increasing number of allergic reactions to animal-derived foods, drugs, and medical devices. Due to a mutated gene coding for α1,3-galactosyltransferase (α1–3GT), humans lack αGal and, therefore, naturally produce anti-α-Gal antibodies (IgM, IgA, and IgG), especially in the context of a xenotransplantation, which can lead to extreme immunological reactivity, including hyperacute rejection of the transplant. Recently, these uncontrollable immune reactions have driven demand for more accurate procedures to better detect αGal in animal-derived foods or bioprosthetics. The currently most widely used α-Gal-specific monoclonal antibody is an IgM antibody (clone M86), developed in Ggta1 KO mice and isolated from hybridoma tissue culture. As the IgM isotype has limited purification properties, specificity, and sensitivity, we aimed to produce a novel IgG antibody with high affinity and extensive applicability. Methods. An experimental murine IgG1 anti-αGal antibody (IgG-αGalomab) was developed by immunization of Ggta1 knockout (KO) mice, and its affinity was evaluated using ELISA, Western blot, flow cytometry, and immunohistochemistry/immunofluorescence. Results. Compared to IgM-M86, IgG-αGalomab demonstrated ~1200-fold higher binding potency and lower cross-reactivity with competitive molecules, i.e., bovine serum albumin, galactobiose, and lactose. Unlike IgM-M86, IgG-αGalomab showed an increasing affinity over time in the binding tests performed on xenogeneic tissues. Notably, high-affinity for αGal was detected by Western blot at high dilution [1:200,000] of IgG-αGalomab compared to IgM-M86 [1:1000]. By flow cytometry, specificity and dose-dependent response were confirmed using in vitro cultures of porcine and human fibroblasts. Finally, in immunofluorescence and immunohistochemistry analysis, αGal was demonstrated to be detectable by IgG-αGalomab at a dilution of [1:1000], while IgM-M86 was demonstrated to be detectable at [1:100]. Conclusions. Altogether, our newly developed antibody showed high sensitivity and specificity for α-Gal in various applications. Based on its potential binding capacity, IgG-αGalomab could have important applications in precision medicine for predicting, treating, and preventing immune-mediated phenomena of patients in different medical areas. Full article

Background: The origin of natural anti-galactose-α-1,3-galactose (anti-Gal) antibodies in humans is only partially understood. The gut microbiome has been proposed as an important source of galactose-α-1,3-galactose (αGal) epitopes that drive the maturation of anti-Gal–reactive B cells. Certain bacteria expressing αGal epitopes, notably Escherichia coli O86:B7, have been shown to elicit anti-Gal antibody responses in α1,3-galactosyltransferase knockout (α3GalT1 KO) mice. In this study, we investigated the interaction between currently widely used bacteria polysaccharide vaccine, the 23-valent pneumococcal polysaccharide vaccine (PPV23), which contains capsular polysaccharides (CPS) from multiple Streptococcus pneumoniae serotypes, and host anti-Gal antibodies. Methods: We conducted a genomic analysis to identify α1,3-galactosyltransferase (α3GalT1) genes in S. pneumoniae strains. Binding of PPV23 to anti-Gal monoclonal antibodies was evaluated by ELISA, and αGal epitope content in PPV23 was estimated using a four-parameter logistic (4PL) model fitted to the ELISA calibration data. To assess in vivo immunogenicity, we immunized α3GalT1 KO mice with PPV23 and measured serum anti-Gal IgG and IgM titers before and after vaccination. Results: Genomic analysis revealed the presence of α3GalT1 genes in S. pneumoniae strains. PPV23 showed specific binding to anti-Gal monoclonal antibodies as detected by ELISA. Quantitative modeling indicated that αGal epitopes are present at low abundance within PPV23, consistent with limited expression of αGal among a minority of included serotypes. Immunization of α3GalT1 KO mice with PPV23 induced a significant rise in anti-Gal IgG titers (mean value from 124 to 384), whereas anti-Gal IgM titers remained relatively unchanged (mean value at the range of 6500–7500). High baseline anti-Gal IgM levels observed in α3GalT1 KO mice are consistent with age-dependent induction by the gut microbiota. Conclusions: These results provide genetic and immunological evidence that αGal epitopes derived from S. pneumoniae are present in PPV23 and can engage pre-existing anti-Gal antibodies. Our findings underscore a complex interplay among bacterial glycosyltransferase genes, vaccine polysaccharide composition, and host anti-Gal antibody repertoires, which may influence vaccine immunogenicity. Consideration of host natural antibody profiles may therefore be important for interpreting responses to carbohydrate-based vaccines and for guiding vaccine design. Full article

Sialyl-Tn (sTn) is a tumor-associated carbohydrate antigen (TACA) abundantly expressed by various types of carcinomas. While conventional antibody-based platforms have traditionally been used for the detection and targeting of sTn, alternative binding scaffolds may offer distinct advantages. Variable lymphocyte receptor B (VLRB), the immunoglobulin-like molecule of jawless vertebrates, offers a promising alternative for glycan recognition. In this study, a phage-displayed VLRB library was utilized to identify sTn-specific binders. Two candidates, designated as ccombodies A8 and B11, were isolated after four rounds of biopanning. Both were expressed and purified using Ni-affinity and FPLC, yielding proteins with apparent molecular weights of ~27 kDa in SDS-PAGE. Sequence analysis revealed a preference for glycan-binding residues in randomized hypervariable regions, with A8 exhibiting an increased aliphatic content. ELISA confirmed selective binding to sTn and other O-glycans containing the core α-GalNAc, with EC₅₀ values of 18.2 and 14.2 nM for A8 and B11, respectively. Vicia villosa lectin inhibited ccombody binding to sTn, indicating shared epitope recognition. Additionally, both ccombodies bound to sTn-positive glycoproteins and carcinoma cell lines HeLa and LS174T. These findings demonstrate that phage display of VLRBs enables the identification of high-affinity, glycan-specific binders, offering a compelling alternative to immunoglobulin-based platforms for future diagnostic and therapeutic applications targeting tumor-associated glycans. Full article

Alpha-gal syndrome (AGS) is an IgE-mediated allergy, triggered by a carbohydrate—galactose-α-1,3-galactose (α-Gal). AGS is marked by a delayed onset of symptoms, typically occurring 3–8 h after the ingestion of red meat or other mammalian-derived products. The primary risk factor is believed to be tick bites, which sensitize individuals through the introduction of α-Gal via tick saliva. Diagnosis of AGS is based on a combination of anamnesis and detection of α-Gal-specific IgE antibodies. We evaluated 28 patients with a history of unexplained anaphylaxis, angioedema, and/or urticaria, in whom the diagnostic work-up included the assessment of serum IgE specific to alpha-gal. Elevated alpha-gal-specific IgE levels were detected in five patients. Among them, four reported anaphylactic episodes following meat consumption. In three cases, symptoms developed during the evening or nighttime, typically 3 to 6 h after the last meal. One patient experienced anaphylaxis within one hour after a meal. Another patient presented angioedema up to 24 h after meat consumption and was also tested positive for specific IgE against beef and pork allergens. The AGS case series showed variability in clinical picture and time to reaction. In patients presenting idiopathic anaphylaxis and nonspecific symptoms after red meat consumption, AGS should be considered as a differential diagnosis. Full article

Since carbohydrate antigen 19-9 (CA 19-9) is a significant biomarker for the clinical diagnosis and treatment of pancreatic cancer, a sensitive sandwich-type immunosensor was proposed with an epoxy functionalized covalent organic framework (EP-COF_TTA-DHTA) as the antibody carrier and an electroactive COF_{TTA-2,6-NA(OH)2} as the signal amplification probe for the sensitive detection of CA 19-9. The flexible covalent linkage between the epoxy-functionalized EP-COF_TTA-DHTA and the antibodies was employed to improve the dynamics of the antigen–antibody interaction significantly. Meanwhile, AuNPs@COF_{TTA-2,6-NA(OH)2} with abundant electroactive sites enhanced the current response of the immunoreaction significantly. After optimizing the incubation time and concentration of the antibody, CA 19-9 was quantitatively detected by differential pulse voltammetry (DPV) based on the sensitive sandwich-type immunosensor with a low detection limit of 0.0003 U/mL and a wide linear range of 0.0009–100 U/mL. The electrochemical immunosensor exhibits high specificity, stability and repeatability, and it provides a feasible and efficient method for the pathologic analysis and treatment of tumor markers. Full article

Background: Herein, we review the Cotton Top Tamarin (CTT), Saguinus oedipus, a unique spontaneous model for colorectal cancer (CRC). Despite its predisposition to inflammatory bowel disease (IBD) and frequent development of CRC, the CTT is adept at avoiding colorectal metastasis in the liver. In contrast, the common marmoset (CM), Callithrix jacchus, is a natural negative control, in that it also contracts IBD, but usually not CRC. We review our findings in these New World monkeys in terms of the expression of CEACAM adhesion models and their related molecules to contrast them with human disease. Methods: Specimens were collected from aforementioned monkey colorectal and other tissues, colonic washings, serum for analysis of tissue extraction, and colonic washings via ELISA, using a battery of antibodies. Fixed tissues were analyzed using immunohistochemistry and CEACAMs were extracted via Western blotting. Serum CEA levels were analyzed using ELISA, and DNA was extracted via a Bigblast genomics sequencing kit. Results: Serum CEA was significantly elevated in CTTs, and one-third of them die from CRC. Unlike others, we were unable to stain for CEA in tissues. The sialylated carbohydrate antigen recognized by monoclonal antibody (MAb) SPAN-1 does stain in 16.7% of CTT tissues, but the anti-aminoproteoglycan MAb, CaCo.3/61, stained 93.3% (OR70·00[CI6.5–754.5] p < 0.0001). The common CEA kits from Abbott and Roche were non-conclusive for CEA. We later adopted a CEA AIA-PACK from Tosoh Medics, which identified a 50 Kda band via Western blotting in humans and CTTs. The CEA levels were higher using the CEA AIA-PACK than the Pharmatrope kit (932 ± 690 versus 432 ± 407 ng/mL (p < 0.05)) in human patient colonic effluent, not statistically significant (NSS) for CTT extracts or effluent (733 ± 325 and 739 ± 401 ng/mL, respectively). It was suggested that the smaller CTT CEA moiety might lack components that facilitate the spread of liver metastasis. Later, using more specific CEA assays and increased numbers of specimens, we were able to show higher CEA serum expression in CTTs than in CMs (632.1 ± 306.1 vs. 81.6 ± 183.6, p < 0.005), with similar differences in the serum samples. Western blotting with the anti-CEA T84.66 MAb showed bands above 100 KDa in CTTs. The profiles in CTTs were similar to human patients with inflammatory bowel disease. We established that the CEA anchorage to the cell was a GPI-linkage, advantageous for the inhibition of differentiation and anoikis. With further CEA DNA analysis, we were able to determine at least five different mechanisms that may inhibit liver metastasis, mostly related to CEA, but later expanded this to seven, and increased the relationships to CEACAM1 and other related molecules. Recently, we obtained CTT liver mRNA transcriptomes that implicated several pathways of interest. Conclusions: With efforts spanning over three decades, we were able to characterize CEA and other changes that allow us to better understand the CTT phenomenon of liver metastasis inhibition. We are in the process of characterizing the CTT liver mRNA transcriptome to compare it with that of the common marmoset. Currently, liver CTT gene expression patterns suggest that ribosomes, lipoproteins, and antioxidant defense are related to differences between CTTs and CMs. Full article

Bronchial asthma remains a serious medical problem, as approximately 10% of patients fail to achieve adequate symptom control with available treatment options. Macrophages play a pivotal role in the pathophysiology of asthma, as well as in some other respiratory disorders. Typically, they are classified into two major classes, M1 and M2; however, recent findings have indicated that in fact there is a whole range of macrophage polarization and functional diversity beyond this bimodal division. The isolation of individual cell sub-populations and the identification of their role and diagnostic/therapeutic significance is still a challenge. Here, we have attempted to assess the differences between patient-derived macrophage populations from bronchoalveolar lavage fluid (BALF) samples in different pulmonary disease conditions, based on their capability to interact with a range of specific and relatively non-specific carbohydrate-based ligands (containing galactose (linear or cyclic form), mannose, trimannose, etc.). Obviously, the main target of these ligands was CD206; however, other minor receptors, able to bind carbohydrates, have also been reported for macrophages. Trimannose binds most specifically to CD206 macrophage receptors, while monomannose has intermediate affinity, and galactose has low affinity and may involve binding to other receptors. This clearly indicates the ligands were chosen based on their predicted binding strength and specificity for CD206, providing the rationale for the study. In some cases, the activated macrophage affinity to galactose base ligands was higher than that to mannose, indicating that complexes of CD206 or other carbohydrate-binding receptors may contribute substantially to macrophage functional features. In addition, variations in receptor clustering and distribution may substantially affect affinity to the same ligand. Interestingly, with a panel of 6–10 different carbohydrate-based ligands with FTIR or fluorescent marker, we were able not only to distinguish between healthy and disease states but also between closely related diseases such as purulent endobronchitis, obstructive bronchitis, pneumonia, and bronchial asthma. For further investigation, specific sub-populations of macrophages, seen as hallmarks to specific diseases, can be isolated and studied separately, likely giving new insights with diagnostic and therapeutic significance for hard-to-treat patients. The group of patients with resistant disease can also be identified with this approach as a fingerprint method to find a more targeted therapeutic strategy, improving their clinical outcomes. As expected, this will provide a large additional array of data for analysis, compared to the work going on in the world. The dataset used by other researchers mainly for known “antibody” ligands is semi-quantitative and insufficient for the purposes of typing as yet unknown and uncomplicated sub-populations. The analysis of the presented data in combination with personalized information from patients’ medical records will be carried out using both traditional methods and machine learning methods. Full article

Detecting IgE sensitizations in the serum of allergic dogs is commonly performed using allergen extracts, but these are difficult to standardize. This article details the development and validation of the Pet Allergy Xplorer (PAX; Nextmune, Stockholm, Sweden), the first multiplex macroarray for the detection of IgE sensitization in dogs using allergen extracts and molecular components; the PAX is derived from the Allergy Xplorer (ALEX²; MacroArray Diagnostics, Vienna, Austria). The selection of allergens, cartridge processing, strategy for identifying and blocking IgE directed against cross-reactive carbohydrate determinants (CCDs), and the method used for determining the positivity threshold are described. The validation of the PAX included evaluations of the specificity of its anti-IgE monoclonal antibody, specificity of IgE binding to target allergens, assay precision, and internal consistency. Additionally, the influence of possible confounding factors, such as sample type, the influence of hemolysis, lipemia, bilirubinemia, and elevated CCD-IgE, was tested. Finally, the sensitization rates of 23,858 European dogs to 145 environmental and Hymenoptera venom allergens were summarized. The PAX is accurate and reproducible and has a unique CCD-detection and blocking strategy; its molecular allergens offer a unique window on allergen cross-reactivity. Full article

A major reason for the failure of the immune system to detect tumor antigens (TAs) is the insufficient uptake, processing, and presentation of TAs by antigen-presenting cells (APCs). The immunogenicity of TAs in the individual patient can be markedly increased by the in situ targeting of tumor cells for robust uptake by APCs, without the need to identify and characterize the TAs. This is feasible by the intra-tumoral injection of α-gal micelles comprised of glycolipids presenting the carbohydrate-antigen “α-gal epitope” (Galα1-3Galβ1-4GlcNAc-R). Humans produce a natural antibody called “anti-Gal” (constituting ~1% of immunoglobulins), which binds to α-gal epitopes. Tumor-injected α-gal micelles spontaneously insert into tumor cell membranes, so that multiple α-gal epitopes are presented on tumor cells. Anti-Gal binding to these epitopes activates the complement system, resulting in the killing of tumor cells, and the recruitment of multiple APCs (dendritic cells and macrophages) into treated tumors by the chemotactic complement cleavage peptides C5a and C3a. In this process of converting the treated tumor into a personalized TA vaccine, the recruited APC phagocytose anti-Gal opsonized tumor cells and cell membranes, process the internalized TAs and transport them to regional lymph-nodes. TA peptides presented on APCs activate TA-specific T cells to proliferate and destroy the metastatic tumor cells presenting the TAs. Studies in anti-Gal-producing mice demonstrated the induction of effective protection against distant metastases of the highly tumorigenic B16 melanoma following injection of natural and synthetic α-gal micelles into primary tumors. This treatment was further found to synergize with checkpoint inhibitor therapy by the anti-PD1 antibody. Phase-1 clinical trials indicated that α-gal micelle immunotherapy is safe and can induce the infiltration of CD4+ and CD8+ T cells into untreated distant metastases. It is suggested that, in addition to converting treated metastases into an autologous TA vaccine, this treatment should be considered as a neoadjuvant therapy, administering α-gal micelles into primary tumors immediately following their detection. Such an immunotherapy will convert tumors into a personalized anti-TA vaccine for the period prior to their resection. Full article

Vertically ordered mesoporous silica films (VMSF) are a class of porous materials composed of ultrasmall pores and ultrathin perpendicular nanochannels, which are attractive in the areas of electroanalytical sensors and molecular separation. However, VMSF easily falls off from the carbonaceous electrodes and thereby impacts their broad applications. Herein, carbon nitride nanosheets (CNNS) were served as an adhesive layer for stable growth of VMSF on the glassy carbon electrode (GCE). CNNS bearing plentiful oxygen-containing groups can covalently bind with silanol groups of VMSF, effectively promoting the stability of VMSF on the GCE surface. Benefiting from numerous open nanopores of VMSF, modification of VMSF’s external surface with carbohydrate antigen 15-3 (CA15-3)-specific antibody allows the target-controlled transport of electrochemical probes through the internal silica nanochannels, yielding sensitive quantitative detection of CA15-3 with a broad detection range of 1 mU/mL to 1000 U/mL and a low limit of detection of 0.47 mU/mL. Furthermore, the proposed VMSF/CNNS/GCE immunosensor is capable of highly selective and accurate determination of CA15-3 in spiked serum samples, which offers a simple and effective electrochemical strategy for detection of various practical biomarkers in complicated biological specimens. Full article

Grain processing produces many by-products, including wheat bran, wheat germ and rice bran, which are rich in carbohydrates, proteins and trace elements. In this study, these grain-derived by-products were used as raw materials to conduct solid-state fermentation using mixed strains of Aspergillus kawachii and Rhizopus oryzae, and the potential immunomodulatory and anti-allergic properties of fermented product were evaluated. Solid-state fermentation of a grain by-product mixture, consisting of rice bran, wheat bran, and wheat germ in a 2:1:1 weight ratio, using both A. kawachii L1 and R. oryzae L1 at 26 °C for 5 days, significantly increased the total phenolic, flavonoid, and amino acid contents. The anti-allergic activity of aqueous extract of the fermented product was evaluated in murine models of food allergy and delayed-type hypersensitivity. Oral administration of the fermented product extract (100–200 mg/kg) notably alleviated allergic symptoms such as diarrhea and histopathological changes in the intestines. Moreover, the extract effectively reduced allergen-specific serum antibodies, suppressed splenic cytokine secretion, and mitigated tissue edema and inflammation induced by allergens. Importantly, the extract induced the production of IL-10 and TGF-β, which are well-known cytokines primarily secreted by regulatory T cells. These results underscore the promising immunomodulatory effects of A. kawachii and R. oryzae fermented grain product, suggesting their potential as functional foods or additives for managing allergic disorders, with implications for future therapeutic and dietary applications. Full article

Apolipoprotein A2-ATQ/AT (apoA2-ATQ/AT) is a new biomarker for diagnosing pancreatic cancer (PC). In this study, the value of blood carbohydrate antigen 19-9 (CA19-9) and apoA2-ATQ/AT levels in diagnosing stage 0 and IA PC was evaluated. During 2014–2021, 12 patients with stage 0 PC and 12 patients with IA PC (average age: 73.8 years) underwent resection at JA Onomichi General Hospital. In addition, the data of 200 healthy controls were collected from a community-based cohort study. Levels of two apoA2-isoforms were measured using enzyme-linked immunosorbent assay (ELISA) with specific antibodies to calculate the apoA2-i Index as a surrogate value for apoA2-ATQ/AT. The cutoff value for the apoA2-i Index was determined to be 62.9 μg/mL. CA19-9 levels were also measured through ELISA. Among all 24 patients with PC, the positivity rates for apoA2-i and CA19-9 were 33.3% and 25.0%, respectively. The positivity rates for apoA2-i and CA19-9 were 16.7% and 8.3% in patients with stage 0 PC and 50.0% and 41.7% in those with stage IA, respectively. For CA19-9-negative patients, the apoA2-i positivity rate was 9.1% in stage 0 and 42.9% in stage IA. The combined positivity rate for both markers was 16.7% in stage 0 and 66.7% in stage IA. Imaging findings in apoA2-i- and CA19-9-positive patients included pancreatic duct dilatation (87.5%/100%), duct stenosis (75.0%/50%), and atrophy (87.5%/66.7%). The imaging findings of this study suggest that apoA2-i may enhance the sensitivity for detecting CA19-9-negative stage 0 and IA PC, and complementary measurements with CA19-9 may be valuable for diagnosing early-stage PC. Therefore, minute PC with pancreatic duct dilation, duct stenosis, and atrophy may exhibit a high positivity rate, aiding differential diagnosis. Full article

Vegetative desiccation tolerance has evolved within the genera Craterostigma and Lindernia. A centre of endemism and diversification for these plants appears to occur in ancient tropical montane rainforests of east Africa in Kenya and Tanzania. Lindernia subracemosa, a desiccation-sensitive relative of Craterostigma plantagineum, occurs in these rainforests and experiences adequate rainfall and thus does not require desiccation tolerance. However, sharing this inselberg habitat, another species, Lindernia brevidens, does retain vegetative desiccation tolerance and is also related to the resurrection plant C. plantagineum found in South Africa. Leaf material was collected from all three species at different stages of hydration: fully hydrated (ca. 90% relative water content), half-dry (ca. 45% relative water content) and fully desiccated (ca. 5% relative water content). Cell wall monosaccharide datasets were collected from all three species. Comprehensive microarray polymer profiling (CoMPP) was performed using ca. 27 plant cell-wall-specific antibodies and carbohydrate-binding module probes. Some differences in pectin, xyloglucan and extension epitopes were observed between the selected species. Overall, cell wall compositions were similar, suggesting that wall modifications in response to vegetative desiccation involve subtle cell wall remodelling that is not reflected by the compositional analysis and that the plants and their walls are constitutively protected against desiccation. Full article

The sensitivity of immunoassays is generally limited by the low signal reporter/recognition element ratio. Nanomaterials serving as the carriers can enhance the loading number of signal reporters, thus improving the detection sensitivity. However, the general immobilization strategies, including direct physical adsorption and covalent coupling, may cause the random orientation and conformational change in proteins, partially or completely suppressing the enzymatic activity and the molecular recognition ability. In this work, we proposed a strategy to load recognition elements of antibodies and enzyme labels using boronic acid-modified metal-organic frameworks (MOFs) as the nanocarriers for signal amplification. The conjugation strategy was proposed based on the boronate ester interactions between the carbohydrate moieties in antibodies and enzymes and the boronic acid moieties on MOFs. Both enzymes and MOFs could catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂, therefore achieving dual signal amplification. To indicate the feasibility and sensitivity of the strategy, colorimetric immunoassays of prostate specific antigen (PSA) were performed with boronic acid-modified Cu-MOFs as peroxidase mimics to catalyze TMB oxidation and nanocarriers to load antibody and enzyme (horseradish peroxidase, HRP). According to the change in the absorbance intensity of the oxidized TMB (_oxTMB), PSA at the concentration range of 1~250 pg/mL could be readily determined. In addition, this work presented a site-specific and oriented conjugation strategy for the modification of nanolabels with recognition elements and signal reporters, which should be valuable for the design of novel biosensors with high sensitivity and selectivity. Full article