Anti-human Serum Research Articles

Comparison of polyclonal and monoclonal antibody assays for serum amyloid A in cats: a study based on an automated turbidimetric immunoassay in a primary care veterinary hospital.

Comparing the utility of the anti-human serum amyloid A (SAA)-specific monoclonal and polyclonal antibodies assays (LZ-SAA) with the pure monoclonal anti-human antibody assays (VET-SAA) during clinical practice in primary care hospital populations by measuring SAA measurement in healthy and diseased domestic cats. 52 healthy and 185 diseased client-owned cats. SAA concentration was measured using different LZ-SAA and VET-SAA measurements for healthy and various diseased cats. Sensitivity, specificity, and accuracy were calculated for each disease. VET-SAA has higher sensitivity than LZ-SAA for the most common diseases presenting to primary care veterinary hospitals, including chronic kidney disease, tumors, and gingivostomatitis. Our results reveal the capability of detecting low SAA concentrations in healthy and diseased cats using VET-SAA in contrast to LZ-SAA, which found elevations of SAA concentrations only in diseased cats. Our findings indicate that switching to the new VET-SAA instead of the conventional LZ-SAA will likely enhance the diagnostic performance in primary care veterinary hospitals.

Implementation of a Macroporous Polyhydroxyethylmethacrylate Cryogel-Based Mini-Bioreactor System to Improve Monoclonal Antibody Production.

Monoclonal antibodies (mAbs) are pioneers in the diagnosis and treatment of many diseases, such as cancer, asthma, poisoning, viral infections, etc. As the market value of mAbs increases in the biopharma industry, the demand for high quantities is met by upscaled production using bioreactor systems. Thus, disposable, porous matrices called cryogels have gained the primary focus for adherent support in the proliferation of hybridoma cells. In this study, a gelatin-immobilized polyhydroxyethylmethacrylate-based cryogel material (disc-shaped, 9 mL bed volume) was synthesized, and a mini-bioreactor set up developed for culturing hybridoma cells to produce mAbs continuously. The hybridoma clone, 1B4A2D5, secreting anti-human serum albumin monoclonal antibodies, was immobilized in the cryogel matrix (2 discs, 18 mL bed volume). The hybridoma cells were attached to the matrix within 12 h after inoculation, and the cells were in the lag phase for seven days, where they were secreted mAb into the circulation medium. During the initial exponential phase, the glucose consumption, lactic acid production, and mAb production were 3.36 mM/day, 3.67 mM/day, and 55.61 µg/mL/day, respectively. The medium was refreshed whenever the glucose in the media went below 50% of the initial glucose concentration. The cryogenic reactor was run continuously for 25 days, and the mAb concentration reached a maximum on the 17th day at 310.59 µg/mL. The cumulative amount of mAbs produced in 25 days of running was 246 µg/mL, 7.7 times higher than the mAbs produced from T-flask batch cultivation. These results demonstrate that the developed polyhydroxyethylmethacrylate-based cryogel reactor can be used efficiently for continuous mAb production.

Engineered IgM and IgG cleaving enzymes for mitigating antibody neutralization and complement activation in AAV gene transfer

Systemic dosing of adeno-associated viral (AAV) vectors poses potential risk of adverse side effects including complement activation triggered by anti-capsid immunity. Due to the multifactorial nature of toxicities observed in this setting, a wide spectrum of immune modulatory regimens are being investigated in the clinic. Here, we discover an IgM cleaving enzyme (IceM) that degrades human IgM, a key trigger in the anti-AAV immune cascade. We then engineer a fusion enzyme (IceMG) with dual proteolytic activity against human IgM and IgG. IceMG cleaves B cell surface antigen receptors and inactivates phospholipase gamma signaling invitro. Importantly, IceMG is more effective at inhibiting complement activation compared with an IgG cleaving enzyme alone. Upon IV dosing, IceMG rapidly and reversibly clears circulating IgM and IgG in macaques. Antisera from these animals treated with IceMG shows decreased ability to neutralize AAV and activate complement. Consistently, pre-conditioning with IceMG restores AAV transduction in mice passively immunized with human antisera. Thus, IgM cleaving enzymes show promise in simultaneously addressing multiple aspects of anti-AAV immunity mediated by B cells, circulating antibodies and complement. These studies have implications for improving safety of AAV gene therapies and possibly broader applications including organ transplantation and autoimmune diseases.

Half-life extension of single-domain antibody-drug conjugates by albumin binding moiety enhances antitumor efficacy.

Single-domain antibody-drug conjugates (sdADCs) have been proven to have deeper solid tumor penetration and intratumor accumulation capabilities due to their smaller size compared with traditional IgG format ADCs. However, one of the key challenges for improving clinical outcomes of sdADCs is their abbreviated in vivo half-life. In this study, we innovatively fused an antihuman serum albumin (αHSA) nanobody to a sdADCs targeting oncofetal antigen 5T4, conferring serum albumin binding to enhance the pharmacokinetic profiles of sdADCs. The fusion protein was conjugated with monomethyl auristatin E (MMAE) at s224c site mutation. The conjugate exhibited potent cytotoxicity against various tumor cells. Compared with the nonalbumin-binding counterparts, the conjugate exhibited a 10-fold extended half-life in wild-type mice and fivefold prolonged serum half-life in BxPC-3 xenograft tumor models as well as enhanced tumor accumulation and retention in mice. Consequently, n501-αHSA-MMAE showed potent antitumor effects, which were comparable to n501-MMAE in pancreatic cancer BxPC-3 xenograft tumor models; however, in human ovarian teratoma PA-1 xenograft tumor models, n501-αHSA-MMAE significantly improved antitumor efficacy. Moreover, the conjugate showed mitigated hepatotoxicity. In summary, our results suggested that fusion to albumin-binding moiety as a viable strategy can enhance the therapeutic potential of sdADCs through optimized pharmacokinetics.

A new trivalent recombinant protein for type 2 diabetes mellitus with oral delivery potential: design, expression, and experimental validation

Glucagon-like peptide-1 (GLP-1) receptor agonists are increasingly used in clinical practice for the management of type 2 diabetes mellitus. However, the extremely short half-life of GLP-1 and the need for subcutaneous administration limit its clinical application. Thus, half-life extension and alternative delivery methods are highly desired. DARPin domains with high affinity for human serum albumin (HSA) have been selected for the half-life extension of therapeutic peptides and proteins. In the present study, novel trivalent fusion proteins as long-acting GLP-1 receptor agonists with potential for oral delivery were computationally engineered by incorporating a protease-resistant modified GLP-1, an anti-human serum albumin DARPin, and an approved cell-penetrating peptide (Penetratin, Tat, and Polyarginine) linked either by rigid or flexible linkers. Theoretical studies and molecular dynamics simulation results suggested that mGLP1-DARPin-Pen has acceptable quality and stability. Moreover, the potential affinity of the selected fusion proteins for GLP-1 receptor and human serum albumin was explored by molecular docking. The recombinant construct was cloned into the pET28a vector and expressed in Escherichia coli. SDS-PAGE analysis of the purified fusion protein matched its molecular size and was confirmed by western blot analysis. The results demonstrated that the engineered fusion protein could bind HSA with high affinity. Importantly, insulin secretion assays using a mouse pancreatic β-cell line (β-TC6) revealed that the engineered trivalent fusion protein retained the ability to stimulate cellular insulin secretion. Immunofluorescence microscopy analysis indicated the CPP-dependent cellular uptake of mGLP1-DARPin-Pen. These findings demonstrated that mGLP1-DARPin-Pen is a highly potent oral drug candidate that could be particularly useful in the treatment of type 2 diabetes mellitus. Communicated by Ramaswamy H. Sarma

COULD A DECELLULARIZATION PROTOCOL BE VALIDATED WITHOUT TALKING ABOUT HUMAN LEUKOCYTE ANTIGEN?

Decellularization techniques have advanced to reduce the risk of immune rejection in transplantation. Validation of these protocols typically relies on Crapo's criteria1, which include the absence of visible nuclei and low DNA content. In our study, five decellularization protocols were compared to determine the optimal approach for human fascia lata (HFL) samples. However, our findings raised questions as to why recipients can still develop immunity despite meeting validation criteria.HFL samples were decellularized using four protocols with SDS-Triton X100-DNase (D1 to D4-HFL) and one protocol using solvent-detergent-based baths (D5-HFL). The decellularized samples (D-HFL) were compared to native samples (N-HFL) using histology, and DNA content was measured. The human leukocyte antigen (HLA) content within the matrix was assessed using western blot analysis. Both D-HFL and N-HFL samples, along with negative control patches, were implanted in the backs of 28 Wistar rats. Anti-human IgG serum levels were evaluated after one month.H&E and Hoechst staining revealed the absence of residual cells in all decellularization protocols. DNA content was consistently below the critical threshold (p<0.05). All implanted D-HFL samples resulted in significantly lower anti-human IgG levels compared to N-HFL (p<0.01). However, 2.5 out of 4 rats developed immunity after being implanted with D1 to D4-HFL, with varying levels of anti-human IgG. Only rats implanted with D5-HFL showed undetectable levels of IgG and were considered non-immunized. Western blot analysis indicated that only D5-HFL had a residual HLA content below 1%.The literature on decellularization has primarily relied on Crapo's criteria, which do not consider the role of HLA mismatch in acute immune rejection. Our results suggest that a residual HLA content below 1% should also be considered to prevent immunization, even if other validation criteria are met. Further research is needed to evaluate the impact of residual HLA levels on human allotransplantation outcomes.

Pharmacological profiles and clinical efficacy of ozoralizumab (Nanozora® 30 ‍mg Syringes for S.C. Injection), the first Nanobody® compound in Japan

Ozoralizumab, a novel TNF inhibitor, is the first NANOBODY® compound in Japan for rheumatoid arthritis. This compound consists of a humanized fusion protein with a trimeric structure having two anti-human TNFα NANOBODY® molecules and one anti-human serum albumin NANOBODY® molecule, and has the unique structure without an Fc portion. Ozoralizumab showed an inhibitory effect on TNFα-induced cell death, and its inhibitory concentration was lower than that of etanercept, adalimumab, and infliximab. Ozoralizumab also showed inhibitory effects on human TNFα-induced cellular infiltration in the murine air pouch model and reduced the arthritis scores in a murine rheumatoid arthritis model. In addition, ozoralizumab showed distinctive pharmacological characteristics different from the traditional IgG antibodies, which may be attributed to its unique structure, such as its ability to rapidly distribute to inflamed joint tissues in a murine rheumatoid arthritis model, and its immune complexes with TNFα do not induce inflammation in a murine subcutaneous inflammation model. In clinical trials, ozoralizumab demonstrated clinical efficacy in rheumatoid arthritis patients with inadequate response to methotrexate, which was observed from day 3 of treatment. Ozolalizumab also showed improvements in clinical symptoms in rheumatoid arthritis patients without methotrexate. The safety profile of the compound was not significantly different from that of current TNF inhibitors. Based on these results, ozoralizumab was approved in September 2022. Ozoralizumab shows early improvement of clinical symptoms in patients with rheumatoid arthritis, and its characteristic structure is expected to be new treatment options for patients who have an inadequate response to current bDMARDs.

DISCOVERY OF NOVEL ANTI-SERUM ALBUMIN VHH AS A BUILDING BLOCK FOR PK PROLONGATION

Abstract Background The serum half-life of endogenous albumin is approximately 19 days in humans and 1.5-2.5 days in rodents. This extended half-life in vivo is primarily due to effective recycling upon internalization mediated by the neonatal Fc receptor (FcRn). Many protein therapeutics smaller than 60 kD have short serum half-life, which can be extended by fusing the proteins of interest with an anti-albumin antibody. The fusion proteins then take advantage of FcRn-mediated recycling. Single domain antibody (VHH) molecules (around 15 kD), derived from camelid heavy-chain-only IgG, are attracting increased attention globally in the field of antibody-based therapies due to several features including small size, high stability, low immunogenicity, good tissue penetration and cost effectiveness in manufacturing. Methods an anti-human/cynomolgus monkey/murine/canine/feline serum albumin VHH lead was discovered from a proprietary, large native phage-displayed VHH library, which was then humanized, and affinity matured. The optimized VHH was fused to bispecific and trispecific antibodies for in vivo PK studies. Results An anti-human serum albumin VHH Ab was discovered, and it also bound to cyno and mouse serum albumin with high affinity without affecting the interaction of HSA with FcRn. The VHH showed good developability and, once fused to protein therapeutics, long half-life and anti-tumor activity in rodents. Conclusions a novel VHH against serum albumin of different species was discovered from native VHH libraries, and it can be easily assembled into bispecific Abs and multispecific Abs to prolong the molecules’ PK profile.

Structural, nonclinical, and clinical features of ozoralizumab: A novel tumour necrosis factor inhibitor.

Tumour necrosis factor (TNF) inhibitors are currently the most widely used biological agents to treat rheumatoid arthritis. Ozoralizumab (OZR), a novel TNF inhibitor, is an antibody using variable heavy-chain domains of heavy-chain antibody (VHHs) and became the first VHH drug approved for the treatment of rheumatoid arthritis in September 2022. VHHs isolated from camelid heavy-chain antibodies can bind antigens with a single molecule. OZR is a trivalent VHH that consists of two anti-human TNFα VHHs and one anti-human serum albumin (anti-HSA) VHH. This review summarizes OZR's unique structural characteristics and nonclinical and clinical data. The clinical data outline the pharmacokinetics, efficacy, relationship between efficacy and pharmacokinetics, and safety of OZR, focusing on a Phase II/III confirmatory study (OHZORA trial).

Antigenic mapping of enterovirus A71 from Taiwan and Southeast Asia

Enterovirus A71 (EV-A71) is a non-enveloped virus possessing 4 capsid proteins: VP1-VP4. The outermost capsid protein, VP1, plays roles in both antigenicity and virulence of the virus. The concept of generating other EV-A71 genotypes of reverse genetics (rg) viruses by replacing VP1 can be made possible with synthetic biotechnology, allowing us to redesign organisms, creating unavailable ones. To determine suitable vaccine candidates against EV-A71 infections, we combined synthetic biotechnology, rg-virus production and high-fidelity determinants to produce genetically stable viruses. With the use of antigenic cartography, we are able to view the antigenic distance among various points. We analyzed and generated various EV-A71 VP1 sequences from Taiwan and Southeast Asian (SEA) countries, which were then used to produce recombinant rg-viruses and the viral proteins were purified for immunization of mice and rabbits. Antisera against various EV-A71 genotypes were used in neutralization assays against various Taiwan and SEA EV-A71 genotypes. Based on neutralization data from mice and rabbit antisera, we found that antisera produced from several genotypes were able to effectively neutralize the various Taiwan and SEA EV-A71 genotypes. Additionally, comparing the antigenic maps produced from mouse, rabbit and human antisera against different EV-A71 genotypes, a difference in clustering was seen and the spacing between points also differed. Based on antigenic mapping and neutralizing activities, B4 7008-HF and C4 M79 may be good potential vaccine candidates against EV-A71.

Discovery and characterization of hydroxylysine O-glycosylation in an engineered IL-2 fusion protein

In the present study, an engineered interleukin-2 (IL-2) fusion protein consisting of an anti-human serum albumin nanobody linked by ASTKG and a (G4S)2 linker to IL-2 was constructed. Liquid chromatography-mass spectrometry (LC-MS) characterization was performed on the intact molecule and at the peptide level. The LC-MS molecular mass analysis for the engineered fusion protein showed the appearance of unreported +340 Da peaks, apart from the expected O-glycosylation-related peaks in the IL-2 domain. Through a combination analysis of a K120R mutated molecule (The lysine at the position of 120 was mutated to arginine while the rest amino acid sequence remain unchanged), the possibility of a non-cleaved valine-histidine-serine signal peptide was ruled out and the presence of hydroxylysine (HyK) O-glycosylation in the ASTKG linker was confirmed. HyK O-glycosylation have been reported in other proteins such as collagen, which occurs in the conserved Gly-Xaa-HyK motif and is catalyzed by lysyl hydroxylase-3 complex. The present study showed high similar conserved motif of HyK-O-glycosylation in collagen, implying the HyK O-glycosylation in the engineered IL-2 possibly was catalyzed by the Chinese hamster ovary homolog of enzymes promoting HyK O-glycosylation in collagen. Bioactivity testing results revealed that HyK-O-glycosylation had no obvious effect on the in vitro activity of engineered IL-2. Our study is the first to report HyK-O-glycosylation modifications in therapeutic proteins through LC-MS characterization and in vitro activity analysis, which expands the scope of post-translational modification knowledge of therapeutic proteins.

Efficacy of Pgp3 vaccination for Chlamydia urogenital tract infection depends on its native conformation.

Urogenital tract infections with Chlamydia trachomatis have frequently been detected among patients diagnosed with sexually transmitted infections, and such infections lead to inflammatory complications. Currently, no licensed chlamydial vaccine is available in clinical practice. We previously reported that immunization with recombinant C. trachomatis plasmid-encoded virulence factor Pgp3 provided cross-serovar protection against C. muridarum genital tract infection. Because Pgp3 is a homotrimer and human antisera only recognize the trimeric form of Pgp3, we compared the effects of the native conformation of Pgp3 (trimer) and heat-denatured Pgp3 (monomer) to determine whether the native conformation is dispensable for the induction of protective immunity against chlamydial vaginal challenge. Both Pgp3 trimer and monomer immunization induced corresponding specific antibody production, but only trimer-induced antibody recognized endogenous Pgp3, and trimer-immunized mouse splenocytes showed the highest IFN-γ production upon restimulation with the chlamydial elementary body or native Pgp3 in vitro. Importantly, only Pgp3 trimer-immunized mice showed shortened lower genital tract chlamydial shedding and decreased upper genital tract pathology. Thus, Pgp3-induced protective immunity against Chlamydia urogenital tract infection is highly dependent on the native conformation, which will guide the design of Pgp3-based polypeptides and multi-subunit chlamydial vaccines.

An Initial Injection and a Crossover Injection of Amniotic Suspension Allograft Following Failed Treatment with Hyaluronic Acid or Saline Are Equally Effective in the Treatment of Moderate Symptomatic Knee Osteoarthritis Over 12 Months

The purpose of this crossover study was to determine the efficacy of amniotic suspension allograft (ASA) for moderate symptomatic knee osteoarthritis following failed treatment with hyaluronic acid (HA) or saline through 12 months' postcrossover injection using patient-reported and safety outcomes. In this multicenter study, 95 patients from a 200-patient single-blind randomized controlled trial were eligible to crossover and receive a single injection of ASA 3 months after failed treatment with HA or saline. Patient-reported outcomes, including Knee Injury and Osteoarthritis Outcome Score (KOOS) and visual analog scale (VAS), were collected out to 12 months postcrossover to determine pain and function. Radiographs and blood were collected for assessment of changes. Statistical analyses were performed using mixed effects model for repeated measures. Treatment with ASA following failed treatment with HA or saline resulted in significant improvements in KOOS and VAS scores compared with crossover baseline. There were no differences in radiographic measures or anti-human leukocyte antigen serum levels compared with baseline and no severe adverse events reported. In addition, more than 55% of patients were responders at months 3, 6, and 12 as measured by the Outcome Measures in Arthritis Clinical Trials-Osteoarthritis Research Society International simplified responder criteria. There were no significant differences between the original ASA randomized group and crossover cohorts at any of the time points evaluated, suggesting that prior failed treatment with HA or saline did not significantly impact outcomes following treatment with ASA. This study showed that patients who previously failed treatment with HA or saline had statistically significant improvements in pain and function scores following a crossover injection of ASA that was sustained for 12 months, as measured by KOOS and VAS. There were no serious adverse events reported, and the injection was safe. II, prospective cohort study.

Statement of Retraction: Combination therapy with Nab-paclitaxel and the interleukin-15 fused with anti-human serum albumin nanobody as a synergistic treatment for colorectal cancer

This article refers to:Combination therapy with Nab-paclitaxel and the interleukin-15 fused with anti-human serum albumin nanobody as a synergistic treatment for colorectal cancer

AbSE Workflow: Rapid Identification of the Coding Sequence and Linear Epitope of the Monoclonal Antibody at the Single-cell Level.

Monoclonal antibody (mAb) has been widely used in immunity research and disease diagnosis and therapy. Antibody sequence and epitope are the prerequisites and basis of mAb applications, which determine the properties of antibodies and make the preparation of antibody-based molecules controllable and reliable. Here, we present the antibody sequence and epitope identification (AbSE) workflow, a time-saving and cost-effective route for rapid determination of antibody sequence and linear epitope of mAb even at the single-cell level. The feasibility and accuracy of the AbSE workflow were demonstrated through the identification and validation of the coding sequence and epitope of antihuman serum albumin (antiHSA) mAb. It can be inferred that the AbSE workflow is a powerful and universal approach for paired antibody-epitope sequence identification. It may characterize antibodies not only on a single hybridoma cell but also on any other antibody-secreting cells.

RETRACTED ARTICLE: Combination therapy with Nab-paclitaxel and the interleukin-15 fused with anti-human serum albumin nanobody as a synergistic treatment for colorectal cancer

Statement of Retraction We, the Editors and Publisher of the journal Bioengineered, have retracted the following article: Lipei Wu, Weiwei Wang, Jiale Tian, Chunrun Qi, Zhengxin Cai, Wenhui Yan, Shihai Xuan & Anquan Shang. Combination therapy with Nab-paclitaxel and the interleukin-15 fused with anti-human serum albumin nanobody as a synergistic treatment for colorectal cancer. Bioengineered. 2022; 13(1): 1942-1951. DOI: 10.1080/21655979.2021.2023997 Since publication, it has been brought to the attention of the Editors and Publisher that this article includes substantial similarity with another published article: Hsu, F-T, Tsai, CL, Chiang, I-T, Lan, K-H, Yueh, P-F, Liang, W-Y, Lin, C-S, Chao, Y, & Lan, K-L. Synergistic effect of Abraxane that combines human IL15 fused with an albumin-binding domain on murine models of pancreatic ductal adenocarcinoma. Journal of Cellular and Molecular Medicine. 2022; 26: 1955– 1968. doi:10.1111/jcmm.17220 The previous article is not referenced at any point within the Bioengineered article. Further investigations conducted by the Publisher have raised significant concerns about the integrity of the data and results reported in the article due to the choice of in vivo and in vitro models, as well as the lack of original data to support the quantifications presented. When approached for an explanation, the authors were unable to provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. The corresponding author listed in this publication has been informed. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Seasonal Prevalence and Host Preference of Some Medically Important Aedes Species of Doon Valley, India

In the recent past, several outbreaks of mosquito-borne diseases in Dehradun city make it necessary for a comprehensive understanding of man-vector contacts, disease transmission and determination of appropriate resting sites for adult mosquitoes. The study was conducted within the periphery of Dehradun city in Doon valley, Uttarakhand state, India consisting of eight (08) locations namely Sahaspur, Premnagar, Raipur, Karanpur, Sahastradhara, Dudhli, Doiwala and Rani Pokhri with the objective to analyze and assess the seasonal prevalence and host choice of the mosquitoes with special reference to Aedes species. Among the eight locations of the current study, Raipur registered the highest mosquito abundance (17%) trailed by Sahaspur (16%) and Karanpur (15%). A low abundance of mosquitoes was registered conjointly from Doiwala and Rani Pokhri areas (9% from each area). In these areas, the profile plots of the 2-way ANOVA displayed the highest variation for the data collected from human habitations followed by bovine sheds, whereas the lowest recorded variation was from the poultry. With regards to seasonal prevalence, the highest mosquito abundance was registered during the monsoon followed by the post-monsoon. In provender analysis, a total of 1912 blood meals were collected for antibody test among which 1851 were found positive, with the highest reaction with human antisera (57.75%), followed by bovine antisera (21.34%) and pigs antisera (4.97%). Ae. aegyptihad the highest Anthropophilic Index (A.I.) with 68.78%, followed by Ae. albopictus (64.89%) and Ae. vittatus (55.55%). However, Human Blood Predominance (H.B.P.) and Pure Human Blood Pervasiveness (P.H.B.P.) were found highest for Ae. albopictus with values 0.43 and 0.44 respectively. The results also show the seasonal prevalence and feeding habits of these vectors. This study would be helpful in formulating season-wise strategies for the control of Aedes vector mosquitoes in different eco-epidemiological situations.

Toward the development of a label-free multiple immunosensor based on thin film transistor microelectrode arrays

A label-free immunosensor using thin film transistor microelectrode arrays (TFT-µEAs) derived from flat-panel technology has been developed for the first time. The TFT-µEAs used here incorporate a large sensing area of 15 mm × 15 mm with a 150 × 150 array of transparent indium tin oxide (ITO) electrodes placed at a 100 μm pixel pitch. Anti-human serum albumin (HSA) IgG was characterized to be immobilized on functionalized ITO electrodes. HSA, which is one of the indexes for liver functions, was successfully detected by recording the change of real-time current based on the change in the electric double layer capacitance before and after conjugation of HSA with anti-HSA IgG. Ionic strength and pH value of HSA solution were optimized. In 0.01 mM phosphate buffered saline with pH 6.0, linear range of HSA was determined as 0.3–300 pM. The present TFT-µEAs are applicable to a label-free multiple immunosensing by patterning antibodies on its surface.

A resource of high-quality and versatile nanobodies for drug delivery

SummaryTherapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs “Duraleukin” and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs.

SERS-based biosensing platform using shape-coded hydrogel microparticles incorporating silver nanoparticles

Biosensors based on surface-enhanced Raman scattering (SERS) represent an efficient tool that can quickly detect even very low concentrations of the target molecule. Recent SERS-based biosensing has been moving toward a multiplex detection platform to analyze samples containing a variety of molecules, such as blood and urine. In this study, shape-coded suspension arrays have been developed for SERS-based multiplex biosensing. The suspension microarrays were fabricated using different shapes of poly(ethylene glycol) (PEG)-based hydrogel microparticles, which were prepared by a simple photopatterning process. The resultant hydrogel particles were coated with silver nanoparticles by a silver mirror reaction and then divided into two groups depending on the shape. As a proof of concept, square-shaped particles were coated with 4-mercaptophenylboronic acid by self-assembly to detect glucose, while circular particles were functionalized with anti-human serum albumin (HSA) to detect HSA. The assay was performed in the concentration range of 1 pg/mL to 1 μg/mL for both glucose and HSA without crosstalk. Target selectivity was also confirmed from Raman mapping, which shows that each group of particles reveals a different pattern. Moreover, we verified the potential of our method in multiplex immunoassays by quantifying two different antigens using SERS nanotags. The performance of the developed SERS-based multiplex immunoassay platform was demonstrated by the detection of a very low concentration of biomolecules with excellent selectivity.