Anti-human IgG Antibody Research Articles

Neutralizing and Non-Neutralizing Anti-FVIII Antibodies in Black and White Hemophilia A Subjects: A Natural History Profile

The most common complication in hemophilia A (HA) treatment, affecting 25-30% of severe HA patients, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 gene haplotypes H1-H5 are defined by combinations of nonsynonymous SNPs encoding FVIII sequence variants D1241E, M2238V and R484H. F8 haplotypes H2-H5 are more prevalent in individuals with black African ancestry, while >90% of the white population has the H1 haplotype. This study used a validated Luminex-based assay to determine total anti-FVIII antibody titers in plasma from 395 HA (189 black, 206 white) and 23 non-HA control subjects, measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3 and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic Bethesda assay with the Nijmegen modification. Linear B-cell epitopes recognized by antibodies in human plasma samples were characterized using commercial peptide microarrays with imprinted 15-mer peptides spanning the FVIII A1, A2, C1 and C2 domains, with binding interactions detected using fluorescent-labeled anti-human IgG antibodies. Neither total nor inhibitory antibody titers correlated with F8 haplotype. FVIII peptides with the D1241E and M3348V polymorphisms showed low antibody reactivity, indicating they do not comprise linear B-cell epitopes. Similarly, antibodies from subjects with H3 and H5 haplotypes, who were necessarily infused with FVIII products having a different haplotype than that of their endogenous, (dysfunctional) F8 sequence, did not show haplotype-correlated differential binding to the three BDD-FVIII or full-length FVIII proteins, indicating the polymorphic M2238V or D1241E sites do not correspond to immunodominant, conformational B-cell epitopes. Interestingly, the BDD-FVIII proteins were significantly more reactive with antibodies in plasma than were two commercial full-length recombinant FVIII products. Overall, results of this study indicated that low-titer FVIII-reactive antibodies are readily detected in most HA subjects and in a majority of healthy non-HA controls. The observed stronger immunoreactivity of BDD-FVIII suggests that B-domain removal exposes novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains. Disclosures Pratt: Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding. Peters:Sanofi: Employment. Mann:Haematologic Technologies: Other: Owner; Stago: Consultancy; Novo Nordisk: Consultancy; Takeda: Consultancy; Shire: Consultancy; Baxalta: Consultancy.

Identification of Dengue Fever Markers by Dot Immunoasay

The aim . Development of diagnostic kit for identifying markers of dengue fever at all stages of the disease. Materials and methods . In blood serum from patients with suspected dengue fever, NS1 antigen and specific IgM and IgG were detected by immune chromatography, dot immunoassay, enzyme-linked immunosorbent assay, using commercial test systems, as well as the “Dengue Spectrum” experimental kit. Results and discussion . A diagnostic kit has been developed for the detection of dengue fever markers, based on the mechanism of simultaneous differential detection of the agent NS1 protein and IgM and IgG class antibodies with the formation of specific complexes between markers from the test sample and known capture immune reagents, in a certain order, discretely fixed on a dense substrate. It was found that the effective detection of specific IgG and IgM to dengue virus can be carried out according to the scheme in which IgG is captured on the total antigen of the virus with detection using labeled anti-human IgG antibodies, and IgM is detected by capture on anti-human IgM antibodies with detection using total viral antigen. Detection of dengue virus NS1 protein can be performed using a substrate with immobilized monoclonal antibodies to NS1 and a gold immune sol bound to antibodies against NS1. This protocol of the dot analysis provides the limit for determining the recombinant analogue of the NS1 protein equal to 100 ng/ml. Comparative testing of the kit against the panel of clinical samples showed a good agreement between the results and the data obtained using imported commercial tests. The developed kit can be used for screening clinical samples, both in laboratory and in the field.

Detection of Intragraft Anti-Blood Group A and B Antibodies Following Renal Transplantation

BackgroundGraft immunocomplex capture fluorescence analysis is an attractive method to detect intragraft donor-specific anti-HLA antibodies. In ABO-incompatible transplantation, anti-A and B antibodies are also considered to be important donor specific antibodies (ABO-DSA). Therefore, it is useful to monitor intragraft ABO-DSAs to assess antibody-mediated rejection. MethodsTo capture A and B antigens, anti-Band III, von Willebrand factor (VW), and plasmalemma vesicle-associated protein (PLVAP) beads were produced. The allograft specimen was homogenized in a lysis buffer. Subsequently, A and B antigens were captured by anti-Band III, VW, or PLVAP beads. The immune complexes were then detected by phycoerythrin-conjugated anti-human IgG antibodies and analyzed using a Luminex system. ResultsAlthough Band III and VW beads yielded false positives and false negatives, PLVAP beads captured A and B antigens with high sensitivity (91.7%) and specificity (100%) when an index > 1.5 was considered positive. The proximity in A and B antigens and PLVAP expression was confirmed using immunohistochemical evaluation. Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis supported that PLVAP is an A and B antigen carrier protein. Case ReportBiopsies were conducted following an ABO-incompatible renal transplant (type A to O) and evaluated for ABO-DSA. Graft immunocomplex capture fluorescence analysis was demonstrated as follows: 3.19 (1 h, serum creatinine [s-Cr] 3.95 mg/dL, titer IgG 1:512, glomerulitis [g] 0, peritubular capillaritis [ptc] 0, complement 4d [C4d] 1); 1.8 (4 d, s-Cr 2.29 mg/dL, titer 1:256, g 0, ptc 0, C4d 3); 1.2 (22 d, s-Cr 1.58 mg/dL, titer 1:128, g 0, ptc 2, C4d 3). This result indicated that the remnant ABO-DSA were adsorbed and subsequently removed from the allograft successfully. ConclusionsThis novel application could be used to detect intragraft ABO-DSAs, which could lead to a correct diagnosis and shed light on the ABO-DSA kinetics following ABO-incompatible transplantation.

Abstract 4521: Flow cytometric measurement of increased erythrocyte bound IgG: A potential screening method for colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) incidence is increasing worldwide. While screening for CRC has led to early detection, thus reducing mortality, checking for fecal occult blood, as the accepted screening method for CRC worldwide, has some shortcomings including false-negative results in early disease stages and/or advanced CRC of the right ascending colon. We have previously reported on the ectopic expression of Band3 (encoded by SLC4A1), an antigen abundant in the cytoplasmic membrane of erythrocytes, in a case of CRC with autoimmune hemolytic anemia (AIHA), and suggested that Band3 expression in CRC might stimulate production of the autoantibody that binds to the erythrocyte membrane. Besides, the amount of erythrocyte-bound IgG may be higher in CRC patients compared with healthy people, even though no symptoms or signs of AIHA are seen. We conducted a prospective observational study to compare the amount of erythrocyte-bound IgG, including anti-Band 3 antibody, between CRC patients and healthy controls. Patients and Methods: From October 2016 to November 2017, 30 CRC patients and six healthy controls were enrolled. Direct Coombs test was carried out, using test tube and column methods, immediately after blood collection. Cell suspensions were incubated with either anti-human IgG antibody or a control isotype conjugated with FITC. Fluorescence was measured with a flow cytometer. Mean fluorescence intensity (MFI) was calculated as the difference in fluorescence intensity between anti-human IgG and control isotypes. Total serum IgG was collected using protein G sepharose. The amount of serum erythrocyte-bound IgG was quantified by ELISA using erythrocyte membrane protein coated 96-well plates and anti-human IgG antibody. Erythrocyte membrane protein was immunoprecipitated and subjected to immunoblotting using anti-Band3 IgG to confirm its proportion to total serum IgG. Results: Clinical AIHA was not observed in any patient, but a direct Coombs test was positive in eight patients (one on the test tube method, five on the column method, and two patients on both methods). The median±SD MFI was 40±17.4 in patients with CRC and 23±11.6 in controls (p=0.005). Moreover, with the MFI cutoff value set at 31, ROC curve analysis showed the sensitivity, specificity, and area under the curve to be 100%, 70% and 0.867, respectively. In CRC patients, the median MFI was not affected by positive or negative results for fecal occult blood, involvement of the left or right colon, and/or disease stage. The serum erythrocyte-bound IgG and the proportion of anti-Band3 to total serum IgG was higher in CRC patients than in controls. Conclusion: Whether flow cytometric measurement of total erythrocyte-bound IgG, including anti-Band 3 antibody, in CRC patients can be a useful screening tool warrants further evaluation in a larger cohort. Citation Format: Akihito Kitao, Shinichiro Kawamoto, Keiji Kurata, Ikuyo Hayakawa, Hiroshi Matsuoka, Yasuo Sumi, Yoshihiro Kakeji, Toyomi Kamesaki, Hironobu Minami. Flow cytometric measurement of increased erythrocyte bound IgG: A potential screening method for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4521.

A rapid point-of-care test for dengue virus-1 based on a lateral flow assay with a near-infrared fluorescent dye

Dengue fever is caused by the dengue virus (DENV), and DENV1 is the prevalent epidemic serotype in south China. A new lateral flow assay (LFA) based on a near-infrared (NIR) fluorescent dye was developed to detect anti-DENV1 IgG antibodies. DyLight-800 was used as the marker conjugated to goat anti-human IgG antibodies, and recombinant dengue type 1 envelope protein was used as the capture protein on the test line. Twenty samples from patients infected with DENV1 and 160 negative controls were analyzed using this new NIR-LFA. The results of the NIR-LFA were compared with the results of Panbio Dengue IgG ELISA and the Dengue Duo IgM/IgG Cassette. Nineteen confirmed DENV1-positive samples were identified by NIR-LFA, giving 95% (19/20) sensitivity. No significant differences existed in the results when the 20 primary clinical samples were analyzed using NIR-LFA, Panbio ELISA, and the Dengue Duo Cassette. However, NIR-LFA had a lower limit of detection than IgG ELISA and Duo IgM/IgG Cassette did when analyzing a 2-fold dilution series of the 19 samples positively identified by NIR-LFA. When incorporated with an NIR POCT device, the new NIR-LFA was rapid, easy to use, and highly sensitive in detecting DENV1, and has potential for application to clinical diagnosis.

Influences of Pre-formed Donor-Specific Anti–Human Leukocyte Antigen Antibodies in Living-Donor Renal Transplantation: Results With Graft Immunocomplex Capture Fluorescence Analysis

BackgroundAdvances in immunosuppressants enable organ transplantation for sensitized patients. However, influences of pre-formed donor-specific anti–human leukocyte antigen (HLA) antibodies (DSA) have not been fully understood in renal transplantation (RT). On the other hand, immunocomplex capture fluorescence analysis (ICFA) is a reliable method to detect donor-specific anti-HLA antibodies and HLA antigen complexes. Graft ICFA can detect DSA in an allograft (g-DSA). MethodsTo elucidate the consequences of pre-formed DSA, 198 patients who underwent living-donor RT were enrolled for this study (observation period: 57.8 ± 34.9 months); 187 patients in the DSA− group (excluding ABO-incompatible cases) and 11 patients in the DSA+ group. Before RT, all DSA+ patients had undergone rituximab administration and plasmapheresis. For a graft ICFA, the biopsy specimen (1 × 105 cells) was dissolved, and HLA antigens were captured by anti-HLA beads. Finally, DSA-HLA complexes were detected by means of PE-conjugated anti-human IgG antibodies and analyzed by use of a Luminex system. A ratio (sample/blank beads, mean of fluorescence intensity) was calculated: ≥1.0 was determined as positive g-DSA. ResultsThere were no significant differences in 5-year graft survival (87.9%/100% in the DSA−/DSA+ groups, respectively). In terms of antibody-mediated rejection (AMR), within 1 month after RT, pathologically determined AMR occurred 3.2% and 63.4% in the DSA− and DSA+ groups, respectively (P < .0001). However, interestingly, more than half of them (57.1%) indicated only subclinical AMR, that is, no fluctuation of S-Cr. As representative of 2 cases of subclinical AMR, g-DSA deposition could be confirmed (1.15 ± 0.04) at 1 hour after reperfusion by graft ICFA. Furthermore, g-DSA shifted to 2.20 ± 0.98 at 3 weeks after transplantation, along with a decline in s-DSA mean of fluorescence intensity (1718-506.5). ConclusionsAlthough pathologically determined AMR occurred more frequently in pre-formed DSA+ recipients, it can be argued that a successful de-sensitization protocol inhibits further production of DSA and graft destruction.

Quantitative Detection of Weak D Antigen Variants in Blood Typing using SPR

Modern techniques for quantifying blood group antibody-antigen interactions are very limited, especially for weaker interactions which result from low antigen expression and/or partial expression of the antigen structure. Surface plasmon resonance (SPR) detection is often used to monitor and quantify bio-interactions. Previously, a regenerable, multi-fucntional platform for quantitative RBC phenotyping of normal antigen expression using SPR detection was reported. However, detection of weaker variants were not explored. Here, this sensitivity study used anti-human IgG antibodies immobilized to a gold sensor surface to two clinically important types of weaker D variants using SPR; weak D and partial D. Positive pre-sensitised cells bind to the anti-human IgG monolayer, and the response unit (RU) is reported (>100 RU). Unbound negative cells are directly eluted (<100 RU). Weak D cells were detected between a range of 180–580 RU, due to a lower expression of antigens. Partial D cells, category D VI, were also positively identified (352–1147 RU), similar to that of normal D antigens. The detection of two classes of weaker D variants was achieved for the first time using this fully regenerable SPR platform, opening up a new avenue to replace the current subjective and arbitrary methods for quantifying blood group antibody-antigen interactions.

Identification of a host cell protein impurity in therapeutic protein, P1

Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification. In the current study, we identified the presence of CHO-catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell proteins. The CHO-catalase protein has been characterized using a combination approach of one-dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8 in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was further confirmed by western blot analysis using a specific anti-catalase antibody.

Graft Immunocomplex Capture Fluorescence Analysis to Detect Donor-Specific Antibodies and HLA Antigen Complexes in the Allograft

ABSTRACTBackground: Immunocomplex capture fluorescence analysis (ICFA) is an attractive method to detect donor-specific anti-HLA antibodies (DSA) and HLA antigen complexes. Currently, antibody-mediated rejection (AMR) due to DSA is usually diagnosed by C4d deposition and serological DSA detection. Conversely, there is a discrepancy between these findings frequently. Thereupon, our graft ICFA technique may contribute to establish the diagnosis of AMR.Methods: Graft samples were obtained by a percutaneous needle biopsy. Then, the specimen was dissolved in PBS by the lysis buffer. Subsequently, HLA antigens were captured by anti-HLA beads. Then, DSA–HLA complexes were detected by PE-conjugated anti-human IgG antibodies, where DSA had already reacted with the allograft in vivo, analyzed by a Luminex system.Results: A ratio (sample MFI/blank beads MFI) was calculated: ≥ 1.0 was determined as positive. We found that DSA–HLA complexes in the graft were successfully detected from only slight positive 1.03 to 79.27 in a chronic active AMR patient by graft ICFA. Next, positive graft ICFA had predicted the early phase of AMR (MFI ratio: 1.38) even in patients with no serum DSA. Finally, appropriate therapies for AMR deleted DSA deposition (MFI ratio from 0.3 to 0.7) from allografts.Conclusions: This novel application would detect early phase or incomplete pathological cases of AMR, which could lead to a correct diagnosis and initiation of appropriate therapies. Moreover, graft ICFA might address a variety of long-standing questions in terms of DSA.Abbreviations: AMR: Antibody-mediated rejection; DSA: Donor-specific antibodies; ICFA: Immunocomplex capture fluorescence analysis.

Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors.

The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG.

Confirmatory analysis of etanercept in equine plasma by LC-MS for doping control.

Etanercept is a protein-based medication for the treatment of human patients with rheumatoid arthritis and other autoimmune-based diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method for confirmation of etanercept in equine plasma. Etanercept was extracted from plasma by anti-human IgG antibody linked to magnetic beads. The analyte was reduced and alkylated, and then digested by trypsin. Tryptic peptides (T1 from human tumour necrosis factor receptor 2 of etanercept, T15 and T27 from human IgG1 of the protein) were employed for detection and confirmation of the analyte. The limit of detection was 5 ng/mL, and the limit of confirmation 10 ng/mL. This method is specific for confirmation of etanercept, as assessed using the results from BLAST and SEQUEST searches. The results from SEQUEST searches also revealed an unexpected unique specificity of product ion spectrum of IgG1 T27 with only a single product ion for identification of etanercept. It is the first report for such a finding, to the authors' knowledge. The method was successful in analyses of the plasma samples collected post administration of etanercept to horses. Etanercept was detected up to 11 days post administration. This method will be helpful for confirmation of etanercept or other protein-based drugs consisting of human IgG1, in equine drug testing. Copyright © 2016 John Wiley & Sons, Ltd.

A simultaneous two-colour detection method of human IgG- and IgE-reactive proteins from lactic acid bacteria

Whole cell extracts of two Lactobacillus strains were tested with primary antibodies from two pooled sera from allergic patients. Fluorescently labelled anti-human IgG and anti-human IgE secondary antibodies applied in Western blotting, together with an appropriate image acquisition protocol facilitated imagining bacterial proteins that reacted with human IgG and IgE.

Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality.

We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL(-1) for MMP-2 and 5.7 pg mL(-1) for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained.

A fluorescent imaging assay of cast in renal disease based on graphene quantum dots and Fe3O4 nanoparticles

BackgroundRenal disease has become a global public health problem. Cast is a useful disease marker of kidney injury and renal failure. Hence, a cast-targeted fluorescent imaging assay is developed for the laboratory diagnosis of renal disease. MethodsFirstly, graphene quantum dots (GQDs) were stripped from graphene oxide sheets and amine-modified. Then, anti-human IgG antibody was conjugated with Fe3O4 nanoparticles to identify the cast in urine. Furthermore, the modified GQDs were linked onto the surface of Fe3O4/anti-IgG nanocomposites. Lastly, this Fe3O4/GQD fluorescent probe was added into the sample to detect the cast through fluorescent imaging. ResultsPreliminary application of this probe in clinical detection showed that the common types of casts in urine (including RBC, WBC, fatty and granular casts) could be detected by this fluorescent imaging assay. The method has the advantages of fast speed, high sensitivity (lowest detection limit to 2casts/ml), good selectivity, and wide linear range (2–2000casts/ml). Regression analysis also showed that there was a good linear relationship (y=0.9495×+10.974, R2=0.9879) between the fluorescent counts and the casts in urine. ConclusionThis cast-targeted fluorescent imaging assay may be a potential method for the laboratory diagnosis of renal disease.

A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4+ and CD8+ T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8+ T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31+ intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

Adjunction of Avidin to a Cysteamine Self-Assembled Monolayer for Impedimetric Immunosensor

In this work an impedimetric immunosensor based on affinity immobilization method of a biotin labelled anti-human IgG antibody, used as a model system, was reported. The experimental procedure involves the growth of a self-assembled monolayer of a thiol (cysteamine) carrying terminal amine groups on gold electrodes. Glutarardehyde, a homobifunctional cross-linker, was used as a coupling reagent for the covalent linking of avidin to the amine groups of cysteamine. The attachment of the biotin labeled antibodies (anti-Human IgG) to the subsequent modified gold electrode was achieved by affinity interactions tacking advantage of the strong avidin-biotin bridge. The stepwise assembly process of the electrode was interrogated by means of cyclic voltammetry, electrochemical impedance spectroscopy and contact angle measurements. The response of the antibody modified electrode to their target antigens was investigated in the presence of BSA (bovin serum albumin) in order to alleviate non-specific adsorption problems. A proposed electrical model was used to analyse the experimental data. The resulting immunosensor has a linear dynamic range of 100 - 900 ng?ml–1 of antigen and a detection limit of 100 ng?ml–1.

Fourier plane colorimetric sensing using broadband imaging of surface plasmons and application to biosensing

We demonstrate an optical technique for refractive index and thickness sensing of sub-wavelength-thick dielectric analytes. The technique utilizes the broadband, multimode, directional leakage radiation arising from the excitation of hybrid mode surface plasmons (SP) on low aspect ratio periodic plasmonic substrates with period ≈λ. The approach requires relaxed fabrication tolerances compared to extra ordinary transmission-based sensing techniques, wherein minor shifts in the fabricated dimensions result in a very large change from the designed resonant wavelength. We show that refractive index perturbations due to about 10-nm-thick dielectric can be captured optically by the usage of carefully designed plasmonic substrates, a halogen lamp source, free-space optical components, polarizers, and a low-end, consumer-grade charge coupled device camera. The plasmonic substrates were designed for converting the signature of hybrid mode SP excitation into a transmission peak by utilizing a thin homogeneous metal layer sandwiched between the periodic plasmonic structures and the substrate. The resonance is highly sensitive to the refractive index and thickness of the analyte superstrate. The excitation of hybrid mode SP results in a polarization rotation of 90° of the leaked radiation at resonant wavelength. In order to eliminate the problem of image registration (i.e., placing the same feature in the same pixel of the image, for comparison before and after a change in refractive index) for sensing, we perform the color analysis in the Fourier plane. The change in color of the bright emitted spot with highest momentum, corresponding to the leakage of fundamental SP mode, was used to measure the changes in refractive index, whereas the number and color of spots of lower momenta, corresponding to higher-order Fabry Perot modes, was used to measure the variation in thickness. We further show that the Fourier plane analysis can also be used to sense the index of thicker dielectrics, where real plane image analysis may fail to sense index perturbations, simply due to superposition of different modes in the real plane images of such substrates. Control experiments and analysis revealed a refractive index resolution of 10–5 RIU. The results were correlated with simulations to establish the physical origin of the change in the fundamental mode and higher-order modes due to the refractive index and thickness of analyte. As a demonstration of an application and to test the limits of sensing, the substrates were used to image the surface functionalization using 2-nm-thick 11-mercaptoundecanoic acid and immobilization of 7-nm-thick mouse anti-human IgG antibody. In biological systems, where a priori knowledge about a process step is available, where accurate chemical composition testing is not necessary or possible, the presented method could be used to study the surface changes using a label-free sensing mechanism.

Naked-eye sensitive ELISA-like assay based on gold-enhanced peroxidase-like immunogold activity.

A naked-eye sensitive ELISA-like assay was developed based on gold-enhanced peroxidase-like activity of gold nanoparticles (AuNPs). Using human IgG (H-IgG) as an analytical model, goat anti-human IgG antibody (anti-IgG) adsorbed on microtiter plate and AuNPs-labeled anti-IgG acted as capture antibody and detection antibody, respectively. Because the surfaces of AuNPs were blocked by protein molecules, the peroxidase-like activity of AuNPs was almost inhibited, evaluated by the catalytic oxidation of peroxidase enzyme substrate 3,3',5,5'-tetramethylbenzidine (TMB), which could produce a bright blue color in the presence of H2O2. Fortunately, the catalytic ability of AuNPs was dramatically increased by the deposition of gold due to the formation of a new gold shell on immunogold. Under optimal reaction conditions, the colorimetric immunoassay presented a good linear relationship in the range of 0.7-100 ng/mL and the limit of detection (LOD) of 0.3 ng/mL calculated by 3σ/S for UV-vis detection, and obtained LOD of 5 ng/mL for naked-eye detection. The obtained results were competitive with conventional sandwich ELISA with the LOD of 1.6 ng/mL. Furthermore, this developed colorimetric immunoassay was successfully applied to diluted human serum and fetal bovine serum samples, and predicted a broad prospect for the use of peroxidase-like activity involving nanomaterials in bioassay and diagnostics.

Human IgG detection in serum on polymer based Mach-Zehnder interferometric biosensors.

We report a new method for detecting human IgG (hIgG) in serum on integrated-optical Mach-Zehnder interferometer biosensors realized in a high index contrast polymer material system. In the linear range of the sensor (5-200 nM) we observed excellent signal recoveries (95-110%) in buffer and serum samples, which indicate the absence of matrix effects. Signal enhancement was reached by using secondary anti-human IgG antibodies, which bind to immobilized target IgGs and allow detecting concentrations down to 100 pM. This polymer based optical sensor is fully compatible with cost-efficient mass production technologies, which makes it an attractive alternative to inorganic optical sensors. Graphical abstract of the hIgG measured on polymer based photonic sensors using a direct binding assay and a signal enhancement strategy with secondary antibodies.

A novel method for measuring TBAb activity in TSAb- and TBAb-positive serum.

We reported a conversion assay in which thyroid blocking antibody (TBAb) function as thyroid stimulating antibody (TSAb). TBAb-bound porcine thyroid cells (PTC) were made by incubating TBAb(+) serum and PTC for 1 hour. When these TBAb-bound PTC were incubated 4 h with rabbit anti-human (h) IgG antibody (Ab), cAMP production was high, but when incubated with normal rabbit serum (NRS) cAMP production was low. TBAb-Mnoclonal Ab (MoAb) (KI-70) showed similar conversion. However, when TSAb-MoAb(M22) was assayed, anti-hIgG Ab-produced cAMP was lower than NRS-produced cAMP. When a mixture of M22 and KI-70 was assayed, anti-hIgG Ab-produced cAMP was higher than NRS. Thus, it is possible to determine existence of TBAb in TSAb(+)serum when anti-IgG Ab-produced cAMP is higher than NRS-produced cAMP. In this assay TBAb activity in TSAb(+)serum was scored as positive, gray zone and negative when the difference [anti-hIgG Ab-produced cAMP(%)-NRS-produced cAMP(%)] was >100%, 50-100% and <±50%, respectively. In TSAb(+)sera of Graves' patients with no treatment or anti-thyroid therapy, positive TBAb was 9% (3/33 )and 6.9% (5/72), and gray zone was 18 % (6/33) and 25% (18/72), respectively. A low prevelance of TBAb and low TBAb activity (<200% as cAMP) was found in these Graves' patients. A radioisotope treated Graves' patient showed existence of both TSAb and TBAb at 5 months (NRS, 800% cAMP and anti-IgGAb,1,350% cAMP), and highly positive TBAb (NRS, 180% cAMP and anti-hIgG Ab, 3,200% cAMP) at 30 months. This conversion assay is useful principally for TBAb determination but is also useful for TBAb determination in TSAb(+)serum.