Specific Antibody Binding Research Articles

Abstract 4820: Pediatric preclinical testing consortium evaluation of the CD123 antibody drug conjugate, IMGN632, against xenograft models of pediatric acute lymphoblastic leukemia

Abstract Introduction: The characteristic expression of CD123 (the alpha subunit of the IL-3 receptor) in multiple hematological malignancies, including acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm and acute lymphoblastic leukemia (ALL) has made this antigen an attractive target for the development of new therapeutics. IMGN632 is a CD123 targeting antibody drug conjugate (ADC) comprised of a novel humanized antiCD123 antibody, G4723A, linked to a DNA monoalkylating payload of the indolindobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. IMGN632 demonstrates potent activity in AML samples at low concentrations with minimal impact on normal bone marrow progenitors, and antileukemia effects in AML xenograft models. A phase I clinical trial of IMGN632 in CD123 positive malignancies is ongoing (NCT033865). Therefore, it was of interest to test the in vivoefficacy of IMGN632 against preclinical models of pediatric ALL. Methods: Pediatric ALL patient-derived xenografts (PDXs) grew in an orthotopic manner in NSG mice. Engraftment was assessed by the % human leukemic blasts in the peripheral blood (%huCD45+). Treatment commenced when the median %huCD45+exceeded 1%, and mice received IMGN632 or non-binding control ADC (240 µg/kg IV weekly x 3) or vehicle. An event was defined as the %huCD45+>25% or leukemia-related morbidity. The Kaplan-Meier method was used to compare event-free survival (EFS) between treated (T) and control (C) groups. Stringent objective response measures were assigned to each mouse and reported as group medians. Cell surface CD123 levels were expressed as specific antibody binding capacity (sABC). IMGN632 was provided by ImmunoGen, Inc. Results: IMGN632 was tested against 8 pediatric ALL PDXs (3 B-ALL; 3 MLLr-ALL; 2 Ph+-ALL) in which median cell surface CD123 expression ranged from 510-3375 sABC. NSG mice tolerated IMGN632 well with maximum average weight losses of 0.0-7.7% across treatment groups compared to 0.0-8.1% in vehicle controls. IMGN632 induced significant differences in EFS distribution compared to control in 8 of 8 PDXs. T-C values ranged from 9.8 to 64.8 days (T/C 3.0-9.4) and Maintained Complete Responses were observed in 6 of 8 PDXs. In 6 of 8 PDXs the median EFS of the IMGN632-treated groups extended >5 weeks beyond the end of treatment. The control ADC did not significantly delay the progression of any PDX. There was no apparent correlation between CD123 sABC levels and in vivo activity with IMGN632 efficacy being observed across all CD123 expression levels. Conclusions: IMGN632 exerted profound in vivo efficacy against PDXs derived from a broad range of ALL subtypes and with a broad range of CD123 antigen levels. These data strongly support the clinical investigation of IMGN632 in acute lymphoblastic leukemia. (Supported by NCI Grants CA199222 & CA199000) Citation Format: Kathryn Evans, Narimanne El-Zein, Connor Jones, Stephen W. Erickson, Yuelong Guo, Beverly A. Teicher, Sharlene Adams, Patrick A. Zweidler-McKay, Malcolm A. Smith, Richard B. Lock. Pediatric preclinical testing consortium evaluation of the CD123 antibody drug conjugate, IMGN632, against xenograft models of pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4820.

Molecular docking explores heightened immunogenicity and structural dynamics of acetaldehyde human immunoglobulin G adduct.

Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.

Deciphering the domain specificity of C. difficile toxin neutralizing antibodies

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. The pathological effects of CDI are primarily attributed to toxins A (TcdA) and B (TcdB). Adequate toxin-specific antibody responses are associated with asymptomatic carriage, whereas insufficient humoral responses are associated with recurrent CDI. While the data supporting the importance of anti-toxin antibodies are substantial, clarity about the toxin domain specificity of these antibodies is more limited. To investigate this matter, combinations of human mAbs targeting multiple domains of TcdB were assessed using toxin neutralization assays. These data revealed that a combination of mAbs specific to all major toxin domains had improved neutralizing potency when compared to equivalent concentrations of a single mAb or a combination of mAbs against one or two domains. The function and toxin domain binding specificity of serum antibodies elicited by immunization of hamsters with a toxoid vaccine candidate was also assessed. Immunization with a toxoid vaccine candidate provoked toxin neutralizing antibodies specific to multiple domains of both TcdA and TcdB. When assessed in a toxin neutralization assay, polyclonal sera displayed greater activity against elevated concentrations of toxins than equivalent concentrations of individual mAbs. These data suggest a potential benefit of any antibody based therapeutic or prophylactic treatment that targets multiple toxin domains.

Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL−1, with a limit of detection (LOD) of 20 and 2 ng mL−1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.

Neochloris oleoabundans cell walls have an altered composition when cultivated under different growing conditions

The impact that environmental factors have on the intracellular components of microalgae has been the focus of research for a number of decades. Despite that, their effects on the cell wall have received very little attention. In this study, we investigated how different growing conditions affect the cell walls of N. oleoabundans. The results revealed that the cell wall composition varied in that the modifications were different in the four cultivation media: freshwater nitrogen-replete (optimum culture) and -depleted conditions, and seawater nitrogen-replete and -depleted conditions. Nitrogen deficiency in freshwater cultivation was the only condition that significantly (p < .05) increased the total content of carbohydrates in the cell wall. The three most abundant components of freshwater-cultivated cell wall polysaccharides were rhamnose, galactose and glucuronic acid whereas in seawater media the main components of cell wall polysaccharides were rhamnose, glucose and galactose. The combined results of the biochemical analyses and monoclonal antibodies epitope-binding revealed that N. oleoabundans cell walls are likely composed of sulphated polysaccharides enriched in mannose, β-(1 → 4)-d-mannans, and glucose as they grow in seawater. Salinity and nitrogen deficiency also had an impact on the nitrogenous components of the cell wall. Under these conditions we observed a decrease in glucosamine in the cell wall. The analysis of specific binding of monoclonal antibodies, revealed that the cell wall of N. oleoabundans is possibly enriched in arabinogalactan proteins (AGPs). Under salinity and nitrogen deficiency N. oleoabundans increased the proportion of the non-polar to polar amino acids in the cell walls. An increase of leucine in the cell walls may suggest that N. oleoabundans contains leucine-rich repeat proteins which are known to play a vital role in stress responses. This report provides new insights into microalgae cell wall biology and how cell walls are remodelled when growing under different conditions.

Multifunctional iron oxide-carbon hybrid nanoparticles for targeted fluorescent/MR dual-modal imaging and detection of breast cancer cells

An efficient method for the highly sensitive and specific detection of cancer cells is crucial for the early diagnosis of cancer. In this work, we propose a one-pot approach to fabricating magnetic-fluorescent iron oxide-carbon hybrid nanomaterials (MCNP) with excellent stable, high quantum yield and excellent magnetic properties for breast cancer cells recognition and detection via magnetic resonance and multicolour fluorescence imaging. MCNPs were efficiently synthesised via one-pot, multi-component reactions of FeCl3, FeCl2•4H2O, citric acid and ethylenediamine in diethylene glycol. The MCNPs showed strong excitation wavelength-dependent fluorescence in the blue-red region with a high quantum yield of 58.4%, and they presented higher stability and T2 relaxivity than pure iron oxide nanoparticles. After conjugating with CD44 monoclonal antibodies, the fabricated targeting nanoprobe, MCNPs-CD44, demonstrated a specific fluorescence/MRI dual imaging contrast effect in 4T1 breast cancer cells. Biological transmission electron microscope imaging showed a significant preferential uptake of the nanoparticle conjugates by the 4T1 cells. By taking advantage of the high binding affinity and specificity of the CD44 antibodies to the overexpressed CD44 on the cancer cell surface, the developed MCNPs-CD44 probe distinguished 4T1 breast cancer cells from normal cells and detected as low as a few hundred cancer cells, thus indicating the potential application of multifunctional nanocomposites in the MR diagnosis and fluorescence positioning of breast cancer at cellular-level resolution.

Label-free biosensing using a microring resonator integrated with poly-(dimethylsiloxane) microfluidic channels.

Microring resonators have shown promising potential for highly sensitive, label-free, real-time detection of biomolecules. Accurate quantitative detection of target molecules through use of photonic integrated circuits has been demonstrated for environmental monitoring and medical diagnostics. Here, we described the design, fabrication, and characterization of a highly sensitive, label-free microring optical resonator integrated with poly-(dimethylsiloxane) microfluidic channels, which consumes only 30 µl of sample solution. The resonance wavelength shifts resulting from the change in the effective refraction index can be measured in situ, and thus the binding events on the resonator surface, including antibody immobilization, blocking of the resonator surface, and the specific binding of antibody and antigen, can be recorded throughout the entire experimental process in real time. We measured the binding events for the detection of human immunoglobulin G. The system had a detection limit of 0.5 µg/ml, a value substantially (14 times) lower than that of a previously reported microring resonator. To verify the usefulness and adaptability of this technique, human epidermal growth factor receptor 2 was used for the detection. The microring optical resonator was able to monitor reactions between biological molecules in real time and thus can be used in quantitative detection and biological sensing with little sample consumption.

A Quick &amp; Simple Single-Antibody Immunoassay for the Student Laboratory

In this exercise students use a simplified version of an immunoassay that relies on the nonspecific binding of proteins to nitrocellulose followed by the specific binding of antibodies to antigen. This antigen–antibody interaction is detected when a chromogenic substrate, catalyzed by the enzyme conjugated to the antibody, produces a color change. The immunoblot is less expensive and faster to perform than plastic-plate-based assays. I have used this assay for over 10 years in undergraduate courses: in ecology to estimate disease incidence; in botany to explore immunological techniques; and in biotechnology as an exercise in product development.

Diagnostic response to a cross-border challenge for the Swiss caprine arthritis encephalitis virus eradication program.

Animal trading between countries with different small ruminant lentivirus infectious status is a potential danger for the reintroduction of eradicated genotypes. This was the case in 2017 with the importation of a large flock of seropositive goats into Switzerland. The handling of this case permitted us to test the preventive measures in place. The coordination between the local veterinarian and the cantonal and federal veterinary authorities worked efficiently and rapidly involved the national reference center in the investigations. This case posed a challenge for the reference center and enabled scrutiny of the applied diagnostic tests. ELISA and western blot provided consistent results and pointed to an unusually high infection rate in the flock. This was confirmed by the isolation of several viruses from different organs and cells, demonstrating that the spleen is particularly well suited for isolation of small ruminant lentiviruses. The SU5-ELISA, designed to predict the subtype of the infecting virus, correctly pointed to a B1 subtype as the infectious agent. We confirmed that with this test it is necessary to analyze a representative number of samples from a flock and not just individual sera to obtain reliable results. This analysis permitted us to identify particular amino acid residues in the SU5 peptides that may be crucial in determining the subtype specificity of antibody binding. Different gag-pol and env regions were amplified by PCR using primers designed for this purpose. The phylogenetic analysis revealed a surprisingly high heterogeneity of the sequences, pointing to multiple infections within single animals and the entire flock. In conclusion, this case showed that the defense of the CAEV negative status of the Swiss goat population with respect to the virulent, prototypic B1 subtype of small ruminant lentiviruses, requires, among other measures, a diagnostic facility capable of performing a thorough analysis of the collected samples.

Human monoclonal antibodies isolated from a primary pneumococcal conjugate Vaccinee demonstrates the expansion of an antigen-driven Hypermutated memory B cell response

BackgroundCommunity-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination.MethodsPlasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity.ResultsThe data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio’s (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge.ConclusionsThe findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.

Unexpected Low Expression of Platelet Fibrinogen Receptor in Patients with Chronic Myeloproliferative Neoplasms: How Does It Change with Aspirin?

INTRODUCTIONPatients affected by Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered at high risk of thrombo-haemorrhagic events, but the role of the platelet count in the assessment of the risk of vascular events is still controversial. A tight correlation was found between the platelet count and plasma sCD40L, which appears to be required for thrombus formation in vivo. However sCD40L is increased both in MPNs and reactive thrombocytosis. Intravascular aggregates of platelets and leukocytes, mediated by P-selectin and CD11b on the former and the latter, respectively, have been observed.Both of this processes should imply a perpetual - and measurable - platelet activation. Several studies on platelet function have been already proposed, nevertheless the mechanisms through which platelets are able to trigger vascular events, are not yet adequately clarified.A refined method for the determination of platelet activation appears to be the use of platelet PAC-1 antibody, able to identify the expression of the fibrinogen receptor of platelet glycoprotein IIb/IIIa. This expression is indeed unique in the process of platelet activation, and yet rarely analyzed. Moreover, since the platelet fibrinogen receptor exposure seems to be influenced by turbulence in blood flow, we have thought that its evaluation could provide important biological evidences to explain some clinical manifestations, such as microvascular disturbances.METHODSBlood samples from 40 consecutive MPNs patients who never received cytoreductive agents, were obtained. 28/40 patients were receiving a continuative antiplatelet prophylaxis with low dose aspirin (ASA, 75-100 mg) at the time of collection, while 12/40 of them were not on such therapy. Our aim was to verify the expression of platelet fibrinogen receptors (PFRs) in the two different groups of patients compared to healthy volunteers, using whole blood flow cytometry. In each experiment sodium citrate and heparin (positive control of platelet activation) tubes were collected from the same patient. Within 10 minutes from blood sampling, 5 ml of whole blood from each tube was incubated for 20 minutes at room temperature in the dark with saturating concentration of CD61 PerCP, CD62P PE and PAC-1 FITC. Positive control was also incubated with PAC-1 in the presence of Arg-Gly-Asp-Ser (RGDS) in order to test the specific antibody binding. Samples were fixed with paraformaldehyde 1% for 30 minutes at 4°C in the dark and analyzed on a flow cytometer.RESULTSSurprisingly, we have been able to verify a very low PAC-1 binding to platelets in patients with MPNs not receiving cytoreduction nor antiplatelet agents (33%) if compared to that observed in healthy subjects (61%; p<0.0001). The use of aspirin seems conversely to restore the expression of platelet fibrinogen receptor, as PAC-1 binding capacity is comparable to that of healthy volunteers (56%). No difference was found with respect to the JAK2 Val617Phe mutation and its allele burden. Interestingly, by focusing on the group of patients under antiplatelet prophylaxis and with no history of thrombosis, it was found that subjects with persistent microcirculatory disorders show a higher PAC-1 binding capacity if compared to the asymptomatic ones (67% vs 52%, p= 0.04).CONCLUSIONIn untreated MPNs, a large amount of platelets are resting in a conformation that is unable to bind fibrinogen, as demonstrated by the low PAC-1 expression in cytofluorimetry. This lack of activity can be reversed by administering ASA, which is also known for its fibrinolytic and hypoprothrombinemic effects. We hypotesize that the hypercoagulable states observed in these patient could depend on a primarly plasma-driven impairment of fibrin turnover and thrombin generation. Further investigations are going to be promoted by our Group. PAC-1 could be at the same time a good marker of aspirin resistance in patients experiencing microcirculatory disorders. [Display omitted] DisclosuresMartinelli:Janssen: Consultancy; Jazz Pharmaceuticals: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Novartis: Speakers Bureau.

Epitomic Characterization of the Specificity of the Anti-Amyloid Aβ Monoclonal Antibodies 6E10 and 4G8.

The monoclonal antibodies 6E10 and 4G8 are among the first anti-amyloid monoclonal antibodies against Aβ and the most widely used antibodies in Alzheimer’s disease research. Although the epitopes for 6E10 and 4G8 have been reported to correspond to residues 1–16 and 17–24, a more recent high-resolution mapping approach indicates that 6E10 maps to residues 4–10 while 4G8 maps to residues 18–23. To characterize the binding specificity of both antibodies in greater detail, we used immunoselection of random sequences from phage display library followed by deep sequencing and analysis of resulting patterns from thousands of immunoselected sequences. We found that the minimum sequence required for 6E10 binding is R-x-D with over half (53%) of the immunoselected sequences conforming to this pattern. The vast majority of these sequences contain an H at position x (R-H-D), corresponding to residues 5–7 of the Aβ target sequences, but Y is also permitted at this position in a minority of sequences. For 4G8 we found that the most frequent pattern is F-x-A contained in approximately 30% of the sequences, followed by F-A, L-x(3)-A, L-x-F, and F-F each accounting for approximately 18% of the sequences. The F-x-A motif also occurs in islet amyloid poly peptide which may explain why 4G8 also recognizes amyloid fibrils of this peptide. Immunoselection of random sequences and deep sequencing may also be a facile and efficient means of determining residues critical for antibody binding and validating the specificity of monoclonal antibodies and polyclonal antisera.

A high-throughput inhibition assay to study MERS-CoV antibody interactions using image cytometry

The emergence of new pathogens, such as Middle East respiratory syndrome coronavirus (MERS-CoV), poses serious challenges to global public health and highlights the urgent need for methods to rapidly identify and characterize potential therapeutic or prevention options, such as neutralizing antibodies. Spike (S) proteins are present on the surface of MERS-CoV virions and mediate viral entry. S is the primary target for MERS-CoV vaccine and antibody development, and it has become increasingly important to understand MERS-CoV antibody binding specificity and function. Commonly used serological methods like ELISA, biolayer interferometry, and flow cytometry are informative, but limited. Here, we demonstrate a high-throughput protein binding inhibition assay using image cytometry. The image cytometry-based high-throughput screening method was developed by selecting a cell type with high DPP4 expression and defining optimal seeding density and protein binding conditions. The ability of monoclonal antibodies to inhibit MERS-CoV S binding was then tested. Binding inhibition results were comparable with those described in previous literature for MERS-CoV spike monomer and showed similar patterns as neutralization results. The coefficient of variation (CV) of our cell-based assay was <10%. The proposed image cytometry method provides an efficient approach for characterizing potential therapeutic antibodies for combating MERS-CoV that compares favorably with current methods. The ability to rapidly determine direct antibody binding to host cells in a high-throughput manner can be applied to study other pathogen-antibody interactions and thus can impact future research on viral pathogens.

A Flow Injection Chemiluminescent Immunosensor for Ultrasensitive Detection of Brombuterol Based on Resin Beads and Enzymatic Amplification

This study described the use of a rapid and sensitive flow injection chemiluminescence immunoassay based on the specific binding of antigens and antibodies for the detection of brombuterol residues in the swine meat and feed samples. Carboxylic resin beads were used as a solid-phase carrier for supporting more coating antigens through acylamide bonds. Under the mechanism of competitive immunity, the brombuterol in solution competed with the coating antigen immobilized on the resin beads for the limited binding sites of antibody. Then, horseradish peroxidase-labeled secondary antibodies were introduced into the immunosensor to combine with polyclonal antibodies. The chemiluminescence intensity of the luminol-p-iodophenol-H2O2 system was catalyzed by horseradish peroxidase, and thus, signal amplification was achieved. Under the optimal conditions of the concentration of coating antigen, antibody and enzymatic secondary antibody, brombuterol could be detected quantitatively. The chemiluminescence intensity decreased linearly with the logarithm of the brombuterol concentration in the range of 0.001 to 300 ng mL−1. The limit of detection (3σ) and the limit of quantitation were 0.33 pg mL−1 and 1.1 pg mL−1, respectively. This method had undergone a series of test conditions and exhibited good specificity, stability, and reproducibility. The immunosensor can also detect real samples and provide prospects for the detection of other small molecule compounds, such as isoproterenol, enrofloxacin, and ofloxacin.

Trace detection of some nitro-explosives using thermal mediated immunochemical defragmented method

A new immunoassay format using thermally induced defragmentation of some nitro-explosives with a high degree of selectivity is reported. Specific antibodies against three widely used explosives, 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN) were generated by designing suitable haptens using geometry optimization modules. These in-house generated antibodies were used in a newly developed thermal mediated immunochemical biosensing technique which involves the binding of specific antibodies to respective nitro-explosives on a microtiter strip, resulting in the formation of specific immunocomplex. Heating the specific immuno-complex formed on microtiter wells resulted in thermal lysis of nitro-explosives to generate nitrite ions. These ions react with Griess reagent to form a colored chromophore which correlates the concentration of individual explosive in the sample. The present work fulfills the need for an improved explosive detecting system that is highly specific and capable of quickly determining the presence of nitrate containing explosives from a mixture pool.

The re-awakening of hCG expression. Its role in the diagnosis of cervical squamous cell carcinoma.

Objectives:To compare immunohistochemical detection of Human chorionic gonadotropin (hCG) expression in paraffin embedded tissue of squamous cell carcinomas (SCC) and high grade squamous intraepithelial lesions (HSIL).Methods:The samples in this retrospective study were obtained from the archives of the Pathology Department at Gaziantep University, Gaziantep, Turkey, over the period from January 2012 to September 2016. The study group consisted of 55 cases of SCC and 45 cases of HSIL. Tissue expression of hCG was detected by specific binding of anti-hCG antibody using an automated immunohistochemistry staining device. The categorical variables of intensity and coverage were analyzed statistically using Pearson Chi-Square test.Results:High grade squamous cell lesions cases showed weak (84.4%, n=38/45) to no (15.6%, n=7/45) staining for hCG. None of the HSIL cases showed strong positivity. Strong positivity for hCG was detected in 90.9% (n=50/55) of SCC cases.Conclusion:Our study supports the association of ectopic hCG expression in cancer pathogenesis by demonstrating strong hCG immunoreactivity only in SCC cases. This finding can be helpful in supporting the diagnosis of invasive carcinoma in small or fragmented biopsies, which can on their own be confusing for the pathologists.

PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes.

The binding specificities of an individual's antibody repertoire contain a wealth of biological information. They harbor evidence of environmental exposures, allergies, ongoing or emerging autoimmune disease processes, and responses to immunomodulatory therapies, for example. Highly multiplexed methods to comprehensively interrogate antibody-binding specificities have therefore emerged in recent years as important molecular tools. Here, we provide a detailed protocol for performing 'phage immunoprecipitation sequencing' (PhIP-Seq), which is a powerful method for analyzing antibody-repertoire binding specificities with high throughput and at low cost. The methodology uses oligonucleotide library synthesis (OLS) to encode proteomic-scale peptide libraries for display on bacteriophage. These libraries are then immunoprecipitated, using an individual's antibodies, for subsequent analysis by high-throughput DNA sequencing. We have used PhIP-Seq to identify novel self-antigens associated with autoimmune disease, to characterize the self-reactivity of broadly neutralizing HIV antibodies, and in a large international cross-sectional study of exposure to hundreds of human viruses. Compared with alternative array-based techniques, PhIP-Seq is far more scalable in terms of sample throughput and cost per analysis. Cloning and expression of recombinant proteins are not required (versus protein microarrays), and peptide lengths are limited only by DNA synthesis chemistry (up to 90-aa (amino acid) peptides versus the typical 8- to 12-aa length limit of synthetic peptide arrays). Compared with protein microarrays, however, PhIP-Seq libraries lack discontinuous epitopes and post-translational modifications. To increase the accessibility of PhIP-Seq, we provide detailed instructions for the design of phage-displayed peptidome libraries, their immunoprecipitation using serum antibodies, deep sequencing-based measurement of peptide abundances, and statistical determination of peptide enrichments that reflect antibody-peptide interactions. Once a library has been constructed, PhIP-Seq data can be obtained for analysis within a week.

P046 Under assessment of DSA strength in single antibody bead assays may lead to poor outcome in kidney transplantation

Solid phase anti-HLA antibody tests have become an important tool in predicting the compatibility of donor-recipient pairs for kidney transplantation. Additionally, single antigen bead (SAB) assays provide valuable information for the management of post-transplant immunotherapy. However, there can be limitations to SAB assays, for example, the presence of donor specific IgM antibodies and/or binding of high levels of compliment may block binding of donor specific IgG antibodies (DSA) to the SAB. This could possibly result in an under estimation of DSA. The following case is an example of this phenomenon. A 48 years old female patient (HLA Type: A1 A29 B53 B72 DR8 DR13) was transplanted with a deceased donor kidney (HLA Type: A2 A33 B53 B58 DR8 DR11 DQB7 DQA04 DQA05 DPB01:01 DPB04:02) in 2017. The patient was presensitized (pregnancy) to a donor antigen A2 (MFI of 4 A2 beads = 3904) with an associated epitope 144TKH (MFI = 5101, includes A68, A69). On day 3 post-transplant, there was no detectable antibodies to donor antigens due to the sponge effect of the transplanted kidney. On day 6 antibodies to A2 (MFI = 5000) and the associated epitope 144TKH (MFI = 8946) reappeared. Additionally, a low level of de novo antibodies to B*58:01 (MFI = 716) and DPB1*04:02 (MFI = 864) was detected. On day 12, antibodies were detected to only A*02:01 (MFI = 615), while a significant increase in B58 antibody was observed (MFI = 9025). At this time, a biopsy indicated antibody mediated graft rejection; creatinine was 10 mg/dl. Accordingly, the patient’s antibody was further evaluated by an AMOS one wash CDC cross match with frozen cells and sera from day 6 and 12. On day 6, CDC-XM was negative, but on day 12 the CDCXM was positive. The level of DSA to class I of MFI 9640 would typically not give a positive CDC cross match. To determine if there was interference of IgM and/or compliment, the 12-day serum was treated at 63C for 13 min to inactivate these molecules. The heat-treated serum demonstrated an MFI to A*02:01 of MFI = 20,567 compared to an MFI of 615 before heat inactivation. Unfortunately, treatment for antibody-mediated rejection was not initiated soon enough to save the kidney. Although it is rare to observe IgM and/or compliment interference with SAB assays (4 out of 3020 cases, 0.1%), post-transplant assessment of DSA should be performed with heat inactivated sera or other methods to eliminate the under estimation of the strength of IgG antibodies. A.J. Norin:2. Consultant; Company/Organization; ICON PLC, Stony Brook Pathologist. 3. Speaker’s Bureau; Company/Organization; immucor, one lambda.

Formaldehyde-mediated peptide coupling for the titration of epitope-specific antibody in an ELISA format

Synthetic peptides are commonly used as an immobilised substrate in ELISA to measure antibody responses in serum. However, in addition to poor adsorption on the plastic surfaces of plates, titration of antibody response to a specific epitope of interest is challenging because the majority of antibodies in serum may be directed to other regions of the antigen. Using Ras peptides containing either the G12V or G12D mutation as a model antigen, we have tested formaldehyde as a crosslinker to immobilise Ras peptides on the plastic surface of microtitre plates in the presence of bovine serum albumin (BSA) as a carrier. Using this method, specific antibody against the G12V mutation was titrated from a simulated rabbit serum. Non-cognate Ras peptides were included in the ELISA reactions to suppress binding against non-mutated regions of the peptide. Our results showed that formaldehyde enhanced in-well peptide immobilisation of peptide approximately 3-fold and this enhancement required addition of BSA as a carrier. Wild type Ras peptide was not ideal as a non-cognate competitor as it tended to inhibit specific antibody binding against G12D or G12V. In the presence of the non-cognate peptide containing G12D, concentrations of the anti-G12V antibody in a simulated serum were determined with approximately 95% accuracy. Our method will be useful to determine a specific antibody response against a certain epitope in various peptide-based immunotherapy trials.

B cell clonal lineage alterations upon recombinant HIV-1 envelope immunization of rhesus macaques.

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs, but is also targeted by binding, non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus, our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here, in addition to serological analysis, we employed high-throughput BCR sequence analysis from the periphery, lymph nodes and bone marrow, as well as B cell- and antibody-isolation and characterization methods, to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1, 2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic, but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003–0.02%) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors.