Human Serum IgG Research Articles

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

BackgroundPhlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens.Methodology/Principal findingsRecombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure.Conclusions/SignificanceThis is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

Copper deposition-induced efficient signal amplification for ultrasensitive lateral flow immunoassay

This work describes a novel signal amplification strategy via gold nanoparticle (AuNP)–induced copper deposition to develop a highly sensitive and accurate immunochromatographic platform. Copper deposition–assisted signal amplification (CDASA) was first evaluated on commercially HCG test strip. A lower limit of detection (LOD) over the well-known silver enhancement confirms the great potential of this new approach for the visual test strip. Under an optimal condition, an LOD of 1 pg mL−1 and a wide dynamic range from 1 pg mL−1 to 100 ng mL−1 were achieved by the CDASA with rabbit IgG as a model analyte. In terms of LOD, 2–3 orders of magnitude improvement is received by the CDASA as compared with the conventional test strip without any signal amplification. Potential of the proposed strategy for practical application is also validated by detection of Rabbit IgG in human serum, a mimic clinical sample. The CDASA renders much superiority such as a higher deposition efficiency, lower cost, and better reagent stability over the silver enhancement method. The results can be qualitative and quantitative analysis by the naked eyes and the combination of a flatbed scanner and a free imageJ respectively, eliminating any lab equipment.

Sialylated Autoantigen-Reactive IgG Antibodies Attenuate Disease Development in Autoimmune Mouse Models of Lupus Nephritis and Rheumatoid Arthritis.

Pro- and anti-inflammatory effector functions of IgG antibodies (Abs) depend on their subclass and Fc glycosylation pattern. Accumulation of non-galactosylated (agalactosylated; G0) IgG Abs in the serum of rheumatoid arthritis and systemic lupus erythematosus (SLE) patients reflects severity of the diseases. In contrast, sialylated IgG Abs are responsible for anti-inflammatory effects of the intravenous immunoglobulin (pooled human serum IgG from healthy donors), administered in high doses (2 g/kg) to treat autoimmune patients. However, whether low amounts of sialylated autoantigen-reactive IgG Abs can also inhibit autoimmune diseases is hardly investigated. Here, we explore whether sialylated autoantigen-reactive IgG Abs can inhibit autoimmune pathology in different mouse models. We found that sialylated IgG auto-Abs fail to induce inflammation and lupus nephritis in a B cell receptor (BCR) transgenic lupus model, but instead are associated with lower frequencies of pathogenic Th1, Th17 and B cell responses. In accordance, the transfer of small amounts of immune complexes containing sialylated IgG Abs was sufficient to attenuate the development of nephritis. We further showed that administration of sialylated collagen type II (Col II)-specific IgG Abs attenuated the disease symptoms in a model of Col II-induced arthritis and reduced pathogenic Th17 cell and autoantigen-specific IgG Ab responses. We conclude that sialylated autoantigen-specific IgG Abs may represent a promising tool for treating pathogenic T and B cell immune responses in autoimmune diseases.

Detection of toxoplasma-specific immunoglobulin G in human sera: performance comparison of in house Dot-ELISA with ECLIA and ELISA.

In the current study, performance of electrochemiluminescence immunoassay (ECLIA) in detection of anti-toxoplasma IgG in human sera was compared with that of enzyme-linked immunosorbent assay (ELISA). Furthermore, performance of an in house Dot-ELISA in detection of anti-toxoplasma IgG was compared with that of ECLIA and ELISA. In total, 219 human sera were tested to detect anti-toxoplasma IgG using Dynex DS2® and Roche Cobas® e411 Automated Analyzers. Discordant results rechecked using immunofluorescence assay (IFA). Then, sera were used in an in house Dot-ELISA to assess toxoplasma-specific IgG. Of the 219 samples, two samples were found undetermined using ECLIA but reactive using ELISA. Using IFA, the two sera were reported unreactive. Furthermore, two samples were found reactive using ECLIA and unreactive using ELISA. These samples were reported reactive using IFA. The overall agreement for the two former methods was 98% (rZ0.98.1; P< 0.001). The intrinsic parameters calculated for in house Dot-ELISA included sensitivity of 79.5, specificity of 78.2, and accuracy of 78.9%, compared to ECLIA and ELISA. Positive and negative predictive values included 82.9 and 74.2%, respectively. A 100% sensitivity was found in in house Dot-ELISA for highly reactive sera in ECLIA and ELISA. ECLIA is appropriate for the first-line serological screening tests and can replace ELISA due to high speed, sensitivity, and specificity, particularly in large laboratories. Dot-ELISA is a rapid, sensitive, specific, cost-effective, user-friendly, and field-portable technique and hence can be used for screening toxoplasmosis, especially in rural fields or less equipped laboratories.

Human anti-dopamine receptor 1 monoclonal autoantibody (mAb) identifies potential mechanisms of neuronal signaling in post-infectious autoimmune-mediated neuropsychiatric disease

Abstract Neuropsychiatric and movement disorders are associated with infections and anti-neuronal autoantibodies against dopamine receptors D1R and D2R. Most characterized of these disorders is Sydenham chorea (SC), an autoimmune neurologic manifestation of streptococcal-induced rheumatic fever where autoantibodies target the basal ganglia. Pediatric autoimmune neuropsychiatric disorder associated with streptococci (PANDAS) have similar autoantibodies that react with D1R and D2R. SC-derived human mAb 24.3.1 or SC derived IgG signal D2R in transfected cells by decreasing cyclic AMP-dependent kinase (cAMP) activity, reflective of an inhibitory receptor. Anti-D2R 24.3.1 increases dopamine release from human neuronal cells. However, signaling of D1R, an activating receptor, can have different biological effects. Our PANDAS-derived anti-D1R mAb, B4C, and PANDAS sera signaled D1R by activation of cAMP in transfected cells. IgG titers against D1R were significantly elevated in SC and PANDAS sera. To further show IgG recognized the D1R receptor, peptides of the extracellular loop of D1R were constructed and a defined sequence reacted significantly with human mAb B4C and PANDAS serum IgG. Human anti-D1R mAb B4C induced cAMP activity levels comparable to dopamine in a dose-dependent manner. Combined with dopamine, mAb B4C sensitized D1R to at least 50X more responsive to dopamine. In neuropsychiatric diseases where both anti-D1R and anti-D2R autoantibodies may be in combination, anti-D1R autoantibodies could sensitize D1R making it more sensitive to excess dopamine induced by anti-D2R autoantibodies; thus, contributing to dopamine signaling abnormalities and neuropsychiatric pathogenesis.

Validation of an automated ultraperformance liquid chromatography IgG N-glycan analytical method applicable to classical galactosaemia.

Background Classical galactosaemia (OMIM #230400) is a rare disorder of carbohydrate metabolism caused by deficiency of the galactose-1-phosphate uridyltransferase enzyme. The pathophysiology of the long-term complications, mainly cognitive, neurological and female fertility problems, remains poorly understood. Current clinical methods of biochemical monitoring lack precision and individualization with an identified need for improved biomarkers for this condition. Methods We report the development and detailed validation of an automated ultraperformance liquid chromatography N-glycan analytical method of high peak resolution applied to galactose incorporation into human serum IgG. Samples are prepared on 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labelling and post-labelling clean-up with solid-phase extraction. Results This method is shown to be accurate and precise with repeatability (cumulative coefficients of variation) of 2.0 and 8.5%, respectively, for G0/G1 and G0/G2 ratios. Both serum and processed N-glycan samples were found to be stable at room temperature and in freeze-thaw experiments. Conclusions This high-throughput method of IgG galactose incorporation is robust, affordable and simple. This method is validated with the potential to apply as a biomarker for treatment outcomes for galactosaemia.

Diagnosis of amphimeriasis by LAMPhimerus assay in human stool samples long-term storage onto filter paper.

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas.

A novel lateral flow immunoassay for the rapid detection of anti-Dsg3 IgG serum autoantibodies in pemphigus vulgaris.

Pemphigus vulgaris (PV) is a severe autoimmune blistering disease of the skin and mucous membranes. As autoantibodies play an essential role in the disease pathogenesis, the serological detection of anti-desmoglein 3 IgG represents a central tool in the diagnosis of the disease. In this study, we show the validation of a novel lateral flow immunoassay (LFIA) which rapidly detects anti-desmoglein 3 (Dsg3) IgG in human serum. In contrast to other diagnostic procedures, the assay is compact and simple to perform and delivers a fast"yes" or "no" answer within 10minutes without additional hardware requirements for test evaluation. For validation, a blinded collection of 200 sera including 100 sera from 14 PV patients, 75 sera from 24 bullous pemphigoid patients and 25 sera from 6 patients with pemphigus foliaceus collected at different time points during disease was used. Presence or non-presence of anti-Dsg3 IgG within sera was confirmed using a commercially available Dsg3-ELISA. For qualitative evaluation, Dsg3-LFIA test results were assessed by two independent groups of human observers. Furthermore, quantitative evaluation using POCScan reader was applied. The Dsg3-LFIA demonstrated reliable test results with a sensitivity and specificity of 78.1% and 97.1%, respectively. Test results from POCScan and human observers showed a substantial agreement. The Dsg3-LFIA represents a new diagnostic tool for the immediate and reliable detection of anti-desmoglein 3 serum IgG autoantibodies that does not require additional hardware. Further prospective trials are warranted to validate the Dsg3 LFIA in pemphigus.

The First Serological Study of Q Fever in Humans in Lebanon.

The aim of this study was to estimate, for the first time, the human seroprevalence of Q fever in Lebanon, by assessing the presence of antibodies against the causative agent, Coxiella burnetii. A total number of 421 serum samples (226 females and 196 males) were collected in February 2015 from hospitals and laboratories dispersed in five Lebanese provinces: Akkar, Bekaa, Mount Lebanon, Nabatieh, and South Lebanon. Serial testing approach was used. Samples were first screened for IgG phase II antibodies against C. burnetii by Enzyme Linked Immunosorbent Assay (ELISA) Kit. Then, both positive and inconclusive sera were reexamined by immunofluorescence assay (IFA) test with the aims to confirm and specify the infection status (past or probably acute infection) by detecting IgG (I/II) and IgM (I/II) in human sera. Screening of 421 samples was estimated to be 38.70% (95% CI 34-43.3) positive samples, 5.90% (95% CI 3.7-8.2) suspect samples (as doubtful results), and 55.40% (95% CI 50.7-60.1) negative samples. Furthermore, all positive and suspect samples by ELISA test were retested by immunofluorescence assay test (IFAT), and the prevalence of positive sample was 37% and the infection case was recorded: 23.75% (95% CI 19.7-27.8) samples resulted from past infection, 1.9% (95% CI 0.6-3.2) probably acute infection characterized by several dominance clinical symptoms as: fever, cough, headache, difficulty breathing, and atypical pneumonia, and 0.23% (95% CI 0-0.7) inconclusive sample accompanied by different symptoms as bone metastasis and lung cancer. The study records the exposition of 37% of 421 patients to C. burnetii distributed in five Lebanese provinces with the highest seroprevalence in Bekaa and Akkar provinces and the lowest reported in Mount Lebanon. This difference may be due to the presence of high density of livestock production and of major agricultural areas in these two provinces.

Characteristics of humoral response in individual IgG subclasses in the course of selected bacterial infections in humans

It has been recognized that human serum IgG is composed of four distinct subclasses. The IgG subclasses differ not only in their distribution in normal serum, but also in their biological properties and the nature of the antigens which elicit their production. In general, bacterial proteins antigens preferentially induce IgG1 antibodies in humans, with minor contributions of IgG3 and IgG4. In contrast, IgG response to polysaccharides is normally restricted to the IgG2 subclass, although some polysaccharides also induce substantial amounts of IgG1.

Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

Rapid, Point-of-Care, Paper-Based Plasmonic Biosensor for Zika Virus Diagnosis.

Zika virus (ZIKV) is an increasing global health challenge. There is an urgent need for rapid, low-cost, and accurate diagnostic tests that can be broadly distributed and applied in pandemic regions. Here, an innovative, adaptable, and rapidly deployable bioplasmonic paper-based device (BPD) is demonstrated for the detection of ZIKV infection, via quantification of serum anti-ZIKV-nonstructural protein 1 (NS1) IgG and IgM. BPD is based on ZIKV-NS1 protein as a capture element and gold nanorods as plasmonic nanotransducers. The BPD displays excellent sensitivity and selectivity to both anti-ZIKV-NS1 IgG and IgM in human serum. In addition, excellent stability of BPDs at room and even elevated temperature for one month is achieved by metal-organic framework (MOF)-based biopreservation. MOF-based preservation obviates the need for device refrigeration during transport and storage, thus enabling their use in point-of-care and resource-limited settings for ZIKV surveillance. Furthermore, the versatile design (interchangeable recognition element) of BPDs more generally enables their ready adaptation to diagnose other emerging infectious diseases.

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans.

BACKGROUNDAmphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing.OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera.METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections.PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana.MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

Antigens Rv0310c and Rv1255c are promising novel biomarkers for the diagnosis of Mycobacterium tuberculosis infection

This study aimed to identify novel immunogenic epitopes from Mycobacterium tuberculosis (MTB) that could be used in tuberculosis (TB) diagnostics. To determine the diagnostic potential of mycobacterial antigens in serodiagnosis of TB, 256 patients were enrolled in a study and divided into two groups: 126 smear-positive pulmonary TB patients (SPPT) and 130 smear-negative pulmonary TB patients (SNPT); 152 bacillus Calmette-Guerin (BCG)-vaccinated healthy people were used as a control. Murine results showed that antigens Rv0310c-E from RD 8 and Rv1255c-E from RD 10 were strongly immunogenic to Th1 cells and induced a great humoral response. Receiver operating characteristic analysis indicated that Rv0310c-E (area under the curve (AUC): 0.800) and Rv1255c-E (AUC: 0.808) performed better than ESAT-6 (AUC: 0.665) and CFP-10 (AUC: 0.623) proteins but were comparable with Rv3425 (AUC: 0.788) protein in a human serum IgG analysis. Rv0310c-E demonstrated the highest diagnostic ability for the SPPT group (Youden index: 0.5602, sensitivity: 69.84%, specificity: 86.18%), while Rv1255c-E demonstrated the highest diagnostic ability for the SNPT group (Youden index: 0.5674, sensitivity: 73.84%, specificity: 82.89%). In addition, combination analysis found that antigen Rv0310c-E, coupled with the Rv3425 protein (Youden index: 0.6098, sensitivity: 87.30%, specificity: 73.68%) had the strongest performance for TB diagnostics of the SPPT group, and the single antigen Rv1255c-E was strongest for the SNPT group. These results suggest that antigens Rv0310c-E and Rv1255c-E are potential antigens for TB serodiagnostic tests, which may facilitate detection of MTB in smear-negative and smear-positive patients.Emerging Microbes & Infections (2017) 6, e64; doi:10.1038/emi.2017.54; published online 12 July 2017

Effects of INCB052793, a Selective JAK1 Inhibitor, in Combination with Anti-Myeloma Agents on Human Multiple Myeloma (MM) In Vitro and In Vivo

Introduction: Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow (BM) of MM patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling in MM, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken.Methods: The human MM cell lines RPMI8226 and U266 were obtained from ATCC and MM1S was kindly supplied by Steven Rosen, MD (Northwestern University, Chicago, IL). BM aspirates were obtained from patients with MM as approved by the Institutional Review Board (Western IRB BIO 001) and informed consent was obtained in accordance with the Declaration of Helsinki. BM mononuclear cells (MCs) were isolated using density-gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). All cells were maintained in RPMI1640 (Omega Scientific, Tarzana, CA) supplemented with 10% fetal bovine serum (FBS), 2mM l-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin, in an atmosphere of 5% carbon dioxide at 37°C. Primary MM BMMCs were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents cyclophosphamide (CY), melphalan (MEL), and bendamustine (BEN), the proteasome inhibitor carfilzomib (CAR), dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). Cells from RPMI8226 or U266 MM cell lines were cultured in the presence of INCB052793 plus CY, MEL, BEN, CAR, DEX, LEN, or POM. After 48 hours, cell viability was assessed using the MTS assay. For the in vivo studies, mice were implanted with a piece of the human MM tumor LAGk-1A. Seven days post-implantation, mice were randomized into treatment groups, and tumor size was measured on a weekly basis. All in vivo studies were approved by the institutional animal care and use committee.Results: In vitro studies demonstrated that combinations of INCB052793 with a broad spectrum of anti-MM agents synergistically inhibited the viability of BMMCs from MM patients. INCB052793 plus the three alkylating agents or CAR synergistically inhibited the viability of these cells. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1 cell line. In vivo, LAGk-1A-bearing mice had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant reduction in tumor growth compared with all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. In a separate study also using the LAGk-1A model, we evaluated the combination of INCB052793 with CAR or bortezomib (BOR). Combinations of INCB052793 + CAR or BOR were superior at inhibiting tumor growth when compared to single agent INCB052793.Conclusion: These in vitro and in vivo preclinical studies demonstrate that the combination of the JAK1 inhibitor INCB052793 with a broad spectrum of anti-MM agents are effective, and provide further support for the clinical evaluation of these drug combinations for treating MM patients. Studies to further understand the mechanistic effects of these combinations on MM signaling and the tumor microenvironment are ongoing. DisclosuresBerenson:Amgen Inc: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract 1339: Effects of INCB052793, a selective JAK1 inhibitor, in combination with standard of care agents in human multiple myeloma (MM) cell lines and xenograft models

Abstract Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow of myeloma patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken. The human MM cell lines, RPMI8226 or U266, were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents, cyclophosphamide (CY), melphalan (MEL), and bendamustine, the proteasome inhibitor, carfilzomib, the corticosteroid, dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). After 48 hours, cell viability was assessed. Combinations of INCB052793 plus the three alkylating agents or carfilzomib synergistically inhibited the viability of both cell lines in vitro. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1S MM cell line. In vivo, mice bearing the human patient derived MM tumor LAGκ-1A had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at Day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant effect on tumor growth compared to all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through Day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. Studies to further understand the mechanistic effects of these combinations on myeloma signaling and the tumor microenvironment are ongoing. In conclusion, these in vitro and in vivo studies demonstrate that the combination of INCB052793 with a broad spectrum of anti-MM agents is effective, and provide further support for the clinical evaluation of these drug combinations in MM patients. Citation Format: Eric Sanchez, Mingjie Li, Cathy Wang, Puja Mehta, George Tang, Haiming Chen, James R. Berenson. Effects of INCB052793, a selective JAK1 inhibitor, in combination with standard of care agents in human multiple myeloma (MM) cell lines and xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1339.

Comparison of non-magnetic and magnetic beads in bead-based assays

Multiplex bead-based flow cytometry is an attractive way for simultaneous, rapid and cost-effective analysis of multiple analytes in a single sample. Previously, we developed various bead-based assays using non-magnetic beads coated with Staphylococcus aureus and Streptococcus pneumoniae antigens for the detection of antibodies. Here, we compared the performance of the assay using non-magnetic beads with one based on the newly developed magnetic beads. We optimized the magnetic beads' coupling procedure and antibody detection assays for S. aureus and S. pneumoniae antigens and we measured IgG in human pooled serum against a series of S. aureus and S. pneumoniae-derived antigens in a singleplex and in a multiplex assay, respectively. For the multiplex assay, the comparison between magnetic and non-magnetic beads showed: i) in the majority of the cases (13 of the 17 tested S. pneumoniae antigens) significantly higher Median Fluorescence Intensity (MFI) values, ii) lower detection limits, iii) lower coefficient of variation (CV: 12% vs. 7% for non-magnetic vs. magnetic beads), so lower inter-assay variation and hence higher reproducibility. Magnetic bead coupling is cost effective, as we used 25% of the normal amount of antigen and only 50% of the beads in comparison to the non-magnetic beads. This optimized magnetic-based assay, which combines ease of use with an improved assay performance, allows detection of antibodies with a low titer that are potentially missed with the non-magnetic-based assay.

A novel chemiluminescent immunoassay for detection of Toxoplasma gondii IgG in human sera

This study describes Toxoplasma gondii IgG chemiluminescent immunoassay (CLIA) based on the use of a novel immunochemical reagents in the form of the conjugates of original acridinium ester (AE) labels attached to antibodies and SAG2-GRA1-ROP1L chimeric antigen and shows that this test is useful for diagnostic purposes.

Immune recruitment or suppression by glycan engineering of endogenous and therapeutic antibodies

Human serum IgG contains multiple glycoforms which exhibit a range of binding properties to effector molecules such as cellular Fc receptors. Emerging knowledge of how the Fc glycans contribute to the antibody structure and effector functions has opened new avenues for the exploitation of defined antibody glycoforms in the treatment of diseases. Here, we review the structure and activity of antibody glycoforms and highlight developments in antibody glycoengineering by both the manipulation of the cellular glycosylation machinery and by chemoenzymatic synthesis. We discuss wide ranging applications of antibody glycoengineering in the treatment of cancer, autoimmunity and inflammation. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Yeast-generated virus-like particles as antigens for detection of human bocavirus 1-4 specific antibodies in human serum.

Human bocaviruses (HBoV) are non-enveloped, single-stranded DNA viruses, classified into the genus Bocavirus in the family Parvoviridae. Self-assembled virus-like particles (VLPs) composed of the major capsid protein VP2 of HBoV1-4 and mosaic VLPs composed of both VP2 and VP1 capsid proteins of HBoV1 were generated in yeast Saccharomyces cerevisiae and used to detect HBoV-specific IgG in human serum. Recombinant HBoV VLPs were similar to native HBoV particles in size and morphology. The prevalence of HBoV infection in a group of Lithuanian patients with clinical symptoms of respiratory tract infection was studied using purified yeast-generated VLPs as antigens in a competitive enzyme immunoassay (EIA). After depletion of cross-reactive antibodies, the seroprevalence of HBoV1 was 44.2% and the seroprevalence of HBoV2-4 was 35.7%. Mosaic VLPs consisting of HBoV1 VP1 and VP2 proteins showed a stronger reactivity with HBoV1 IgG-positive human serum specimens, and two equivocal serum specimens were reinterpreted as positive. Thus, mosaic VLPs offer a more sensitive tool for HBoV1 serology than currently available serodiagnostics tests based on VP2 VLPs. In conclusion, yeast S. cerevisiae represents an efficient expression system for generating recombinant HBoV1-4 VLPs of diagnostic relevance.