Specific Mouse Monoclonal Antibody Research Articles

Validation of a fibrinogen γ’ enzyme-linked immunosorbent assay using a new monoclonal antibody: effects of bariatric surgery

BackgroundFibrinogen γ’ is a naturally occurring 20-amino-acid splice variant of the fibrinogen γ chain. Animal studies link variations in fibrinogen to obesity, but it is unknown how fibrinogen γ’ is associated with obesity in humans. ObjectivesTo develop and validate an enzyme-linked immunosorbent assay (ELISA) for fibrinogen γ’ quantification in human plasma and analyze fibrinogen γ’ before and after bariatric surgery. MethodsWe generated C-terminal fibrinogen γ’ specific mouse monoclonal antibodies and developed a γ’ ELISA. Validation included measures of accuracy, sensitivity, and precision. Fibrinogen γ’ and total fibrinogen were measured in 60 individuals before and 6 months after bariatric surgery and in 19 normal-weight controls and 120 blood donors. ResultsHighly specific fibrinogen γ’ monoclonal antibodies were produced and successfully used in the ELISA. Recovery was 88%, and limits of detection and quantification were 0.003 mg/mL and 0.014 mg/mL, respectively. Coefficients of variation were 3% for repeatability and 7% for within-laboratory variation. The fibrinogen γ’ reference interval was 0.25 to 0.80 mg/mL. Fibrinogen γ’ concentrations were reduced after bariatric surgery and were higher in individuals with obesity than in those with normal weight. The fibrinogen γ’/total fibrinogen ratio was unchanged after surgery but was higher than the ratio in normal-weight individuals. ConclusionWe developed a precise and sensitive ELISA for fibrinogen γ’. Levels of fibrinogen γ’, but not the fibrinogen γ’/fibrinogen ratio, were reduced 6 months after bariatric surgery. Absolute and relative levels of fibrinogen γ’ were increased in individuals with obesity compared to normal-weight individuals.

A new strategy: high level expression and immunogenicity analysis of triplicate repeated multigenes in Mycoplasma hyopneumoniae.

Highly immunogenic nucleotide fragments from 3 genes of Mycoplasma hyopneumoniae strain 232 were selected using information software technology. After repeating each fragment three times, a total of 9 nucleotide fragments were joined together to form a new nucleotide sequence called Mhp2321092bp. Mhp2321092bp was directly synthesized and cloned into a pET100 vector and expressed in Escherichia coli. After purification, the proteins were successfully validated by SDS-PAGE and Western blotting using mouse His-tag antibody and pig anti-Mhp serum. BALB/c mice were intraperitoneally injected with purified proteins in the high-dose (100 μg), medium-dose group (50 μg) and low-dose (10 μg) groups. Mice in each group were injected on day 1, day 8 and day 15 of feeding, respectively. Serum samples were collected from all mice on the day before immunization and on day 22 after immunization. The antibody level in the mouse serum was detected using western blotting using purified expressed proteins as antigens. IL-2, TNF-α and IFN-γ were simultaneously detected in the mouse serum by ELISA. The results showed that the 60 kDa protein was successfully expressed and reacted specifically with the specific serum Mhp His-Tag mouse monoclonal antibody and pig anti-Mhp serum. From day 0 to day 22 of immunization, IFN-γ increased from 269.52 to 467.74 pg/mL, IL-2 increased from 14.03 to 145.16 pg/mL, and TNF-α increased from 6.86 to 12.37 pg/mL. The IgG antibody in mice increased significantly from 0 day to day 22 after immunization. This study suggests that the expressed recombinant protein may serve as one of the novel vaccine candidates for Mhp.

Diagnostic performance of a novel antigen-capture ELISA for the detection of SARS-CoV-2

Background and aimsThe coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. MethodAn antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. ResultsThe limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01–75.12) and 100% (95% CI: 97.57–100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17–96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19–88.71) for samples with Ct values < 30. ConclusionOur newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus.

Plasma Kallikrein Cleaved H-kininogen: An End-Point Marker for Contact Activation in vitro and ex vivo.

ObjectivesThe contact system consists of coagulation factor XII (FXII), prekallikrein, and H-kininogen (HK) and plays important roles in many diseases. Plasma kallikrein (PKa) cleaved HK (cHK) is a marker of contact activation. Presently, we developed a specific and precise enzyme-linked immunosorbent assay (ELISA) for determination of cHK in vitro and ex vivo.MethodsCleaved HK specific mouse monoclonal antibodies (mAbs) were generated using a peptide corresponding to the PKa cleavage site on HK as immunogen. ELISA, surface plasmon resonance analysis, and immunoprecipitation established the specificity of the antibody, which subsequently was used in a sandwich ELISA. The analytical imprecision and the concentration of cHK in a reference population and in women receiving oral contraceptives (OC) were determined. cHK was assessed in vitro in plasma exposed to polytetrafluoroethylene, silicone, and glass tubes.ResultsThe selected mAb showed excellent specificity towards cHK. The intra-assay and inter-assay CV of the ELISA were 3.6 and 6.0%, respectively. The reference population (60 women, 60 men) displayed a median cHK plasma concentration of 1.38 μg/mL and a reference interval of 0.82 – 2.56 μg/mL. Women receiving OC had significantly higher concentrations, p < 0.001. cHK was significantly elevated in plasma exposed to polytetrafluoroethylene, p = 0.001, and glass, p < 0.0001.ConclusionThe ELISA showed excellent precision and specificity. cHK assessment ex vivo demonstrated ongoing contact activation in healthy individuals, augmented by OC. The cHK antibody and the ELISA could be promising tools in contact activation related diseases and in vitro investigations of the plasma compatibility of blood contacting biomaterials.

Expression and immunogenicity study of a novel mhp183 gene fragment of Mycoplasma hyopneumoniae.

A highly immunogenic nucleotide fragment (195bp) was selected from the Mhp183 gene of Mycoplasma hyopneumoniae using information software technology and was named Mhp183195bp. Three Mhp183195bp were linked to form a new nucleotide sequence called Mhp183615bp. Mhp183615bp was directly synthesized and cloned into a pET100 vector and expressed in Escherichia coli. After purification, the proteins were successfully validated using SDS-PAGE and Western blot. BALB/c mice were injected with purified proteins on the first, eighth, and fifteenth days of feeding, respectively; serum samples were collected from mice on the day of immunization and on the 22nd day after immunization. The antibody level in mouse serum was detected by Western blotting using purified expressed proteins as antigens. IL-2, TNF-α and IFN-γ were simultaneously detected in mouse serum by ELISA. The 30 kDa protein was successfully expressed and reacted specifically with the specific serum Mhp His-Tag mouse monoclonal antibody and pig antibody. The expressed recombinant protein was immunogenic. The expression levels of IFN-γ, IL-2 and TNF-α were found to be significantly higher on day 22 than in the control group. This study suggests that the expressed recombinant protein could be used as one of the novel vaccine candidates for Mhp.

High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions

We recently demonstrated that capturing human monoclonal antibodies (hmAbs) using high affinity anti-human Fc (AHC) antibodies allows reliable characterization of antibody-antigen interactions. Here, we characterized six human Fc specific mouse monoclonal antibodies (mAbs) and compared their binding profiles with three previously characterized goat AHC polyclonal antibodies (pAbs), exhibiting properties of a good capture reagent. All six mouse AHC mAbs specifically bound with high affinity to the Fc region of hIgG1, hIgG2, hIgG4 and to 43 different hIgG variants, containing substitutions and/or mutations in the hinge and/or Fc region, that have been reported to exhibit modified antibody effector function and/or pharmacokinetics. Biacore sensor surfaces individually derivatized with mouse AHC mAbs exhibited >2.5-fold higher hIgG binding capacity compared to the three goat AHC pAb surfaces and reproducibly captured hIgG over 300 capture-regeneration cycles. The results of the capture kinetic analyses performed on 31 antibody-antigen interactions using surfaces derivatized with either of the two highest affinity AHC mAbs (REGN7942 or REGN7943) were in concordance with those performed using goat AHC pAb surfaces. Our data demonstrate that AHC mAbs such as REGN7942 and REGN7943 that have properties superior than the three goat AHC pAbs are highly valuable research reagents, especially to perform capture kinetic analyses of antibody-antigen interactions on optical biosensors.

Development and characterization of SARS-CoV-2 variant-neutralizing monoclonal antibodies

Vaccination and administration of monoclonal antibody cocktails are effective tools to control the progression of infectious diseases and to terminate pandemics such as COVID-19. However, the emergence of SARS-CoV-2 mutants with enhanced transmissibility and altered antigenicity requires broad-spectrum therapies. Here we developed a panel of SARS-CoV-2 specific mouse monoclonal antibodies (mAbs), and characterized them based on ELISA, Western immunoblot, isotyping, and virus neutralization. Six neutralizing mAbs that exhibited high-affinity binding to SARS-CoV-2 spike protein were identified, and their amino acid sequences were determined by mass spectrometry. Functional assays confirmed that three mAbs, F461G11, F461G15, and F461G16 neutralized four variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta) These mAbs are promising candidates for COVID-19 therapy, and understanding their interactions with virus spike protein should support further vaccine and antibody development.

CCNE1 mRNA and cyclin E1 protein expression as predictive biomarkers for efficacy of palbociclib plus fulvestrant versus capecitabine in the phase III PEARL study.

1014 Background: The randomized PEARL trial found no superiority of palbociclib plus endocrine therapy over capecitabine in patients (pts) with metastatic HR-positive, HER2-negative breast cancer resistant to prior aromatase inhibitors (Martin M, Ann Oncol 2020). Gene expression analysis showed high CCNE1 mRNA ( CCNE1) conferring relative resistance to palbociclib in the PALOMA-3 trial (Turner N, JCO 2019), but further validation is needed. Cyclin E1 protein (cyclin E1) expression in this context has not been studied in randomized trials. We explored CCNE1 and cyclin E1 as predictive biomarkers in tumor samples from the PEARL study. Methods: Formalin-fixed paraffin-embeded tumor samples were retrieved from pts enrolled in PEARL cohort 2 (palbociclib (PAL) + fulvestrant (FUL) vs capecitabine (CAPE)). We measured CCNE1 using the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics). Cyclin E1 immunohistochemistry (IHC) staining was performed using specific mouse monoclonal antibody HE12 (Abcam) and scored as percentage of invasive nuclei stained (0-100%). CCNE1 and cyclin E1 correlations were explored using Pearson coefficients. Cox regression models were used for progression free-survival (PFS) analyses using expression levels split by median, to define high ( &gt; median values) vs. low expression. Site of disease and prior chemotherapy were used as confounders in multivariate models. Results: Analyses were conducted in 219 pts (47% receiving PAL + FUL and 53% CAPE) with available tumors, with the analysed patients representative of the overall study. Most samples were from the archival primary (72%), obtained &gt; 5 years before this analysisº (74%). CCNE1 and cyclin E1 were only moderately correlated (r = 0.5). Median CCNE1 was higher in metastatic vs primary (7.37 vs 6.94, p &lt; 0.01), and in luminal B and non-luminal subtypes compared to luminal A (p &lt; 0.001). In patients with high CCNE1 expression, median PFS on CAPE was 10.35 and PAL + FUL was 5.68 (HR = 1.63, 95% CI 1.02-2.59, p = 0.042). In patients with low CCNE1 expression, median PFS on CAPE was 9.43 and PAL + FUL was 8.97 (adjusted HR = 0.93, 95% CI 0.59-1.48, p = 0.762, interaction p = 0.072). Median cyclin E1 protein was higher in luminal B and non-luminal subtypes compared to luminal A (p &lt; 0.01). Cyclin E1 protein expression was not predictive of treatment effect (high cyclin E1 expression CAPE vs PAL + FUL HR = 1.17, low cyclin E1 expression CAPE vs PAL + FUL HR = 1.21, interaction p = 0.977). Conclusions: High tumor CCNE1 mRNA expression identified patients with relative resistance to palbociclib plus fulvestrant, validating prior observations although without statistical significance for interaction. Assessment of Cyclin E1 protein expression did not show predictive value. Investigation treatments to enhance CDK4/6 inhibitor efficacy in tumors with high CCNE1 expression is warranted. Clinical trial information: NCT02028507 .

Anti-Vascular Endothelial Growth Factor C Antibodies Efficiently Inhibit the Growth of Experimental Clear Cell Renal Cell Carcinomas.

Despite improvement during the last ten years in the longevity of patients with metastatic clear cell renal cell carcinoma (mccRCC) the disease remains incurable. Hence, new therapeutic strategies are urgently needed. Relapse following anti-angiogenic treatment depends on the over-expression of vascular endothelial growth factor C (VEGFC), one of the main drivers of lymphangiogenesis. Therefore, we developed specific mouse monoclonal antibodies and evaluated their therapeutic efficacy in vitro and in vivo. Immunization of mice with the domain of VEGFC that stimulates the VEGF receptor 3 (VEGFR3) led to the selection of one hybridoma producing specific anti-VEGFC monoclonal antibodies. The selected 1E9 antibodies were sequenced, and the corresponding variable light and heavy chains were subcloned into expression vectors in frame with sequences encoding the human IgG1 constant heavy and light chains. CHO cells were stably transfected and cloned to produce chimeric antibodies. These antibodies inhibited the activation of VEGFR3 signaling, and therefore the proliferation and migration of VEGFC-stimulated endothelial cells. Moreover, they inhibited the proliferation of VEGFC-expressing renal cancer cells through NRP2 signaling. 1E9 antibodies inhibited the growth of experimental RCC, and their therapeutic efficacy was enhanced by the anti-VEGF antibody bevacizumab. Hence, our results suggest that targeting VEGFC could have a relevant therapeutic impact on mccRCC that relapse following anti-angiogenic treatment.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken

Abstract Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed a sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast was used to immunize the mice. Five mAbs which specifically recognize chIFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing assay to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were identified as optimum pairs of antibodies for detection of chIFN-α. The sandwich ELISA effectively detected native chicken IFN-α in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus (VSV) was evaluated in chicken embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize the activity of chicken IFN-α against VSV were identified. The newly developed antigen capture sandwich ELISA for the detection of chIFN-α will be a useful tool to monitor IFN-α production in chickens during viral infections.

Enzyme Immunoassay-Based Platform for Accurate Detection of Serum Pathological α-Synuclein in Parkinson's Disease Patients.

An assay for accurately diagnosing early stage Parkinson's Disease (PD) is currently unavailable, and therefore, there is an urgent and unmet need. Such a diagnostic assay will enable prompt institution of appropriate supportive management measures to prevent rapid deterioration of disease and improve both quality of life and life expectancy of PD patients. A reliable assay platform will also be of great benefit to drug discovery and drug development in the area of PD. To this end, we describe the development of two indirect, competitive, semiquantitative enzyme immunoassays (EIAs), each employing a disparate singularly specific mouse monoclonal antibody (ssMAb) against pathological aggregates of human α-Synuclein (αSynagg), a well-established biomarker pathognomonic of PD. Our results demonstrate that these EIAs in tandem accurately discriminated between αSynagg serum concentrations from PD patients and age-matched healthy control (HC) individuals (PD = 1700 ± 220 ng/mL; HC = 870 ± 120 ng/mL with an overall sensitivity of 56%, specificity of 63%, positive predictive value of 60%, and negative predictive value of 59%). The limits of detection of αSynagg were 400 and 300 pg/mL for ssMAbs 3C5 and 5H6, respectively. These tandem EIAs have the potential to add to the repertoire of tools for earlier diagnosis of this debilitating disorder, as well as for drug development strategies.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken

Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast were purchased from Kingfisher Biotech, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing test to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were determined and a standard curve was established using recombinant chIFN-α. The sandwich ELISA effectively detected an increased IFN-α production in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid (poly I:C), and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus was characterized in avian embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize its activity were identified. The newly developed antigen sandwich ELISA developed in this study will be a useful tool to monitor IFN-α production in chickens.

Rituximab in dermatology: Revisited

Rituximab (RTX) is a specific mouse and human chimeric monoclonal antibody that has found to have numerous applications in dermatology. Though initially approved by the US FDA for treating rheumatoid arthritis and non-Hodgkin's lymphoma; currently its approval has extended to involve the pemphigus group of disorders; both as a first line drug as well as a therapeutic strategy for recalcitrant cases of pemphigus, unresponsive to conventional therapy. Apart from pemphigus, there have been a number of cutaneous disorders where rituximab has been found to be useful. This review will give a bird's eye view of the applications of rituximab in dermatology.

Mouse Monoclonal Antibodies Generated from Full Length Human Cereblon: Detection of Cereblon Protein in Patients with Multiple Myeloma.

Immunomodulatory drugs (IMiDs) are profoundly active compounds in the treatment of patients with multiple myeloma (MM). However, despite the fact that treatment with IMiDs has dramatically improved survival for patients with MM, the majority of MM patients develop IMiDs resistance over time. We have found that expression of functional cereblon is required for IMiDs′ action. In addition, it has been reported that cells expressing high levels of cereblon are resistant to proteasome inhibitor, implying that patients with high levels of cereblon should be resistant to proteasome inhibitor. If the above conclusions are correct, cereblon could be considered as a biomarker to determine which standard regimens should be used to treat patients with MM. Unfortunately, the conclusions mentioned above have not been clinically confirmed. In order to confirm these conclusions, we have generated three highly specific mouse monoclonal antibodies (mAbs) against full-length human cereblon. These mAbs can be used to do western blot, immunoprecipitation and immunohistochemistry staining. In addition, their epitopes have been precisely determined and the peptides covering their epitopes completely blocked the antibody binding to cereblon in western blot analysis or in immunohistochemistry staining of MM patients′ specimens.

Abstract 23: Antibody targeting of ADAM8 for treatment of triple-negative breast cancer

Abstract Triple-Negative Breast Cancer (TNBC) accounts for 25% of breast cancer deaths. Current therapeutic options are restricted to chemotherapy (CT) and radiation, which fail to block disease progression. Recently, we identified the cell surface protein ADAM8 as a critical driver of TNBC tumor growth and of metastasis in half of all breast cancers (EMBO Mol. Med. 6:278, 2014). Elevated ADAM8 levels correlate with poor breast cancer patient outcome. One third of TNBC patient samples have high ADAM8 levels, while it is undetectable in normal breast tissue. ADAM8 Metalloproteinase (MP) and Disintegrin (DI) domains promote angiogenesis needed for tumor growth, and cancer cell adhesion to and invasion through the endothelium needed for dissemination, respectively. Using a prototype reagent in mice, we validated a unique antibody-based strategy that simultaneously targets ADAM8 MP/DI activities as an effective therapeutic intervention in TNBC. Our group has now prepared a panel of pre-clinical, highly specific anti-human ADAM8 mouse monoclonal antibodies (ADPs) of either IgG1 or IgG2 subclass with dual MP/DI antagonist activity. The top 6 ADPs bind ADAM8 with low dissociation constants of 1.3 x 10-9 M to 7.23 x 10-8 M. The 4 most effective inhibitory ADPs in cell-based assays were screened in a single-dose efficacy study (10 mg/kg 3x/wk) against pre-existing MDA-MB-231 cell-derived tumors (50-75 mm3). ADP2 and ADP13 treatment resulted in significant tumor volume reduction, of 47% and 52%, respectively. Dose-response curves are in progress. ADP13 was also tested in a neo-adjuvant model for its ability to reduce metastases. Antibody treatment was initiated as above and continued for 6 weeks after tumors were resected at a volume of ~200 mm3. Metastases in dissected organs were examined using biophotonic imaging. ADP13 substantially decreased metastasis to multiple organs. Lastly, a neo-adjuvant survival experiment was performed, as above, except that following surgical resection of tumors mice were treated for 12 weeks. Recurrence at the primary site was assessed using palpation. Mice were sacrificed when recurrent tumors reached 900 mm3. ADP13 treatment resulted in significantly improved disease-free survival (P=0.05) and overall survival (P=0.03). More recently, we noted that ADAM8 regulates miRNAs associated with resistance to CT and showed that ADAM8 inhibition sensitizes TNBC cells to Cisplatin, Paclitaxel, and Doxorubicin. In vivo testing of a combinatorial anti-ADAM8 + CT approach is in progress and may present a promising strategy for early entry into the clinic. Conclusions: A new anti-ADAM8 antibody therapeutic shows efficacy against TNBC tumor growth and dissemination, and improves survival in pre-clinical testing. We hypothesize that addition of an anti-ADAM8 antibody to current CT standards of care will enhance tumor killing, reduce metastasis, and increase survival of patients with ADAM8-expressing TNBC. Citation Format: Sonia G. Das, Stefania Pianetti, Gail E. Sonenshein, Nora D. Mineva. Antibody targeting of ADAM8 for treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 23. doi:10.1158/1538-7445.AM2017-23

Characterization of a Novel Specific Mouse Monoclonal Antibody Targeted to Envelope Protein E1/E2 of Hepatitis C Virus

Characterization of a Novel Specific Mouse Monoclonal Antibody Targeted to Envelope Protein E1/E2 of Hepatitis C Virus

The effects of pulsed ultraviolet light, cold atmospheric pressure plasma, and gamma-irradiation on the immunoreactivity of soy protein isolate

Abstract This study investigates the effect of nonthermal processing technologies on soy immunoreactivity. Soy protein isolate was treated with pulsed ultraviolet (PUV) light, direct and remote cold atmospheric pressure plasma (CAPP), and gamma-irradiation (3–100 kGy). Sample weight, surface temperature, hydrogen peroxide content, and pH value have been measured. SDS-PAGE analysis revealed reduced protein intensity bands corresponding to major soy allergens β-conglycinin (Gly m5) and glycinin (Gly m6). Sandwich ELISA using specific mouse monoclonal anti-Gly m5 antibodies (mAbs) confirmed a loss of soy immunoreactivity following PUV light, direct CAPP, and gamma-irradiation with increasing dose and time. The maximum reduction in immunoreactivity (91–100%) in the soluble protein fraction was achieved by direct CAPP as well as PUV light and gamma-irradiation treatment. A decreased immunoreactivity up to 89% was observed for samples treated with remote CAPP. These innovative technologies might have great potential for industrial application due to their effectiveness in reduction of soy immunoreactivity.

A novel IgE-binding epitope of cat major allergen, Fel d 1

Information on the antigenic repertoire, especially the IgE-binding epitopes of an allergen is important for understanding the allergen induced immune response and cross-reactivity, as well as for generating the hypoallergenic variants for specific component resolved immunotherapy/diagnosis (CRIT and CRD). Data on the IgE-binding epitopes of cat allergens are scarce. In this study, a novel IgE-binding epitope of the cat major allergen, Fel d 1, was identified. Mouse monoclonal antibody (MAb) specific to the Fel d 1 was produced. Computerized intermolecular docking was used for determining the residues of the Fel d 1 bound by the specific MAb. The presumptive surface exposed residues of the Fel d 1 intrigued by the MAb are located on the chain 1. They are: L34 and T37 (helix 1); T39 (between helices 1 and 2); P40, E42 and E45 (helix 2); R61, K64, N65 and D68 (helix 3); and E73 and K76 (helix 4). The MAb competed efficiently with the cat allergic patients' serum IgE for Fel d 1 binding in the competitive IgE binding assay, indicating allergenicity of the MAb epitope. The newly identified allergenic epitope of the Fel d 1 is useful in a design of the CRIT and CRD for cat allergy.

A Highly Specific Monoclonal Antibody for Botulinum Neurotoxin Type A-Cleaved SNAP25.

Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A’s location, distribution, and movement within a cell. Due to BoNT/A’s high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25197) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25197, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25197 rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25197 in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25197-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.

Blinatumomab: A First-in-Class Bispecific T-Cell Engager for Precursor B-Cell Acute Lymphoblastic Leukemia.

To review the clinical pharmacology, efficacy, and safety of blinatumomab for the treatment of pediatric and adult precursor B-cell acute lymphoblastic leukemia (B-ALL). A literature search of EMBASE (1947 to April 2015), Medline (1946 to April 2015), PubMed (1996 to April 2015), the U.S. National Institutes of Health Clinicaltrials.gov, the Food and Drug Administration, and relevant meeting abstracts was conducted using the terms blinatumomab, BiTE, bispecific T-cell engager, MT103, MEDI-538, and Blincyto. Human and animal studies describing the pharmacology, pharmacokinetics and pharmacodynamics, efficacy, and safety of blinatumomab for precursor B-ALL were identified. Blinatumomab is a first-in-class bispecific T-cell engager (BiTE) antibody derived from a B-lineage specific antitumor mouse monoclonal antibody that binds to both CD19 of B-cells and CD3 of T-cells. A pivotal phase II trial demonstrated that response rates were high in a refractory or relapsed patient population, with 43% achieving complete remission (CR). Median relapse-free survival was 5.9 months for those with CR or CR with incomplete hematological recovery. Median overall survival was 6.1 months, and 60% of patients achieved minimal residual disease (MRD) negativity. The most common adverse events included pyrexia, neurological events, headache, febrile neutropenia, peripheral edema, nausea, hypokalemia, constipation, and anemia. Blinatumomab is a novel BiTE therapeutic monoclonal antibody that has shown promising results in patients with relapsed or refractory ALL or those achieving a CR with persistent MRD. Phase III clinical trials should define the optimal place in therapy of blinatumomab.