Mouse Monoclonal Antibody Research Articles

An ELISA-based antigenicity test of rabies recombinant glycoprotein cannot predict its protective potency in vivo

The potency of human and veterinary rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, variable, and requires sacrifice of large numbers of mice. ELISA-based methods quantifying rabies glycoprotein (rGP) are being developed as potential alternatives to the NIH potency test for release of rabies vaccines. The aim of the current study was focused on the evaluation of in vitro- and in vivo-based assays in order to assess their concurrence for adequate and reliable assessment of immunogenicity and protective potency of a plant-derived recombinant rGP. The recombinant rGP of strain ERA.KK was engineered, expressed and purified from Nicotiana benthamiana plants. The recombinant rGP excluded the transmembrane and intracytoplasmic domains. It was purified by chromatography (≥90%) from the plant biomass, characterized, and mainly presented as high molecular weight forms, most likely soluble aggregates, of the rGP ectodomain. It was well-recognized and quantified by an ELISA, which utilizes two mouse monoclonal antibodies, D1-25 and 1112-1, and which should only recognize the native trimeric form of the rGP. However, in mice, the recombinant rGP did not induce the production of anti-rabies virus neutralizing antibodies and did not confer protection after intracerebral viral challenge. Similar immunogenicity was observed in guinea pigs and rabbits. Our results demonstrate that use of the ELISA method described here is not predictive of performance in vivo. These data highlight the critical need to develop in vitro potency assays that reliably define the antigen content that can induce a protective response.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Neutralization or enhancement of SARS-CoV-2 infection by a monoclonal antibody targeting a specific epitope in the spike receptor-binding domain

Neutralizing antibodies (NAbs) are believed to be promising prophylactic and therapeutic treatment against the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we reported two mouse monoclonal antibodies 7 Eb-4G and 1Ba–3H that specifically recognized the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein without exhibiting cross-reactivity with the S proteins of SARS-CoV and MERS-CoV. The binding epitopes of 7 Eb-4G and 1Ba–3H were respectively located in the regions of residues 457–476 and 477–496 in the S protein. Only 1Ba–3H exhibited the neutralizing activity for preventing the pseudotyped lentivirus from binding to the angiotensin-converting enzyme 2 (ACE2)-transfected HEK293T cells. The competitive ELISA further showed that 1Ba–3H interfered with the binding between RBD and ACE2. Epitope mapping experiments demonstrated that a single alanine replacement at residues 480, 482, 484, 485, and 488–491 in the RBD abrogated 1Ba–3H binding. 1Ba–3H exhibited the neutralizing activity against the wild-type, Alpha, Delta, and Epsilon variants of SARS-CoV-2, but lost the neutralizing activity against Gamma variant in the plaque reduction assay. On the contrary, 1Ba–3H enhanced the cellular infection of Gamma variant in a dose-dependent manner. Our findings suggest that the antibody-dependent enhancement of infection mediated by the RBD-specific antibody for different SARS-CoV-2 variants must be considered while developing the NAb.

Moesin: A novel receptor on NK lymphocytes binds to TOMM40 on K562 leukemia cells initiating cytolysis

Natural killer lymphocytes (NK cells) are the first line of defense (innate immunity) against viral infections and leukemia since they do not require activation to deliver a lethal hit to infected/aberrant cells. In contrast, T lymphocytes require stimulation by a foreign/neo – antigen, which may take days before they are active against the pathogen (adaptive immunity). A number of receptors on activated NK cells that kill the prototypical leukemia target cell line, K562, have been identified. To date, the receptor(s) by which freshly isolated unstimulated NK cells (naïve, nNK) kill K562 has not been fully elucidated. We provide peptide sequence and immune-blot data from ligand pull down experiments that moesin, a protein that typically links the inner leaf of the plasma membrane to the cytoskeleton, additionally, in NK cells, localizes to the cell surface where it may bind to its ligand, TOMM40 (aka Haymaker, HYMKR), on leukemia cells thereby initiating their destruction. Flow cytometry experiments with a mouse monoclonal antibody (Mab) to a moesin peptide (554 to 565) were performed. Moesin was detected on the surface of CD3–, CD16+nNK cells but was not detected on the surface of freshly isolated unstimulated CD3+, CD16– T cells or CD19+, CD16– B cells from healthy subjects. Moesin, is therefore another marker that distinguishes unstimulated CD3–, CD16+ NK cells from other non-activated lymphocytes. The anti –moesin peptide Mab was highly effective (>95% inhibition) in blocking target cell cytolysis by CD16+ lymphocytes demonstrating that moesin–HYMKR interaction appears to be necessary for most of the observed cell death of K562 caused by unstimulated NK cells.

PET in vivo generators 134Ce and 140Nd on an internalizing monoclonal antibody probe

The in vivo-generator radionuclides 140Nd (t1/2 = 3.4 d) and 134Ce (t1/2 = 3.2 d) were used to trace a urokinase-type plasminogen activator (uPA)-targeting mouse monoclonal antibody, ATN-291, in U87 MG xenograft tumor-bearing mice. ATN-291 is known to internalize on the uPA/uPA-receptor pair, making it an appropriate targeting vector for investigating the fate of in vivo generator daughters on internalizing probes. Ante-mortem and post-mortem PET imaging at 120 h post-injection gave no indication of redistribution of the positron emitting daughter nuclides 134La and 140Pr from tumor tissue (p > 0.5). The lack of redistribution indicates that the parent radionuclides 134Ce and 140Nd could be considered as long-lived PET-diagnostic matches to therapeutic radionuclides like 177Lu, 161Tb and 225Ac when internalizing bioconjugates are employed.

Matrix Metalloproteinase-9 as Prognostic Factor for the Treatment of HER-2 Enriched Breast Cancer.

Background:MMP-9 plays a significant role in invasion and migration of tumor cells and metastasis. A combination MMP-9 biomarker with HER2 and Ki-67 is expected to provide more specificity in prognosis for better breast cancer (BC) treatment options. Methods: A retrospective cohort of 34 patients with HER2 enriched breast cancer were studied from January 2016 until December 2020. Assessment MMP-9 by IHC using Monoclonal Mouse Anti-Human MMP-9 Antigen with semiquantitative immunoreactive scores methods was done. Results:The samples included patients aged 29 to 66 years. Patients’ educational level was mostly high school graduation (n=17; 50%). Distant metastases occurred in 10 (29.4%) patients, histopathological Grade II was found in 29 (85.7%) patients, positive LVI was found in 18 (52.9%) patients, and high proliferation rate (Ki67 > 20%) was found in 32 (94.1%) patients. All the patients underwent MRM with a history of chemotherapy in 29 (85.3%) patients, radiotherapy in 14 (41.2%) patients, and targeted therapy in only seven (20.6%) patients. Most of the patients had locally advanced stage III (n=21; 61.8%). The MMP-9 High expression was found in 20 (58.8%) patients and 14 (41.2%) patients had a low expression. A significant relationship was found between MMP-9 expression and DFS (p=0.023); while a significant relationship was found with OS (p=0.093). The mean DFS for High expression MMP-9 was 37.3 months and 45.3 months for low expression. The mean OS was 37.6 months for High-intensity MMP-9 and 42.7 months for Low-intensity. Conclusions:The MMP-9 expression (p=0.037), age group<40 years old (p=0.024), and the history of radiotherapy (p=0.035) were significantly related to he occurrence of distant metastases. The MMP-9 High expression had a 7.5 times greater risk of distant metastases. Impacts:The MMP-9 can be used as a prognostic factor for distant metastasis in HER2 Enriched BC.

The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has become a worldwide pandemic, and there is a pressing need for the rapid development of novel therapeutic strategies. SARS-CoV-2 viral entry is mediated by interaction between the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein and host cellular receptor, human angiotensin converting enzyme 2 (ACE2). The lack of a high throughput screening (HTS) platform for candidate drug screening means that no targeted COVID-19 treatments have been developed to date. To overcome this limitation, we developed a novel, rapid, simple, and HTS binding assay platform to screen potential inhibitors of the RBD-ACE2 complex. Three “neutralizing” mouse monoclonal antibodies capable of blocking the RBD-ACE2 interaction were identified using our binding assay and pseudovirus neutralization assay followed by further validation with the Focus Reduction Neutralization Test (FRNT), which analyzes the neutralization capacity of samples in the presence of live SARS-CoV-2. Furthermore, the consistency of our binding assay and FRNT results (R2 = 0.68) was demonstrated by patients' serum, of which were COVID-19 positive (n = 34) and COVID-19 negative (n = 76). Several small molecules selected for their potential to inhibit the Spike-ACE2 complex in silico were also confirmed with the binding assay. In addition, we have evaluated vaccine efficacy using binding assay platform and validated through pseudovirus neutralization assay. The correlation between binding assay & psuedovirus assay of the post vaccinated serum showed well correlated (R2 = 0.09) Moreover, our binding assay platform successfully validated different Spike RBD mutants. These results indicate that our binding assay can be used as a platform for in vitro screening of small molecules and monoclonal antibodies, and high-throughput assessment of antibody levels after vaccination. When conducting drug screening, computer virtual screening lacks actual basis, construction of pseudoviruses is relatively complicated, and even FRNT requires a P3 laboratory. There are few methods to determine the competitiveness of the target drug and SRBD or ACE2. Our binding assay can fill this gap and accelerate the process and efficiency of COVID-19 drug screening.

Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen

Structural analysis and detection of optimal cell surface localization of labyrinthin, a pan-adenocarcinoma target, was studied with respect to adenocarcinoma specificity vs. normal and non-adenocarcinoma cells. Patient-derived tissue microarray immunohistochemistry (IHC) was performed on 729 commercially prepared tissue blocks of lung, colon, breast, pancreas, prostate, and ovary cancers combined, plus a National Cancer Institute (NCI) tissue microarray derived from another 236 cases. The results confirmed that anti-labyrinthin mouse monoclonal MCA 44-3A6 antibody recognized adenocarcinomas, but not normal or non-adenocarcinoma cancer cells. The consensus of multiple topology analysis programs on labyrinthin (255 amino acids) estimate a type II cell membrane associated protein with an N-terminus signal peptide. However, because the labyrinthin sequence is enveloped within the 758 amino acids of the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH), a purported tumor associated antigen, standard IHC methods that permeabilize cells can expose common epitopes. To circumvent antibody cross-reactivity, cell surface labyrinthin was distinguished from intracellular ASPH by FACS analysis of permeabilized vs non-permeabilized cells. All permeabilized normal, adeno-and non-adenocarcinoma cells produced a strong MCA 44-3A6 binding signal, likely reflecting co-recognition of intracellular ASPH proteins along with internalized labyrinthin, but in non-permeabilized cells only adenocarcinoma cells were positive for labyrinthin. Confocal microscopy confirmed the FACS results. Labyrinthin as a functional cell-surface marker was suggested when: 1) WI-38 normal lung fibroblasts transfected with labyrinthin sense cDNA displayed a cancerous phenotype; 2) antisense transfection of A549 human lung adenocarcinoma cells appeared more normal; and 3) MCA44-3A6 suppressed A549 cell proliferation. Collectively, the data indicate that labyrinthin is a unique, promising adenocarcinoma tumor-specific antigen and therapeutic target. The study also raises a controversial issue on the extent, specificity, and usefulness of ASPH as an adenocarcinoma tumor-associated antigen.

Characterization of a Novel Monoclonal Antibody for Serine-129 Phosphorylated α-Synuclein: A Potential Application for Clinical and Basic Research.

The Lewy bodies (LBs) are the pathological hallmark of Parkinson's disease (PD). More than 90% of α-synuclein (α-syn) within LBs is phosphorylated at the serine-129 residue [pSer129 α-syn (p-α-syn)]. Although various studies have revealed that this abnormally elevated p-α-syn acts as a pathological biomarker and is involved in the pathogenic process of PD, the exact pathophysiological mechanisms of p-α-syn are still not fully understood. Therefore, the development of specific and reliable tools for p-α-syn detection is important. In this study, we generated a novel p-α-syn mouse monoclonal antibody (C140S) using hybridoma technology. To further identify the characteristics of C140S, we performed several in vitro assays using recombinant proteins, along with ex vivo assays utilizing the brains of Thy1-SNCA transgenic (Tg) mice, the preformed fibril (PFF)-treated neurons, and the brain sections of patients with PD. Our C140S specifically recognized human and mouse p-α-syn proteins both in vitro and ex vivo, and similar to commercial p-α-syn antibodies, the C140S detected higher levels of p-α-syn in the midbrain of the Tg mice. Using immunogold electron microscopy, these p-α-syn particles were partly deposited in the cytoplasm and colocalized with the outer mitochondrial membrane. In addition, the C140S recognized p-α-syn pathologies in the PFF-treated neurons and the amygdala of patients with PD. Overall, the C140S antibody was a specific and potential research tool in the detection and mechanistic studies of pathogenic p-α-syn in PD and related synucleinopathies.

Immunological characterization of chicken tumor necrosis factor-α (TNF-α) using new sets of monoclonal antibodies specific for poultry TNF-α

Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of the TNF superfamily. TNF-α is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-α (chTNF-α) is a long-missed avian ortholog, and its immunological properties remain largely unknown compared to those of its mammalian counterparts. Here, we report the functional characterization and immunomodulatory properties of chTNF-α using a panel of newly developed anti-chTNF-α mouse monoclonal antibodies (mAbs). Using anti-chTNF-α mAbs, we determined the tissue expression of chTNF-α in lymphoid and non-lymphoid organs. A chTNF-α-specific antigen-capture sandwich ELISA was developed using compatible mAb partners by screening and validation of ten different mAbs. Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-α in the circulation of Clostridium perfringens, Eimeria, or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expression of chTNF-α in primary immune cells or cell lines derived from chickens was validated by immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Notably, both 3G11 and 12G6 neutralized chTNF-α-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results enhance our understanding of the functional characteristics of chTNF-α, and these anti-chTNF-α mAbs will serve as valuable immune reagents to inform on inflammatory responses and disease pathogenesis in the fundamental and applied studies of avian species.

Quantifying Circulating IgY Antibody Responses against Select Opportunistic Bacterial Pathogens and Correlations with Body Condition Factors in Wild American Alligators, Alligator mississippiensis.

Simple SummaryImmunoglobulin Y (IgY) was purified from American alligator, Alligator mississippiensis, serum and used to develop a monoclonal antibody (mAb AMY-9) specific for the heavy chains of IgY. This antibody tool was then used to develop an ELISA for quantifying serum antibody responses against whole bacterial pathogens in alligators sampled in Florida, USA and South Carolina, USA. Antibody responses against some of the bacteria were very robust and varied by location and year, and in general these antibody responses correlated well with body condition factors, such as body-mass-indices (BMI). A novel mAb is now available to the scientific community interested in disease ecology of alligators.Little is known about the disease ecology of American alligators (Alligator mississippiensis), and especially how they respond immunologically to emerging infectious diseases and zoonotic pathogens. In this study, we examined serum samples collected from wild alligators in Florida (2010–2011) and South Carolina (2011–2012, 2014–2017) for antibody responses to multiple bacteria. Immunoglobulin Y (IgY) was purified from serum to generate a mouse monoclonal antibody (mAb AMY-9) specific to the IgY heavy chain. An indirect ELISA was then developed for quantifying antibody responses against whole cell Escherichia coli, Vibrio parahaemolyticus, Vibrio vulnificus, Mycobacterium fortuitum, Erysipelothrix rhusiopthiae, and Streptococcus agalactiae. In Florida samples the primary differences in antibody levels were between January–March and late spring through summer and early fall (May-October), most likely reflecting seasonal influences in immune responses. Of note, differences over the months in antibody responses were confined to M. fortuitum, E. rhusiopthiae, V. vulnificus, and E. coli. Robust antibody responses in SC samples were observed in 2011, 2014, and 2015 against each bacterium except E. coli. All antibody responses were low in 2016 and 2017. Some of the highest antibody responses were against V. parahaemolyticus, M. fortuitum, and E. rhusiopthiae. One SC alligator estimated to be 70+ years old exhibited the highest measured antibody response against V. parahaemolyticus and M. fortuitum. By combining data from both sites, we show a clear correlation between body-mass-indices (BMI) and antibody titers in all six of the bacteria examined. Our study provides a critical antibody reagent and a proof-of-concept approach for studying the disease ecology of alligators in both the wild and in captivity.

Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

BackgroundHypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first- or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy.MethodsParental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma, we obtained 25 clones. Each clone was tested for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system.ResultsBased on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity.ConclusionCA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

Quantitative CK19 biomarker detection in breast cancer cell lines.

Cytokeratin19 (CK19) was detected as the most related marker for circulating tumor cells, which was assessed in specific cell lines. MCF7, SKBR3, T47D, and MDA-MB-231, and HeLa cell line as negative control were used. CK19 expression was confirmed by using mouse monoclonal anti-human CK19 antibody. CK19 detection in MDA-MB-231 was not observed. CK19 marker expression was compared in T47D, MCF7, and SKBR3 cell lines. T47D and MCF7 belonged to the luminal subtype of breast cancer (BC) that CK19 expression regulated with an ER marker. SKBR3 belonged to the HER2 positive subtype of BC. However, MDA-MB-231 belonged to the claudin-low subtype of BC that lack of CK19 expression strongly is related to negative ER, PR, and HER2. Therefore, there are not only quantitative differences in CK19 expression, but its expression could also link to the other markers of BC that should be considered in the molecular classification of breast carcinoma. Different expression levels related to cell classification could be useful in the prognosis and treatment of cancers with epithelial origins.

A universal in silico V(D)J recombination strategy for developing humanized monoclonal antibodies

BackgroundHumanization of mouse monoclonal antibodies (mAbs) is crucial for reducing their immunogenicity in humans. However, humanized mAbs often lose their binding affinities. Therefore, an in silico humanization method that can prevent the loss of the binding affinity of mAbs is needed.MethodsWe developed an in silico V(D)J recombination platform in which we used V(D)J human germline gene sequences to design five humanized candidates of anti-tumor necrosis factor (TNF)-α mAbs (C1–C5) by using different human germline templates. The candidates were subjected to molecular dynamics simulation. In addition, the structural similarities of their complementarity-determining regions (CDRs) to those of original mouse mAbs were estimated to derive the weighted interatomic root mean squared deviation (wRMSDi) value. Subsequently, the correlation of the derived wRMSDi value with the half maximal effective concentration (EC50) and the binding affinity (KD) of the humanized anti-TNF-α candidates was examined. To confirm whether our in silico estimation method can be used for other humanized mAbs, we tested our method using the anti-epidermal growth factor receptor (EGFR) a4.6.1, anti-glypican-3 (GPC3) YP9.1 and anti-α4β1 integrin HP1/2L mAbs.ResultsThe R2 value for the correlation between the wRMSDi and log(EC50) of the recombinant Remicade and those of the humanized anti-TNF-α candidates was 0.901, and the R2 value for the correlation between wRMSDi and log(KD) was 0.9921. The results indicated that our in silico V(D)J recombination platform could predict the binding affinity of humanized candidates and successfully identify the high-affinity humanized anti-TNF-α antibody (Ab) C1 with a binding affinity similar to that of the parental chimeric mAb (5.13 × 10−10). For the anti-EGFR a4.6.1, anti-GPC3 YP9.1, and anti-α4β1 integrin HP1/2L mAbs, the wRMSDi and log(EC50) exhibited strong correlations (R2 = 0.9908, 0.9999, and 0.8907, respectively).ConclusionsOur in silico V(D)J recombination platform can facilitate the development of humanized mAbs with low immunogenicity and high binding affinities. This platform can directly transform numerous mAbs with therapeutic potential to humanized or even human therapeutic Abs for clinical use.Graphical

Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

InstructionThe human amphoterin-induced gene and open reading frame (AMIGO) was identified as a novel cell adhesion molecule of type I transmembrane protein. AMIGO2 is one of three members of the AMIGO family (AMIGO1, 2, and 3), and the similarity between them is approximately 40% at the amino acid level. We have previously shown that AMIGO2 functions as a driver of liver metastasis. Immunohistochemical analysis of AMIGO2 expression in colorectal cancer (CRC) using a commercially available anti-AMIGO2 mouse monoclonal antibody clone sc-373699 (sc mAb) correlated with liver metastasis and poor prognosis. However, the sc mAb was found to be cross-reactive with all three molecules in the AMIGO family.MethodsWe generated a rat monoclonal antibody clone rTNK1A0012 (rTNK mAb) for human AMIGO2. The rTNK mAb was used to re-evaluate the association between AMIGO2 expression and liver metastases/clinical outcomes using the same CRC tissue samples previously reported with sc mAb.ResultsWestern blot analysis revealed that a rTNK mAb was identified as being specific for AMIGO2 protein and did not cross-react with AMIGO1 and AMIGO3. The rTNK mAb and sc mAb showed higher AMIGO2 expression, which correlates with a high frequency of liver metastases (65.3% and 47.5%, respectively), while multivariate analysis showed that AMIGO2 expression was an independent prognostic factor for liver metastases (p = 7.930E-10 and p = 1.707E-5). The Kaplan-Meier analyses showed that the rTNK mAb (p = 0.004), but not sc mAb (p = 0.107), predicted worse overall survival in patients with high AMIGO2 expression. The relationship between AMIGO2 expression and poor disease-specific survival showed a higher level of significance for rTNK mAb (p = 0.00004) compared to sc mAb (p = 0.001).ConclusionsThese results indicate that the developed rTNK1A0012 mAb is an antibody that specifically recognizes AMIGO2 by immunohistochemistry and can be a more reliable and applicable method for the diagnostic detection of liver metastases and worse prognosis in patients with high AMIGO2-expressing CRC.

Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma.

Plasma thrombopoietin (TPO) measurements help distinguish between different types of thrombocytopenia but are not feasible in routine clinical practice. We developed a fully automated quantitative chemiluminescent enzyme immunoassay (CLEIA) for measuring TPO (TPO-CLEIA), which is a one-step sandwich-type assay. This assay utilizes a mouse monoclonal capture antibody, which has the neutralizing epitope of the interaction between TPO and the TPO receptor, and a newly generated rabbit monoclonal detector antibody. In analytical performance studies, this assay showed good linearity over the measuring range and high sensitivity. The limit of quantification (LoQ) of this assay was 3.4 pg/mL; low TPO concentration values of almost all healthy individuals exceeded the LoQ value. In clinical validation studies, TPO levels obtained from patients with aplastic anemia (AA) significantly increased, whereas those of patients with immune thrombocytopenia (ITP) were normal or slightly increased. The cutoff value for TPO-CLEIA corresponding to the previously reported values was useful for distinguishing between ITP and AA. These results suggest that TPO-CLEIA can quantify human plasma TPO levels with high accuracy and sensitivity and has the potential to facilitate routine clinical measurement of TPO in patients with various types of thrombocytopenia.

Differential tumor inhibitory effects induced by HER3 extracellular subdomain-specific mouse monoclonal antibodies.

The therapeutic potential of targeting the human epidermal growth factor receptor-3 (ErbB3/HER3) has long been ignored due to impaired tyrosine kinase function and low expression level in tumor cells compared with EGFR and HER2. Although recent investigations have explored the potential benefit of HER3 targeting and several anti-HER3 agents have been developed, there is still a critical need to design and produce more efficient therapeutics. This study was designed to develop tumor inhibitory monoclonal antibodies (MAbs) against different extracellular subdomains of HER3. Distinct extracellular subdomains of HER3 (DI+II and DIII+IV) were utilized to produce MAbs by hybridoma technology. Biochemical and functional characteristics of these MAbs were then investigated by various methodologies, including immunoblotting, flow cytometry, cell proliferation, cell signaling, and enzyme-linked immunosorbent assays. Four anti-DI+II and six anti-DIII+IV MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-DI+II and four anti-DIII+IV MAbs recognized the native form of HER3 by immunoblotting. Four MAbs recognized the membranous HER3 by flow cytometry leading to induction of different levels of receptor internalization and subsequent degradation. Results of cell proliferation assays using these MAbs indicated that they differentially inhibited proliferation of HER3-expressing cancer cells and showed considerable synergistic effects in combination with trastuzumab. Selected MAb with the highest inhibitory effect significantly inhibited the phosphorylation of AKT and ERK1/2 molecules. Some of the anti-HER3 MAbs produced in this study displayed tumor inhibitory function and may be considered promising candidates for future HER3-targeted cancer therapy.

The role of endometrial staining for CD138 as a marker of chronic endometritis in predicting live birth.

Chronic endometritis (CE) is diagnosed via endometrial biopsy and staining for plasma cells. A threshold plasma cell count that identifies CE and predicts pregnancy outcomes has not been established, and the prevalence of plasma cells in the general infertile population is unknown. The purpose of this study was to determine the prevalence of plasma cells in the general infertile population and whether a threshold exists which predicts live birth. Endometrial samples were obtained prospectively from 80 women undergoing IVF, embedded in paraffin, and stained for plasma cells using mouse mono-clonal antibody for CD138. Slides were reviewed at 20× magnification and 10 random images captured. Three reviewers graded each image for plasma cells. Participants underwent single, euploid, and frozen blastocyst transfer. Forty-nine percent of samples had ≥1 plasma cell across 10 HPFs, 11% had ≥5 cells across 10 HPFs, and 4% had ≥10 cells across 10 HPFs. There was no difference in prevalence between those who did and did not achieve live birth. Using thresholds of 1, 5, and 10 plasma cells per 10 HPFs, there were no differences in implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates between patients with and without CE. Endometrial plasma cells are present in half the general infertile population and do not predict implantation, clinical pregnancy, clinical pregnancy loss, or live birth rates at low levels.

Heterogeneity of Fecal Calprotectin Reflecting Generation of Neutrophil Extracellular Traps (NETs) in the Gut: New Immunoassays Are Available

Background: We aimed at obtaining more information on the structure of fecal calprotectin (CP) as a basis for establishing improved quantitative assays and detection of Neutrophil Extracellular Traps (NETs) in stools. Commercial fecal CP assays produce different results, probably due to differences in antibodies, extraction procedures, and standards used. In addition, the structure of fecal CP may be different from that in the standard so that rules for immunoassays are violated. We aimed at solving these problems by studying the structure of fecal CP and developing new antibodies and assay procedures including some for NETs in stools. Methods and Findings: Stool samples from children with abdominal symptoms were extracted by a conventional and a new procedure. Some extracts were run on anion exchange and size exclusion chromatography, and fractions were tested on ELISAs by use of ten new mouse monoclonal antibodies against the CP subunit S100A9. Hybrid ELISAs (named HELISA) were established using anti-DNA or anti-histones for coating of microwells, and enzyme labelled anti-CP was used for development. By ion exchange chromatography, five to ten fecal CP subfraction peaks differing in net electric charge were found, all of which contained the major chromatin components. The presence of DNA and histones followed calprotectin in the chromatographic fractions suggesting that NETs are generated in the gut lumen. The new CP monoclonals reacted very differently against the subfractions so that a mixture of them (called MiMo) must be used to obtain reliable assay values for fecal CP. A new method called FELISA was developed where standards and samples are applied directly in Nunc (Denmark) MaxiSorp plates, without any catching antibody. It takes advantage of the property of CP to bind strongly to the plastic in wells. This method has a higher sensitivity because it will detect CP molecules with only one antigenic epitope available. It will give more reliable estimates and more efficient selection of patients for complex diagnostic procedures. We also developed an alternative to the FELISA: a competitive ELISA where S100A9 coated in microwells will compete with CP in standards and samples for binding to a properly diluted HRP-anti-CP solution. In this method, the presence of other proteins in extraction or dilution buffers will not interfere. Using the HELISA, about 65% of the patients had detectable fecal NETs in concentrations between 150 and 1500 ng/mL; however, the values correlated poorly with CP values. Extraction of fecal samples with a simple buffer of TBS, and pH 5 with 5 mM EDTA, gave a yield of about 90%, while the yields of commercial kits are not specified or lie around 50%. A fecal CP standard will bring methods in accordance with the requirements for immunoassays that the structure of CP in the standard and sample must be the same. A mixture of fecal anion exchange fractions as a standard may be a solution to this problem. The principle worked in the first trial by giving the same values after storage of such a standard at 5C for four months. Conclusions: Fecal CP consists of at least five subfractions containing NETs or degradation products thereof. Commercial kits should not be accepted for clinical use unless it has been shown that they can detect all subfractions which may require the use of a mixture of monoclonals. The methods presented here can be used for such a quality control. The HELISA methods can be used for assays on NETs in stools and to study their possible pathogenic effects in the gut. Use of the FELISA and the S100A9 competitive method may give increased sensitivity, higher precision, and better selection of patients for more complex procedures.

Generation, characterization, and protective ability of mouse monoclonal antibodies against the HA of A (H1N1) influenza virus.

Influenza virus infections pose a continuous threat to human health. Although vaccines function as a preventive and protective tool, they may not be effective due to antigen drift or an inaccurate prediction of epidemic strains. Monoclonal antibodies (mAbs) have attracted wide attention as a promising therapeutic method for influenza virus infections. In this study, three hemagglutinin (HA)-specific mAbs, named 2A1, 2H4, and 2G2, respectively, were derived from mice immunized with the HA protein from A/Michigan/45/2015(H1N1). The isolated mAbs all displayed hemagglutination inhibition activityand the 2G2 mAb exhibited the strongest neutralization effect. Two amino acid mutations (A198E and G173E), recognized in the process of selection of mAb-resistant mutants, were located in antigenic site Sb and Ca1, respectively. In prophylactic experiments, all three mAbs could achieve 100% protection in mice infected with a lethal dose of A/Michigan/45/2015(H1N1). A dose of 1 mg/kg for 2H4 and 2G2 was sufficient to achieve a full protective effect. Therapeutic experiments showed that all three mAbs could protect mice from death if they received the mAb administration at 6 h postinfection, and 2G2 was still protective after 24 h. Our findings indicate that these three mAbs may have potential prevention and treatment value in an H1N1 epidemic, as well as in the study of antigen epitope recognition.