Monoclonal Enzyme-linked Immunosorbent Assay Research Articles

Clinical and Analytical Comparison of Monoclonal and Polyclonal Immunoassays for Fecal Pancreatic Elastase.

Numerous immunoassays have been commercialized to determine pancreatic elastase (PE) in feces in screening for exocrine pancreatic insufficiency (EPI), but how the different assays compare to one another is controversial, especially in the context that all methods use the same cut-off values for interpreting the results obtained on the presence or absence of EPI or the degree of insufficiency if it is present. Our aim was to analytically verify a new method for determining PE, compare the results with a previous method, and verify the declared cut-off values for interpretation of the results. PE in the stool was assayed using a previous monoclonal enzyme-linked immunosorbent assay ("ScheBo ELISA") and a new polyclonal particle-enhanced turbidimetric immunoassay ("Bühlmann PETIA"). The direct method comparison of two immunoassays was performed in 40 samples. Clinical comparisons were conducted against each other for the binary determination of "abnormal/normal" elastase levels and the three-way determination of "severe/moderate/no" EPI in 56 samples. The indirect comparison method used external quality assessment (EQA) data to compare the monoclonal and polyclonal immunoassays for PE, and additionally compare the monoclonal ScheBo ELISA to a monoclonal chemiluminescence immunoassay ("DiaSorin CLIA"). Precision in the series and intra-laboratory precision for Bühlmann PETIA met the manufacturer's specifications for the concentration range of limit/lower values and the range of normal values. The Bühlmann PETIA immunoassay on different analytical platforms yielded comparable results and nearly perfect agreement in the case of three-way classification (kappa = 0.89 with 95%CI from 0.79 to 1.00. ScheBo ELISA tends to generate higher values of pancreatic elastase than the Bühlmann PETIA; agreement between the methods was moderate in the case of binary classification (kappa = 0.43; 95% CI 0.25 to 0.62), and substantial in the case of three-way classification (kappa = 0.62; 95% CI 0.50 to 0.75). EQA data analysis showed a statistically significant difference between ScheBo ELISA and Bühlmann PETIA peer groups (p = 0.031), as well as the DiaSorin CLIA and ScheBo ELISA peer groups (p = 0.010). The ScheBo ELISA and Bühlmann PETIA do not appear to be commutable in the analytical and clinical context. Our data address a discordance between different mono- and polyclonal immunoassays for pancreatic elastase and the potential of misclassification using its universal cut-off values in screening suspected patients for exocrine pancreatic insufficiency.

Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology.

Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

Single-domain antibodies against SARS-CoV-2 RBD from a two-stage phage screening of universal and focused synthetic libraries

BackgroundCoronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobodies, as well as other single-domain antibodies (sdAbs), have been recognized as a potential diagnostic and therapeutic tool for infectious diseases. High-throughput screening techniques such as phage display have been developed as an alternative to in vivo immunization for the discovery of antibody-like target-specific binders.MethodsWe designed and constructed a highly diverse synthetic phage library sdAb-U (single-domain Antibody - Universal library ) based on a human framework. The SARS-CoV-2 receptor-binding domain (RBD) was expressed and purified. The universal library sdAb-U was panned against the RBD protein target for two rounds, followed by monoclonal phage ELISA (enzyme-linked immunosorbent assay) to identify RBD-specific binders (the first stage). High-affinity binders were sequenced and the obtained CDR1 and CDR2 sequences were combined with fully randomized CDR3 to construct a targeted (focused) phage library sdAb-RBD, for subsequent second-stage phage panning (also two rounds) and screening. Then, sequences with high single-to-background ratios in phage ELISA were selected for expression. The binding affinities of sdAbs to RBD were measured by an ELISA-based method. In addition, we conducted competition ELISA (using ACE2 ectodomain S19-D615) and SARS-CoV-2 pseudovirus neutralization assays for the high-affinity RBD-binding sdAb39.ResultsSignificant enrichments were observed in both the first-stage (universal library) and the second-stage (focused library) phage panning. Five RBD-specific binders were identified in the first stage with high ELISA signal-to-background ratios. In the second stage, we observed a much higher possibility of finding RBD-specific clones in phage ELISA. Among 45 selected RBD-positive sequences, we found eight sdAbs can be well expressed, and five of them show high-affinity to RBD (EC50 < 100nM). We finally found that sdAb39 (EC50 ~ 4nM) can compete with ACE2 for binding to RBD.ConclusionOverall, this two-stage strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD sdAb with potential therapeutic activity, and this two-stage strategy can potentially be used for rapid discovery of sdAbs against other targets.

Efficient Hapten-Specific Biopanning Strategy Based on the Fe3O4@ENR-Functionalized Core-Shell Magnetic Nanoparticles.

The biopanning of target-specific phages is one of the most critical steps in the preparation of single-domain antibodies. In the traditional biopanning of haptens, the nonspecific binding of library phages to macromolecular proteins is one of the most challenging problems in preparing single-domain antibodies. In this research, Fe3O4@ENR-functionalized core-shell magnetic nanoparticles (FMNPs) were silylated and aminated by tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane, and target enrofloxacin was coupled onto the surface by the carbodiimide method. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, particle size distribution, zeta potential, transmission electron microscopy observation, and indirect enzyme-linked immunosorbent assay (ELISA). A biopanning strategy based on Fe3O4@ENR FMNPs was then established to solve the problem in the traditional solid-phase biopanning process. The results showed that a considerable number of enrofloxacin (ENR)-positive phages were screened by only one round of biopanning. Finally, two ENR-specific shark-derived single-domain genes were identified and validated by monoclonal phage ELISA, gene sequencing, and biolayer interferometry technology. Our study provides a new biopanning strategy based on Fe3O4@ENR FMNPs for efficiently providing phages specific to haptens.

Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines.

At present, quality control of diphtheria vaccines by both manufacturers and national control laboratories relies heavily on in vivo assays to confirm potency. As part of the VAC2VAC project we have developed a monoclonal antibody (mAb) enzyme-linked immunosorbent assay (ELISA) to measure the relative amount and quality of diphtheria toxoid (DTxd) in Diphtheria-Tetanus based vaccines, and believe this test has the potential to play a key role in a control strategy no longer including an in vivo potency test. The mAb ELISA is highly specific, has good dilutional linearity and is suitable for detecting DTxd in a range of different human vaccine products. We have demonstrated the ability of the assay to discriminate between batches of different content and quality using vaccine batches that had been prepared to contain differing amounts of DTxd or altered by exposure to heat or oxidative stress. We have also demonstrated successful transfer of the method to other laboratories and have shown that different diphtheria antigen materials may be able to serve as a reference antigen for local standardisation of the method. The assay is ideally suited for incorporation into a consistency approach for routine diphtheria vaccine quality control testing and may be suitable to serve as the stability indicating test in replacement of the current in vivo potency test.

Development of a human scFv antibody targeting the lethal Iranian cobra (Naja oxiana) snake venom

Snakebite is one of the health concerns worldwide. Naja oxiana is one of the venomous snakes with a high mortality rate. Anti-serum therapy is the only treatment of the victims. However, in some cases, antiserum injection leads to some side effects in host like serum sickness and anaphylactic shock. It is crucial to develop a neutralizing agent with low side effects. The human antibody library (non-immunized library) was used to isolate specific antibodies against N.oxiana venom components. Four rounds of biopanning were performed to enrich scFv-displaying phages against the venom of N. oxiana. Enrichment of scFv-displaying phages against N. oxiana venom was analyzed by polyclonal Enzyme-Linked Immunosorbent Assay (ELISA). Specific antibody fragments against N. oxiana venom were selected through monoclonal ELISA, and were expressed in E. coli bacterial cells. Purification of the selected clones was performed by using nickel affinity chromatography. Neutralization and protective capacity of specific antibody fragments were analyzed in C57BL/6 mice (i.v. injection). Results of biopanning and polyclonal ELISA demonstrate a successful enrichment process. Five specific antibody fragments with the highest signal in monoclonal ELISA were selected, expressed, and purified. The purity of expressed antibody fragments was monitored by SDS-PAGE and Western blot. The selected antibody fragments were able to neutralize two LD50 of N. oxiana venom and protected all mice when injected 15 min post-envenomation. The data indicate that such selected antibodies are promising tools for further studies and in the development of novel protective agents against N. oxiana venom.

Intralaboratory Reliability and Variability for Allergen-Specific Immunoglobulin Type E Serology Testing.

Atopic dermatitis is a very common condition affecting dogs and often managed with allergen-specific immunotherapy, which requires accurate identification of causative allergens. Serology testing is used commonly. Serum was collected from 35 atopic dogs and separated into three samples each (1, 2, and 3). Samples 1 and 2 were sent to IDEXX Laboratories the same day; sample 3 was stored at -80°C and submitted ∼30 days later. Specific immunoglobulin type E reactivity to various allergens were determined using monoclonal anti-canine enzyme-linked immunosorbent assay (ELISA) and expressed as ELISA absorbance units. Percent difference ranged from 14.30 to 127.34% for samples 1 and 2. These values increased when comparing samples a month apart (21.78 to 129.65%). Between samples 1 and 2, for each allergen there were differences in interpretation 15.18% of the time; 32 of 35 dogs (91.4%) had at least one allergen with a different interpretation. Comparing sample 3 and the average of samples 1 and 2, differences in interpretation increased to 22.32%; all dogs had at least one allergen that was interpreted differently. These differences in interpretation can alter immunotherapy. Overall, results show the need for better reliability for allergen-specific immunoglobulin type E serology testing using monoclonal anti-canine ELISA.

Preparation and identification of the nano antibodies of anti-programmed death receptor-1

Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen, and to provide experimental basis for subsequent functional studies. Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times. The peripheral blood was collected and the lymphocytes were isolated. Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH), and to construct a phage display library. The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA). The PD-1 antigen, which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml), was coated in an ELISA plate, and the phage display library was subjected to 3 rounds of affinity selection. Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA. According to the results of DNA sequencing, three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector, and transformed into E. coli BL21(DE3) competent cells, and then induced by isopropyl-β-D-thiogalactoside. The recombinant VHH antibody protein was purified by nickel column affinity chromatography, and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA. Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc, high titer specific antibody was stimulated, and the immune serum titer reached 1∶32 000. A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes. After 3 rounds of affinity selection, 46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA. Among them, three clones of VHH-B7, VHH-H5 and VHH-H12 had higher repeats, indicating that significant enrichment was obtained. The results of Western Blot and ELISA showed that the purified B7, H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity. Their affinity constants were 1.19×1011 and 1.63×1011, 1.59×1011 L/mol, respectively. Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library, which can bind to the PD-1 antigen with high affinity. This study can provide an experimental basis for subsequent functional studies. Key words: PD-1; Nano antibodies; Heavy chain antibody variable region; Identification; Phage display

Clinical study of the Liangxue-Zhuyu decoction combined with montelukast in the treatment of henoch schonlein purpura

Objective To study the efficacy of Liangxue-Zhuyu decoction combined with montelukast in the treatment of henoch schonlein purpura (HSP) and its influence on immunologic function. Methods A total of 95 HSP children treated in department of pediatrics from February 2014 to December 2016 were enrolled, and then were randomly divided into observation group (n=48) and control group (n=47), meanwhile another 48 healthy children admitted to a medical examination were selected as the healthy group. The patients in the control group were given the treatment of oral montelukast sodium chewable tablets before sleeping (the dose:2-6 years old 4 mg/d, 6-14 years old 5 mg/d), and the patients in the observation group accepted the therapy of Liangxue-Zhuyu decoction based on the treatment of the control group. The course of the treatment in the two groups were 30d. The time of clinical symptom regression and adverse reactions were recorded, and the clinical efficacy was evaluated. Monoclonal antibody immunofluorescence method and Enzyme-linked immunosorbent assay (ELISA) were used to detect the levels of serum T lymphocyte subsets and INF-γ and IL-10 before and after treatment, and their differences among three groups were compared. Results The effective rates of the observation group was 95.8% (46/48), which was significantly higher than 76.6% (36/47) of control group (χ2=7.441, P<0.05). The subsided time of the symptoms, including purpura (5.0 ± 1.3 d vs. 6.6 ± 2.6 d, t=3.816), abdominal pain (3.1 ± 2.1 d vs. 4.1 ± 2.6 d, t=2.139), joint pain (4.3 ± 1.5 d vs. 5.4 ± 1.9 d, t=3.416), fecal occult blood (4.5 ± 1.5 d vs. 6.1 ± 1.9 d, t=4.404) of the observation group were significantly shorter than those of the control group (P<0.05). After the treatment, the CD4 (45.14% ± 9.32% vs. 41.02% ± 9.97%), CD8 (25.10% ± 4.69% vs. 21.52% ± 5.71%) of the observation group was significantly higher than those of the control group, and the CD4/CD8 (1.62 ± 0.32 vs. 1.95 ± 0.35) and IL-10 (33.07 ± 1.86 pg/ml vs. 53.92 ± 2.98 pg/ml) of the observation group was significantly lower than this of the control group (P<0.05). Conclusions The efficacy of the Liangxue-Zhuyu decoction combined with montelukast for HSP was definite, and the effect was rapid with little side effects. The mechanism might be related to regulating the secretion of T lymphocyte and IL-10, thereby to correct Th1/Th2 cell balance and disturbance of immune function. Key words: Purpura, schoenlein-henoch; Liangxue-Zhuyu decoction; Montelukast sodium chewable tablets; T-Lymphocytes; Interferon-gamma; Interleukin-10

Generation and screening of efficient neutralizing single domain antibodies (VHHs) against the critical functional domain of anthrax protective antigen (PA)

Since anthrax is an acute infectious disease, detection and neutralization of the toxins of pathogenic Bacillus anthracis are of great importance. The critical role of protective antigen (PA) component of tripartite anthrax toxin in toxin entry into the host cell cytosol provided a great deal of effort to generate monoclonal antibodies against this constitute. Regarding the importance of anthrax detection/neutralization and unique physicochemical and pharmacological features of VHHs as single domain antibodies, the present study aimed to generate VHHs against the receptor binding domain of PA, termed PAD4. After camel immunization, a gene repertoire of VHH fragments with a diversity of 4.7×108 clones was produced, followed by constructing a VHH phage display library. A stringent successive biopanning was then carried out to isolate the phages displaying high affinity VHHs against PAD4.Polyclonal and monoclonal Enzyme-linked immunosorbent assay (ELISA) verified binding specificity of phages to the target protein. Modeling of VHHs together with the docking simulation studies, illustrated the binding site of antibodies on antigen. Docking analysis revealed that all selected VHHs potently cover the key functional residues of PAD4. Since the selected VHHs could cover and block the receptor binding loops of PA, they could be proposed as hopeful anti-Anthrax candidates.

Antiproliferative and Apoptotic Effects of Novel Anti-ROR1 Single-Chain Antibodies in Hematological Malignancies

Receptor tyrosine kinase–like orphan receptor (ROR) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including cell survival, differentiation, cell migration, cell communication, cell polarity, proliferation, metabolism, and angiogenesis. ROR1 has recently been shown to be expressed in various types of cancer cells but not normal cells. Pharmacokinetics and pharmacodynamics of single-chain Fragment variable (scFv) antibodies provide potential therapeutic advantages over whole antibody molecules. In the present study, scFvs against a specific peptide from the extracellular domain of ROR1 were selected using phage display technology. The selected scFvs were further characterized using polyclonal and monoclonal phage enzyme-linked immunosorbent assay (ELISA), soluble monoclonal ELISA, colony PCR, and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies were also evaluated in lymphoma and myeloma cancer cell lines using MTT and annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the ROR1 peptide. Colony PCR confirmed the presence of full-length VH and Vκ inserts. The percentages of cell growth after 24 h of treatment of cells with individual scFv revealed that the scFv significantly inhibited the growth of the RPMI8226 and chronic lymphocytic leukemia (CLL) cells in comparison with the untreated cells (p < 0.05). Interestingly, 24-h treatment with specific scFv induced apoptosis cell death in the RPMI8226 and CLL cells. Taken together, our results demonstrate that targeting of ROR1 using peptide-specific scFv can be an effective immunotherapy strategy in hematological malignancies.

Identification of highly selective MMP‐14 inhibitory Fabs by deep sequencing

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50 values of 10-4,000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions. Biotechnol. Bioeng. 2017;114: 1140-1150. © 2017 Wiley Periodicals, Inc.

Detection of Mycobacterial Lipoarabinomannan with A Monoclonal Antibody Qualitative ELISA in Urine of Tuberculous Meningitis Patients

BACKGROUND: Tuberculous meningitis is the most severe manifestation of tuberculosis. The diagnostic approach of tuberculous meningitis is difficult. Combination of clinical, laboratory and radiological criteria were used in diagnostic approach of tuberculous meningitis. Urinary mycobacterial lipoarabinomannan (LAM) antigen detection is a promising diagnostic tool. Detection of mycobacterial antigen in concentrated urine sample is predicted to improve the positivity rate of the qualitative enzyme-linked immunosorbent assay (ELISA) diagnostic tool. The purpose of this study is to examine the detection ability of a monoclonal antibody qualitative ELISA in concentrated and unconcentrated urine of tuberculous meningitis patients.METHODS: This research is a descriptive, crosssectionally designed. The study was conducted in the Clinical Pathology Department laboratory of Dr. Hasan Sadikin Hospital, in July-October 2014. A total of 27 patients diagnosed as tuberculous meningitis patients were included and the subjects were classified into possible and probable criteria according to consensus criteria. The subjects were classified as definite if the cerebrospinal fluid culture was positive for Mycobacterial tuberculosis growth. The subjects were examined for the presence of LAM in unconcentrated and concentrated urine with a monoclonal antibody qualitative ELISA method.RESULTS: Unconcentrated urinary LAM examination positivity was 0% while in concentrated urine was 14.8%. The positivity of concentrated urinary LAM were higher among the definite criteria group.CONCLUSION: Concentrating urine sample increase the positivity rate of urinary LAM detection with ELISA method as high as 14.8%. The urinary antigen detection is higher among the definite tuberculous meningitis patients.KEYWORDS: LAM, concentrated urine, tuberculous meningitis, qualitative ELISA

Association of Biofilm Formation, Psl Exopolysaccharide Expression, and Clinical Outcomes in Pseudomonas aeruginosa Keratitis: Analysis of Isolates in the Steroids for Corneal Ulcers Trial.

Bacterial virulence factors are increasingly recognized as important in the understanding of clinical infections. To determine whether 2 potential virulence factors, in vitro biofilm formation and Psl exopolysaccharide (EPS) expression, influence clinical presentation or outcomes in Pseudomonas aeruginosa keratitis. Laboratory investigation using P aeruginosa clinical isolates from the double-blind Steroids for Corneal Ulcers Trial (SCUT), which included patients at Aravind Eye Hospital, Proctor Foundation, University of California, San Francisco, and Dartmouth-Hitchcock Medical Center. SCUT was conducted from September 1, 2006, through February 22, 2010. All data used in this study were obtained during this period. Pseudomonas aeruginosa clinical isolates from SCUT were evaluated for in vitro biofilm formation, and Psl EPS expression was assessed using an anti-Psl monoclonal antibody (mAb) enzyme-linked immunosorbent assay. Planktonic growth kinetics and the susceptibility to anti-Psl mAb-mediated opsonophagocytic killing (OPK) were also evaluated in a subset of isolates. Linear regression assessed associations between SCUT patients' visual acuity and their corresponding biofilm formation and Psl EPS expression. Generalized estimating equation regression models were used to assess whether the change in visual acuity among SCUT patients was associated with Psl EPS expression or biofilm formation. Biofilm formation, Psl production, OPK, and visual acuity. The P aeruginosa SCUT strains produced a mean (SD) in vitro biofilm score of 1.06 (0.32) (range 0.17-2.12). A 1-unit increase in biofilm was associated with a worse visual acuity of 2 lines measured in SCUT patients at baseline (0.20 logMAR; 95% CI, -0.03 to 0.44; P = .09) and 3 months (0.21 logMAR; 95% CI, 0.003 to 0.44; P = .047). Of 101 confirmed P aeruginosa SCUT isolates, 100 expressed Psl EPSs. In addition, all Psl-positive strains evaluated in the OPK assay were susceptible to anti-Psl mAb-mediated OPK. The ability of P aeruginosa keratitis isolates to form biofilms in vitro was correlated with worse vision at presentation and after 3 months in SCUT. Ninety-nine percent of P aeruginosa keratitis isolates from SCUT produced Psl EPSs, and 100% of these evaluated Psl-positive isolates were susceptible to anti-Psl mAb-mediated OPK. These data indicate that biofilm formation and Psl EPSs may be candidate targets for novel therapeutics against P aeruginosa keratitis.

Development of ELISA and Colloidal Gold-PAb Conjugate-Based Immunochromatographic Assay for Detection of Abrin-a.

When abrin-a was combined with several polyclonal antibodies (PAb), the detection limit could be increased. In this way, a monoclonal antibody (capture) and polyclonal antibody (detection) sandwich enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-PAb conjugate-based immunochromatographic assay for detection of abrin-a were developed. The ELISA had a detection limit of 3.9 ng/mL for abrin-a in standard solution and 7.8 ng/mL in soybean milk, and was more sensitive than polyclonal antibody (capture) and monoclonal antibody (detection) ELISA, which had a detection limit of 15.6 ng/mL. The test strip had a detection range of 50 to 500 ng/mL for abrin-a and a detection limit in standard solution or soybean milk samples of 50 ng/mL. However, the test strip had a reduced detection capability compared with a colloidal gold-monoclonal antibody conjugate-based immunochromatographic assay test strip, which had a lower detection limit of 10 ng/mL. The developed ELISAs and test strip show the specificity towards abrin-a and have no cross-reactivity towards abrin-b, -c, -d, ricin, or the agglutinins from either castor beans or rosary peas.

Effectiveness of 14-days course of clarithromycin-based triple therapy as first line therapy for h.pylori infection in egyptian elderly patients

The aim of the study is to assess the effectiveness of 14-days course of Clarithromycin-based triple therapy as first line therapy for H. pylori infection in Egyptian elderly patients with normal liver and kidney functions. Methods: The study wasexperimental study carried out on 34 elderly patients having H. pylori gastritis with normal liver and kidney functions. We tested for H. pylori stool antigen using a quantitative monoclonal enzyme-linked immunosorbent assay (ELISA) assay before and 4 weeks after the treatment to confirm eradication. Patients received Clarithromycin-based triple therapy(Clarithromycin 500mg twice daily, Amoxicillin 1000mg twice daily, and Omeprazole 40mg twice daily) for H. pylori eradication for 14 days. Results: The cure rate of Clarithromycinbased triple therapy was 88.2% (30/34) and only 11.8% (4/34) was having persistent infection. None of the patients discontinued the drugs indicating good tolerability.Conclusion: The use of 14-days course of Clarithromycin-based triple therapy as first line therapy for H. pylori infection is highly effective and tolerable in Egyptian elderly patients with normal liver and kidney functions.

Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the Cronobacter sakazakii. A pair of monoclonal antibodies (mAbs) selected from mAbs produced by 11 murine hybridomas was selected for the sandwich ELISA procedure. Targets of two mAbs were 100 kDa and 42 kDa protein extracted from the bacteria, respectively, which were proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. The limit of detection of this method was established as 1 × 104 cfu/mL, and the linear range from 1 × 105 to 1 × 108 cfu/mL. In the real sample test, 1 cfu/g C. sakazakii was detected in artificially contaminated powdered infant formula with 4 h enrichment.

Application of mouse monoclonal antibody (mAb) 4C10-based enzyme-linked immunosorbent assay (ELISA) for amandin detection in almond (Prunus dulcis L.) genotypes and hybrids

Amandin presence in 108 almond genotypes/hybrids and 80 almond marketing varieties grown in different locations was determined using murine monoclonal antibody 4C10-based sandwich ELISA. The results indicated that amandin was present in all the tested samples. The ELISA immunoreactivity variations were up to 8 fold among genotypes/hybrids and 2.5 fold among the almond marketing varieties. Amandin content variations were also confirmed using Western blot and dot blot. No correlation was observed between almond seed size and total soluble protein or amandin content.

Drug-induced immune thrombocytopenia: incidence, clinical features, laboratory testing, and pathogenic mechanisms

Abstract Drug-induced immune thrombocytopenia (DIIT) is a relatively uncommon adverse reaction caused by drug-dependent antibodies (DDAbs) that react with platelet membrane glycoproteins only when the implicated drug is present. Although more than 100 drugs have been associated with causing DIIT, recent reviews of available data show that carbamazepine, eptifibatide, ibuprofen, quinidine, quinine, oxaliplatin, rifampin, sulfamethoxazole, trimethoprim, and vancomycin are probably the most frequently implicated. Patients with DIIT typically present with petechiae, bruising, and epistaxis caused by an acute, severe drop in platelet count (often to &lt;20,000 platelets/µL). Diagnosis of DIIT is complicated by its similarity to other non–drug-induced immune thrombocytopenias, including autoimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness, and must be differentiated by temporal association of exposure to a candidate drug with an acute, severe drop in platelet count. Treatment consists of immediate withdrawal of the implicated drug. Criteria for strong evidence of DIIT include (1) exposure to candidate drug–preceded thrombocytopenia; (2) sustained normal platelet levels after discontinuing candidate drug; (3) candidate drug was only drug used before onset of thrombocytopenia or other drugs were continued or reintroduced after resolution of thrombocytopenia, and other causes for thrombocytopenia were excluded; and (4) reexposure to the candidate drug resulted in recurrent thrombocytopenia. Flow cytometry testing for DDAbs can be useful in confirmation of a clinical diagnosis, and monoclonal antibody enzyme-linked immunosorbent assay testing can be used to determine the platelet glycoprotein target(s), usually GPIIb/IIIa or GPIb/IX/V, but testing is not widely available. Several pathogenic mechanisms for DIIT have been proposed, including hapten, autoantibody, neoepitope, drug-specific, and quinine-type drug mechanisms. A recent proposal suggests weakly reactive platelet autoantibodies that develop greatly increased affinity for platelet glycoprotein epitopes through bridging interactions facilitated by the drug is a possible mechanism for the formation and reactivity of quinine-type drug antibodies. Immunohematology 2014;30:55–65.

Synthesis of furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) and novel haptens for the development of a sensitive enzyme-linked immunosorbent assay (ELISA)

A sensitive and specific monoclonal enzyme-linked immunosorbent assay (ELISA) for the determination of tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. Three different haptens of AMOZ were synthesized. The haptens (I, III) were derived from 4-carboxybenzaldehyde and ethyl 4-bromobutyrate, respectively. Hapten II was based on hapten I but a CN bond in the parent structure was reduced. Corresponding immunogens and coating antigens were prepared to improve the assay sensitivity. The hybridomas 4 × 108 secreting antibodies against CPAMOZ were obtained from immunogens I-BSA by monoclonal antibody (mAb) technology. The antibody indicated that: (1) the small molecular AMOZ cannot induce the specific response against AMOZ; (2) the weakened conjugation effect improved the recognition of AMOZ. Assessment of twelve coating antigen/antibody combinations consequently resulted in the development of an indirect competitive enzyme-linked immunosorbent assay (icELISA). The results showed that the sensitivity was highly improved about fifteen-fold for the novel heterologous coating antigen at low hapten density. The IC50 value of the ELISA for CPAMOZ was 0.13 ng mL−1. The cross reactivity values of the assay with NPAMOZ, AMOZ and FTD were 118%, 2.3% and 16.3%. Recoveries from AMOZ fortified animal tissues were in a range of 82.6–108.4% and the CV values were less than 12.5%. The immunoassay was further validated by a LC-MS/MS method and the two methods showed good correlation (r2 = 0.9908). The proposed icELISA could be used for an accurate monitoring of AMOZ in animal tissues.