A general strategy to control antibody specificity against targets showing molecular and biological similarity: Salmonella case study

Abstract The control of antibody specificity plays pivotal roles in key technological fields such as diagnostics and therapeutics. During the development of immunoassays (IAs) for the biosensing of pathogens in food matrices, we have found a way to rationalize and control the specificity of polyclonal antibodies (sera) for a complex analytical target (the Salmonella genus), in terms of number of analytes (Salmonella species) and potential cross-reactivity with similar analytes (other bacteria strains). Indeed, the biosensing of Salmonella required the development of sera and serum mixtures displaying homogeneous specificity for a large set of strains showing broad biochemical variety (54 Salmonella serovars tested in this study), which partially overlaps with the molecular features of other class of bacteria (like specific serogroups of E. coli). To achieve a trade-off between specificity harmonisation and maximization, we have developed a strategy based on the conversion of the specificity profiles of individual sera in to numerical descriptors, which allow predicting the capacity of serum mixtures to detect multiple bacteria strains. This approach does not imply laborious purification steps and results advantageous for process scaling-up, and may help in the customization of the specificity profiles of antibodies needed for diagnostic and therapeutic applications such as multi-analyte detection and recombinant antibody engineering, respectively.

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Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽

10.3390/toxins13040254 ◽

2021 ◽

Vol 13 (4) ◽

pp. 254

Author(s):

Shelby S. Szteiter ◽

Ilse N. Diego ◽

Jonathan Ortegon ◽

Eliana Salinas ◽

Abcde Cirilo ◽

...

Keyword(s):

Snake Venom ◽

Polyclonal Antibody ◽

Polyclonal Antibodies ◽

Cross Reactivity ◽

Snake Envenomation ◽

Disintegrin Domain ◽

The Common ◽

Neutralization Ability ◽

New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

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Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants

Biochemical Journal ◽

10.1042/bj2590847 ◽

1989 ◽

Vol 259 (3) ◽

pp. 847-853 ◽

Cited By ~ 17

Author(s):

I Benveniste ◽

A Lesot ◽

M P Hasenfratz ◽

F Durst

Keyword(s):

Cytochrome C ◽

Rat Liver ◽

Higher Plants ◽

Polyclonal Antibodies ◽

Reductase Activity ◽

Jerusalem Artichoke ◽

Cross Reactivity ◽

Cytochrome C Reductase ◽

Nadph Cytochrome ◽

Cytochrome P 450

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

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Rapid immunoassays for detection of anabolic nortestosterone in dietary supplements

Czech Journal of Food Sciences ◽

10.17221/507/2012-cjfs ◽

2013 ◽

Vol 31 (No. 5) ◽

pp. 514-519 ◽

Cited By ~ 2

Author(s):

B. Holubová ◽

S. Göselová ◽

L. Ševčíková ◽

M. Vlach ◽

M. Blažková ◽

...

Keyword(s):

Dietary Supplements ◽

Rapid Detection ◽

Visual Detection ◽

Limit Of Detection ◽

Polyclonal Antibodies ◽

Cross Reactivity ◽

Rapid Analysis ◽

Experimental Conditions ◽

Immunochromatographic Strip ◽

Strip Test

An enzyme immunoassay (ELISA) and an immunochromatographic strip were designed for a rapid detection of nortestosterone in dietary supplements. Two polyclonal antibodies and two types of nortestosterone-protein coating conjugates were tested to develop the most appropriate method. Under optimal experimental conditions, the most sensitive ELISA achieved the IC<sub>50 </sub>and the limit of detection values of 6.41 and 0.09 ng/ml, respectively. The assay specificity was tested measuring cross-reactivity of several steroids. The interference with the assay was negligible (< 0.1%), except for cross-reactivity with another frequently abused steroid testosterone (23%). The optimised gold particle-based immunochromatographic strip provided in semi-quantitative test a visual detection limit of 1 ng/ml. None of these methods showed the interference using a filtrate of the suspension of non-contaminated sample. After the validation for particular matrices, the ELISA and the strip test could be useful tools for a rapid analysis of nortestosterone in crude extracts of dietary supplements.

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Structural heterogeneity in farm structures: a configurational approach

Journal of Agribusiness in Developing and Emerging Economies ◽

10.1108/jadee-12-2018-0183 ◽

2019 ◽

Vol 10 (1) ◽

pp. 65-83

Author(s):

Bruno Varella Miranda ◽

Anna Grandori

Keyword(s):

Structural Heterogeneity ◽

System Level ◽

Large Set ◽

Legal Form ◽

Content Type ◽

Property Rights Protection ◽

Farm Structures ◽

Development Paths ◽

And Control ◽

Corporate Farm

Purpose The purpose of this paper is to provide a multidimensional framework for the identification, description and comparative analysis of alternative farm structures and their properties for economic development. Design/methodology/approach Integrating previous typologies and considering a large set of examples, the authors identify six attributes that are necessary to characterize and compare farm structures: size; strategy; organizational form; legal form; who the owners are; and degree of separation of ownership and control. They also discuss potential complementarities between those organizational attributes and specific features of the institutions of developing and emerging countries, such as contract enforcement and property rights protection regime, and developed capital markets and corporate law. Findings Conceptually and empirically, effective farm structures can deviate from the templates traditionally considered – “small family-owned farm” or “large factory-like corporate farm,” combining structural attributes in diverse ways. The dimensionalization of farm structures also helps in revealing complementary institutional traits at the regional or larger system level that may foster development processes. Research limitations/implications The paper is limited to theory building and case-based evidence. Nevertheless, it provides dimensions that can be measured on a larger scale and by quantitative studies. Originality/value This paper sheds light on organizational diversity in agriculture and on a wider set of feasible development paths.

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A blocking antibody against canine CSF-1R maturated by limited CDR mutagenesis

Antibody Therapeutics ◽

10.1093/abt/tbaa018 ◽

2020 ◽

Vol 3 (3) ◽

pp. 193-204

Author(s):

Breno C B Beirão ◽

Teresa P Raposo ◽

Louise M Imamura ◽

Max Ingberman ◽

Ted Hupp ◽

...

Keyword(s):

Inflammatory Diseases ◽

Antibody Fragment ◽

Cross Reactivity ◽

Antibody Engineering ◽

Model Systems ◽

Native Form ◽

Blocking Antibody ◽

Macrophage Activity ◽

Beneficial Effects ◽

Variable Heavy

Abstract CSF-1R is a receptor mostly associated with the mononuclear phagocytic system. However, its expression within tumors has been linked with poor prognosis in both humans and dogs. Accordingly, several reports have demonstrated the beneficial effects of blocking CSF-1R in model systems of cancer. In this study, we generated a monoclonal antibody that could block CSF-1R in dogs as the first step to develop an anticancer drug for this species. Initially, an antibody was raised by the hybridoma methodology against the fragment responsible for receptor dimerization. mAb3.1, one of the resulting hybridoma clones, was able to bind macrophages in fixed tissues and was shown to inhibit cells of the mononuclear phagocytic line. Nevertheless, mAb 3.1 could not bind to some glycoforms of the receptor in its native form, while also demonstrating cross-reactivity with other proteins. To enhance binding properties of the mAb, five amino acids of the complementarity-determining region 2 of the variable heavy chain of mAb3.1 were mutated by PCR, and the variant scFv clones were screened by phage display. The selected scFv clones demonstrated improved binding to the native receptor as well as increased anti-macrophage activity. The resulting scFv antibody fragment presented here has the potential for use in cancer patients and in inflammatory diseases. Furthermore, this work provides insights into the use of such restricted mutations in antibody engineering.

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Dysregulation of Placental Functions and Immune Pathways in Complete Hydatidiform Moles

International Journal of Molecular Sciences ◽

10.3390/ijms20204999 ◽

2019 ◽

Vol 20 (20) ◽

pp. 4999 ◽

Cited By ~ 1

Author(s):

Jennifer R. King ◽

Melissa L. Wilson ◽

Szabolcs Hetey ◽

Peter Kiraly ◽

Koji Matsuo ◽

...

Keyword(s):

Gene Expression ◽

First Trimester ◽

Receptor Interaction ◽

Rna Seq ◽

Bioinformatics Analyses ◽

Immune Genes ◽

Molecular Features ◽

Immune Pathways ◽

And Control ◽

Hydatidiform Moles

Gene expression studies of molar pregnancy have been limited to a small number of candidate loci. We analyzed high-dimensional RNA and protein data to characterize molecular features of complete hydatidiform moles (CHMs) and corresponding pathologic pathways. CHMs and first trimester placentas were collected, histopathologically examined, then flash-frozen or paraffin-embedded. Frozen CHMs and control placentas were subjected to RNA-Seq, with resulting data and published placental RNA-Seq data subjected to bioinformatics analyses. Paraffin-embedded tissues from CHMs and control placentas were used for tissue microarray (TMA) construction, immunohistochemistry, and immunoscoring for galectin-14. Of the 14,022 protein-coding genes expressed in all samples, 3,729 were differentially expressed (DE) in CHMs, of which 72% were up-regulated. DE genes were enriched in placenta-specific genes (OR = 1.88, p = 0.0001), of which 79% were down-regulated, imprinted genes (OR = 2.38, p = 1.54 × 10−6), and immune genes (OR = 1.82, p = 7.34 × 10−18), of which 73% were up-regulated. DNA methylation-related enzymes and histone demethylases were dysregulated. “Cytokine–cytokine receptor interaction” was the most impacted of 38 dysregulated pathways, among which 17 were immune-related pathways. TMA-based immunoscoring validated the lower expression of galectin-14 in CHM. In conclusion, placental functions were down-regulated, imprinted gene expression was altered, and immune pathways were activated, indicating complex dysregulation of placental developmental and immune processes in CHMs.

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On the correlation of statistical and automatic process control

Proceedings of the Institution of Mechanical Engineers Part B Journal of Engineering Manufacture ◽

10.1243/095440503762502314 ◽

2003 ◽

Vol 217 (1) ◽

pp. 99-109 ◽

Cited By ~ 1

Author(s):

J Harris

Keyword(s):

Process Control ◽

Automatic Control ◽

Fault Tree Analysis ◽

Automatic Process ◽

General Strategy ◽

Statistical Control ◽

Automatic Process Control ◽

Intervention Protocol ◽

And Control ◽

Fault Information

A process control strategy is proposed based upon the twin themes of statistical and automatic process control. The main categories of product fault are identified and related to the capabilities of statistical and automatic control. Statistical control is supported by process fault information from a process-specific fault tree analysis, which provides the basis for a corrective intervention protocol. Application is discussed in terms of fuzzy automatic control, which offers a greater generality than conventional automatic control modelling. Prior publications that fuzzify statistical control zones are arguably incomplete in the application of logic propositions and also in the identification of process faults. The present work proposes a general strategy, which may be adapted to specific processes. Both control by variables and control by attributes may be included within this treatment.

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Human Kallikrein 13: Production and Purification of Recombinant Protein and Monoclonal and Polyclonal Antibodies, and Development of a Sensitive and Specific Immunofluorometric Assay

Clinical Chemistry ◽

10.1373/49.1.77 ◽

2003 ◽

Vol 49 (1) ◽

pp. 77-86 ◽

Cited By ~ 48

Author(s):

Carl Kapadia ◽

Albert Chang ◽

Georgia Sotiropoulou ◽

George M Yousef ◽

Linda Grass ◽

...

Keyword(s):

Ovarian Cancer ◽

Amniotic Fluid ◽

Polyclonal Antibodies ◽

Biological Fluids ◽

Expression System ◽

Immunohistochemical Analysis ◽

Cross Reactivity ◽

Yeast Expression System ◽

Tissue Extracts ◽

Cancer Tissues

Abstract Background: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids. Methods: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies. A sandwich-type immunoassay was developed with these antibodies. The assay was used to measure hK13 in various biological fluids and tissue extracts. Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections. Results: The hK13 immunoassay had a detection limit of 0.05 μg/L and showed no cross-reactivity with homologous kallikreins. The assay was linear at 0–20 μg/L, and within-and between-run CVs were <10% (n = 12). hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate. hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues. hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate. hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues. Recovery of active enzyme added to milk or amniotic fluid was 70–98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors. In fluids and tissue extracts, hK13 was found in its free (∼30 kDa) form. Conclusions: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.

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Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879600800111 ◽

1996 ◽

Vol 8 (1) ◽

pp. 68-75 ◽

Cited By ~ 31

Author(s):

H. E. Jensen ◽

B. Aalbaek ◽

P. Lind ◽

H. V. Krogh ◽

P. L. Frandsen

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Fungal Species ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Immunohistochemical Techniques ◽

Immunohistochemical Diagnosis ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

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Fluorescence Polarization Immunoassay for Determination of Enrofloxacin in Pork Liver and Chicken

Molecules ◽

10.3390/molecules24244462 ◽

2019 ◽

Vol 24 (24) ◽

pp. 4462

Author(s):

Xing Shen ◽

Jiahong Chen ◽

Shuwei Lv ◽

Xiulan Sun ◽

Boris B. Dzantiev ◽

...

Keyword(s):

Fluorescence Polarization ◽

Limit Of Detection ◽

Polyclonal Antibodies ◽

Screening Method ◽

Sample Pretreatment ◽

High Sensitivity ◽

Cross Reactivity ◽

Fluorescence Polarization Immunoassay ◽

Pork Liver ◽

Sensitivity Specificity

Enrofloxacin (ENR) is a widely used fluoroquinolone (FQ) antibiotic for antibacterial treatment of edible animal. In this study, a rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for monitoring ENR residues in animal foods. First, ENR was covalently coupled to bovine serum albumin (BSA) to produce specific polyclonal antibodies (pAbs). Three fluorescein-labeled ENR tracers (A, B, and C) with different spacers were synthesized and compared to obtain higher sensitivity. Tracer C with the longest arm showed the best sensitivity among the three tracers. The developed FPIA method showed an IC50 (50% inhibitory concentration) of 21.49 ng·mL−1 with a dynamic working range (IC20–IC80) of 4.30–107.46 ng·mL−1 and a limit of detection (LOD, IC10) of 1.68 ng·mL−1. The cross-reactivity (CR) of several structurally related compounds was less than 2%. The recoveries of spiked pork liver and chicken samples varied from 91.3% to 112.9%, and the average coefficients of variation were less than 3.83% and 5.13%, respectively. The immunoassay took only 8 min excluding sample pretreatment. This indicated that the established method had high sensitivity, specificity, and the advantages of simplicity. Therefore, the proposed FPIA provided a useful screening method for the rapid detection of ENR residues in pork liver and chicken.

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