Diagnosis of schistosomiasis using recombinant fructose-1,6-bisphosphate aldolase from a Formosan strain ofSchistosoma japonicum

AbstractSchistosoma japonicumobtained from Taiwan is a zoophilic strain that only infects domestic and small animals. Recombinant fructose-1,6-bisphosphate aldolase (FBPA) derived from this strain was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human schistosomiasis. The full-length DNA sequence of FBPA was found to be 1092 bp, encoding a protein of 363 amino acid residues, with a molecular mass of 39.6 kDa. A total of 120 participants were recruited from China and Taiwan to evaluate the diagnostic value of this recombinant protein. In these participants, 34 were found to be infected withS. japonicum, 16 withAscaris lumbricoides, 15 with hookworm, 13 withParagonimus westermaniand 13 withClonorchis sinensis, whereas 29 had no ova on faecal examination. Western blot analysis showed that the recombinant FBPA reacts strongly with schistosome ova-positive sera. The sensitivity and specificity of ELISA with FBPA were found to be 85.3% and 93.0%, respectively. These results indicate that FBPA derived from the Formosan strain ofS. japonicumcan be used for the diagnosis of human schistosomiasis.

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Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

Scientific Reports ◽

10.1038/s41598-021-96466-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Patamalai Boonserm ◽

Songchan Puthong ◽

Thanaporn Wichai ◽

Sajee Noitang ◽

Pongsak Khunrae ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

3D Structure ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Variable Regions ◽

Complementarity Determining Regions ◽

Crucial Part ◽

Major Amino Acid

AbstractIt is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

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Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Toxins ◽

10.3390/toxins12040273 ◽

2020 ◽

Vol 12 (4) ◽

pp. 273

Author(s):

Caixia Zhang ◽

Weiqi Zhang ◽

Xiaoqian Tang ◽

Qi Zhang ◽

Wen Zhang ◽

...

Keyword(s):

Amino Acid ◽

Ochratoxin A ◽

Limit Of Detection ◽

Basic Knowledge ◽

Affinity Constant ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Assay Sensitivity ◽

Coating Antigen ◽

Relative Standard

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

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Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.998-999.210 ◽

2014 ◽

Vol 998-999 ◽

pp. 210-213

Author(s):

Chun Ling Zhao ◽

Wen Jing Yu ◽

Ji Yu Ju

Keyword(s):

Amino Acid ◽

Recombinant Protein ◽

Active Form ◽

Cloning And Expression ◽

Amino Acid Residues ◽

Gene Encoding ◽

Reading Frame ◽

Arenicola Cristata ◽

Fibrin Plate ◽

Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

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Molecular Expression of a Cysteine Proteinase of Clonorchis sinensis and Its Application to an Enzyme-Linked Immunosorbent Assay for Immunodiagnosis of Clonorchiasis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.2.411-416.2004 ◽

2004 ◽

Vol 11 (2) ◽

pp. 411-416 ◽

Cited By ~ 49

Author(s):

Isao Nagano ◽

Fuquan Pei ◽

Zhiliang Wu ◽

Jun Wu ◽

Huier Cui ◽

...

Keyword(s):

Recombinant Protein ◽

Adult Worm ◽

Immunosorbent Assay ◽

Fasciola Hepatica ◽

Cysteine Proteinase ◽

Clonorchis Sinensis ◽

Recombinant Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Predicted Amino Acid Sequence ◽

Crude Extracts

ABSTRACT We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.

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Antigenic Evolution of Vaccine-Derived Polioviruses: Changes in Individual Epitopes and Relative Stability of the Overall Immunological Properties

Journal of Virology ◽

10.1128/jvi.80.6.2641-2653.2006 ◽

2006 ◽

Vol 80 (6) ◽

pp. 2641-2653 ◽

Cited By ~ 39

Author(s):

Maria L. Yakovenko ◽

Elena A. Cherkasova ◽

Gennady V. Rezapkin ◽

Olga E. Ivanova ◽

Alexander P. Ivanov ◽

...

Keyword(s):

Amino Acid ◽

Rational Design ◽

Driving Forces ◽

Oral Poliovirus Vaccine ◽

Enzyme Linked Immunosorbent Assay ◽

Human Populations ◽

Amino Acid Residues ◽

Specific Amino Acid ◽

Antigenic Sites ◽

Neutralizing Capacity

ABSTRACT The Sabin oral poliovirus vaccine (OPV) readily undergoes changes in antigenic sites upon replication in humans. Here, a set of antigenically altered descendants of the three OPV serotypes (76 isolates) was characterized to determine the driving forces behind these changes and their biological implications. The amino acid residues of OPV derivatives that lie within or close to the known antigenic sites exhibited a marked tendency to be replaced by residues characteristic of homotypic wild polioviruses, and these changes may occur very early in OPV evolution. The specific amino acid alterations nicely correlated with serotype-specific changes in the reactivity of certain individual antigenic sites, as revealed by the recently devised monoclonal antibody-based enzyme-linked immunosorbent assay. In comparison to the original vaccine, small changes, if any, in the neutralizing capacity of human or rabbit sera were observed in highly diverged vaccine polioviruses of three serotypes, in spite of strong alterations of certain epitopes. We propose that the common antigenic alterations in evolving OPV strains largely reflect attempts to eliminate fitness-decreasing mutations acquired either during the original selection of the vaccine or already present in the parental strains. Variability of individual epitopes does not appear to be primarily caused by, or lead to, a significant immune evasion, enhancing only slightly, if at all, the capacity of OPV derivatives to overcome immunity in human populations. This study reveals some important patterns of poliovirus evolution and has obvious implications for the rational design of live viral vaccines.

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Structure-based identification of inositol polyphosphate 1-phosphatase fromEntamoeba histolytica

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714021245 ◽

2014 ◽

Vol 70 (11) ◽

pp. 3023-3033 ◽

Cited By ~ 2

Author(s):

Khaja Faisal Tarique ◽

Syed Arif Abdul Rehman ◽

Christian Betzel ◽

Samudrala Gourinath

Keyword(s):

Amino Acid ◽

Metal Binding ◽

Three Dimensional ◽

Protozoan Parasite ◽

Amino Acid Residues ◽

Inositol Polyphosphate ◽

Inositol Monophosphatase ◽

Specific Amino Acid ◽

Fructose 1,6 Bisphosphate ◽

Hydrolysis Of

Inositol polyphosphate 1-phosphatase fromEntamoeba histolytica(EhIPPase) is an Mg2+-dependent and Li+-sensitive enzyme that catalyzes the hydrolysis of inositol 1,4-bisphosphate [Ins(1,4)P2] intomyo-inositol 1-monophosphate and PO43−. In the present work, EhIPPase has been biochemically identified and its crystal structure has been determined in the presence of Mg2+and PO43−at 2.5 Å resolution. This enzyme was previously classified as a 3′(2′),5′-bisphosphate nucleotidase in the NCBI, but its biochemical activity and structural analysis suggest that this enzyme behaves more like an inositol polyphosphate 1-phosphatase. The ability of EhIPPase to hydrolyze the smaller Ins(1,4)P2 better than the bulkier 3′-phosphoadenosine 5′-phosphate (PAP) is explained on the basis of the orientations of amino-acid residues in the binding site. This structure is the first of its class to be determined from any protozoan parasite, and is the third to determined among all organisms, following its rat and bovine homologues. The three-dimensional fold of EhIPPase is similar to those of other members of the inositol monophosphatase superfamily, which also includes inositol monophosphatase, 3′(2′),5′-bisphosphate nucleotidase and fructose-1,6-bisphosphate 1-phosphatase. They all share conserved residues essential for metal binding and substrate hydrolysis, with the motif D-Xn-EE-Xn-DP(I/L)DG(S/T)-Xn-WD-Xn-GG. The structure is divided into two domains, namely α+β and α/β, and the substrate and metal ions bind between them. However, the ability of each enzyme class to act specifically on its cognate substrate is governed by the class-specific amino-acid residues at the active site.

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Expression and Characterization of Recombinant Human Protein S in Heterologous Cells - Studies of the Interaction of Amino Acid Residues Leu-608 to Glu-612 with Human C4b-Binding Protein

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648487 ◽

1992 ◽

Vol 67 (05) ◽

pp. 526-532 ◽

Cited By ~ 9

Author(s):

Glenn T G Chang ◽

Hans K Ploos van Amstel ◽

Martin Hessing ◽

Pieter H Reitsma ◽

Rogier M Bertina ◽

...

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Recombinant Protein ◽

Plasma Protein ◽

Binding Protein ◽

Protein S ◽

Full Length ◽

Human Protein ◽

Amino Acid Residues ◽

Amino Terminal

SummaryMouse C127 epithelioid cells were genetically engineered to produce biologically active ³-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of ³-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully ³-carboxylated and fully p-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gin, respectively and this variant was designated C variant. The binding of these variants to C4BP was the same as wild type recombinant protein S. This suggests that amino acid residues leu-608, asp-609, asp-611 and glu-612 are not essential for binding of the intact full length protein to C4BP.

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A Novel Monoclonal Antibody against FbaA Can Inhibit the Binding of the Complement Regulatory Protein Factor H to Group A Streptococcus

Clinical and Vaccine Immunology ◽

10.1128/cvi.00438-10 ◽

2011 ◽

Vol 18 (4) ◽

pp. 552-558 ◽

Cited By ~ 7

Author(s):

Cuiqing Ma ◽

Yiyang Guo ◽

Haiyan Gu ◽

Ling Zhang ◽

Hainan Liu ◽

...

Keyword(s):

Amino Acid ◽

Competitive Inhibition ◽

Group A Streptococcus ◽

Regulatory Protein ◽

Factor H ◽

Microbial Pathogens ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Protein Factor ◽

Group A

ABSTRACTSome microbial pathogens utilize human complement regulatory proteins, such as factor H (FH) and factor H-like protein 1 (FHL-1), for immune evasion. FbaA is an FHL-1 and FH binding protein expressed on the surface of group A streptococcus (GAS), a common agent of pharyngeal, skin, and soft tissue infections. In this study, we prepared monoclonal antibodies (MAbs) against FbaA, assayed them for specificity, and located their binding domains in FbaA. We found an MAb called FbaA MAb2, which demonstrated the highest affinity to GAS among all of the MAbs. Based on the binding with component peptides, the detected epitope, which was specific for FbaA MAb2, was the amino acid residues 95 to 118 of FbaA; on the other hand, it did not bind with the truncated protein of the internally deleted residues of the segment from 95 to 118 of FbaA. Furthermore, the predominant amino acids specific for FbaA MAb2 screened by phage display epitope library were I, T, P, D, and L, corresponding to the amino acid residues 101, 103, 105, 106, and 110 of FbaA, respectively. The binding location of FbaA with FH and FHL-1 was a 16-amino-acid region corresponding to amino acid residues 97 to 112 of FbaA, which overlapped the FbaA MAb2 binding domain, as confirmed by competitive inhibition enzyme-linked immunosorbent assay and immunofluorescence microscopy. Based on the results of the invasion assay, FbaA MAb2 can inhibit the binding of FH to GAS.

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Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.4.552-557.2003 ◽

2003 ◽

Vol 10 (4) ◽

pp. 552-557 ◽

Cited By ~ 25

Author(s):

Tetsuro Ikegami ◽

Masahiro Niikura ◽

Masayuki Saijo ◽

Mary E. Miranda ◽

Alan B. Calaor ◽

...

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Immunosorbent Assay ◽

Rapid Detection ◽

Ebola Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Specific Detection ◽

Antigen Capture ◽

Cynomolgus Macaques

ABSTRACT Antigen capture enzyme-linked immunosorbent assay (ELISA) is one of the most useful methods to detect Ebola virus rapidly. We previously developed an antigen capture ELISA using a monoclonal antibody (MAb), 3-3D, which reacted not only to the nucleoprotein (NP) of Zaire Ebola virus (EBO-Z) but also to the NPs of Sudan (EBO-S) and Reston Ebola (EBO-R) viruses. In this study, we developed antigen capture ELISAs using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques.

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Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

10.21203/rs.3.rs-310107/v2 ◽

2021 ◽

Author(s):

Fangyu Wang ◽

Ning Li ◽

Yunshang Zhang ◽

Xuxefeng Sun ◽

Yali Zhao ◽

...

Keyword(s):

Amino Acid ◽

Scfv Antibody ◽

Phage Library ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Single Chain ◽

Single Chain Antibody ◽

Relative Standard ◽

Directional Evolution ◽

Scfv Library

Abstract A recombinant anti-enrofloxacin single-chain antibody (scFv) was produced for the detection of enrofloxacin. An immunized mouse phage display scFv library with a capacity of 2.35×109 CFU/mL was constructed and used for anti-enrofloxacin scFv screening. After four rounds of bio-panning, 10 positives were isolated and identified successfully. The highest positive scFv was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residues Leu121 were the key residues for the binding of ScFv to ENR. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Leu121 to Asn. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant ScFv were established for enrofloxacin respectively. The IC50 value of the assay established with the ScFv mutant was 1.63 ng/mL, while the parental ScFv was 21.08 ng/mL, this result showed highly increased affinity with up to 12.9-folds improved sensitivity. The mean recovery for ENR ranged from 71.80% to 117.35% with 10.46% relative standard deviation between the intra-assay and the inter-assay. The results indicate that we have obtained a highly sensitive anti-ENR scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of ENR residue in animal derived edible tissues and milk.

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