Development of an ic-ELISA and immunochromatographic strip based on IgG antibody for detection of ω-conotoxin MVIIA.

ω-conotoxin MVIIA(ω-CTX MVIIA) is a peptide consisting of 25 amino acid residues secreted mainly by Conus magus. In view of the toxin threat to humans and animals and defined application in analgesic therapy, it is necessary to develop a rapid, effective and accuracy method for the quantification and analysis of ω-CTX MVIIA in real samples. In the present study, a hybridoma cell named 2E5 stable secreting IgG antibody against ω-CTX MVIIA was selected successfully, and the subtype of Mab 2E5 was IgG1. The purified monoclonal antibody(Mab) 2E5 has high affinity (about 2.79 × 109 L/mol), and shows high specificity to ω-CTX MVIIA antigen. The linear range of ic-ELISA to detect ω-CTX MVIIA was 0.20˜7.22 µg/mL, with a lower detection limit (LOD) of 0.14 ng/mL. The average recovery of intra- and inter-assay were (85.45 ±â€¯2.28)% and (88.03 ±â€¯4.80)% respectively, with a coefficient of variation from 2.59% to 5.42%. The LOD of colloidal strip by naked eye was 1 µg/mL, and the detection time was less than 10 min without any equipment. The developed ELISA and colloidal test strips based on this IgG antibody could be used to detect ω-CTX MVIIA residue in real Conus samples.

Screening of a ScFv Antibody With High Affinity for Application in Human IFN-γ Immunoassay.

Interferon gamma (IFN-γ), a signal proinflammatory cytokine secreted by immune cell, and plays a critical role in the pathogenesis and progression of many diseases. It has been regarded as an important marker for determination of disease-specific immune responses. Therefore, it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real blood samples. Until now, the immunoassay based on singe chain variable fragment (scFv) antibody for human IFN-γ is still not reported. In the present study, an scFv antibody named scFv-A8 with high specificity was obtained by phage display and biopanning, with the affinity 2.6 × 109 L/mol. Maltose binding protein (MBP) was used to improve the solubility of scFv by inserting an linker DNA between scFv and MBP tag, and the resulted fusion protein (MBP-LK-scFv) has high solubility and antigen biding activity. The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive enzyme-linked immunosorbent assay (ELISA) (ic-ELISA) for detection of human IFN-γ, and the result indicated that the linear range to detect IFN-γ was 6-60 pg/mL with IC50 of 25 pg/mL. The limit of detection was 2 pg/mL (1.3 fm), and the average recovery was 85.05%, further demonstrating that the detection method based on scFv has higher recovery and accuracy. Hence, the developed ic-ELISA can be used to detect IFN-γ in real samples, and it may be further provided a scientific basis for disease diagnosis.

Detection of okadaic acid (OA) using ELISA and colloidal gold immunoassay based on monoclonal antibody.

Okadaic Acid (OA), a small seafood-borne toxin secreted by Dinophysis and Prorocentrum dinoflagellates, is generally distributed in various species of shellfish and has caused diarrhetic shellfish poisoning (DSP). In view of OA toxin threat to humans and animals, it is essential to develop a rapid, accurate and sensitive method for the detection and quantification of OA in real samples. In this study, a monoclonal antibody named 10E8 was screened by cells fusion of Sp2/0 with spleen cells isolated from immunized mouse, and the isotype of McAb 10E8 was belonged to IgG1. The resulted McAb 10E8 displayed higher specificity to OA antigen, with the highest affinity of 2.66×109L/moL until now. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect OA was 20-750ng/mL. The limit of detection (LOD) was 12pg/mL, and the recovery average was (84.04±5.08)%. The LOD of colloidal gold immunoassay by naked eye and strip reader was 1ng/mL and 100pg/mL, respectively, with an average recovery of (88.0275±4.4225)%. Therefore, the developed ELISA and colloidal gold immunoassay based on this McAb can be used for OA detection in real samples.

Screening and Molecular Evolution of a Single Chain Variable Fragment Antibody (scFv) against Citreoviridin Toxin.

Citreoviridin (CIT), a small food-borne mycotoxin produced by Penicillium citreonigrum, is generally distributed in various cereal grains and farm crop products around the world and has caused cytotoxicity as an uncompetitive inhibitor of ATP hydrolysis. A high affinity single chain variable fragment (scFv) antibody that can detect the citreoviridin in samples is still not available; therefore, it is very urgent to prepare an antibody for CIT detection and therapy. In this study, an amplified and assembled scFv from hybridoma was used to construct the mutant phage library by error-prone PCR, generating a 2 × 108 capacity mutated phage display library. After six rounds of biopanning, the selected scFv-5A10 displayed higher affinity and specificity to CIT antigen, with an increased affinity of 13.25-fold (Kaff = 5.7 × 109 L/mol) compared to that of the original wild-type scFv. Two critical amino acids (P100 and T151) distributed in H-CDR3 and L-FR regions that were responsible for scFv-5A10 to CIT were found and verified by oligonucleotide-directed mutagenesis, and the resulting three mutants except for the mutant (P100K) lost binding activity significantly against CIT, as predicated. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect CIT was 25-562 ng/mL with IC50 at 120 ng/mL. The limit of detection was 14.7 ng/mL, and the recovery average was (90.612 ± 3.889)%. Hence, the expressed and purified anti-CIT MBP-linker-scFv can be used to detect CIT in corn and related samples.

Corrigendum: The pathogenesis, detection, and prevention of Vibrio parahaemolyticus.

[This corrects the article on p. 144 in vol. 6, PMID: 25798132.].

The pathogenesis, detection, and prevention of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a Gram-negative motile bacterium that inhabits marine and estuarine environments throughout the world, is a major food-borne pathogen that causes life-threatening diseases in humans after the consumption of raw or undercooked seafood. The global occurrence of V. parahaemolyticus accentuates the importance of investigating its virulence factors and their effects on the human host. This review describes the virulence factors of V. parahaemolyticus reported to date, including hemolysin, urease, two type III secretion systems and two type VI secretion systems, which both cause both cytotoxicity in cultured cells and enterotoxicity in animal models. We describe various types of detection methods, based on virulence factors, that are used for quantitative detection of V. parahaemolyticus in seafood. We also discuss some useful preventive measures and therapeutic strategies for the diseases mediated by V. parahaemolyticus, which can reduce, to some extent, the damage to humans and aquatic animals attributable to V. parahaemolyticus. This review extends our understanding of the pathogenic mechanisms of V. parahaemolyticus mediated by virulence factors and the diseases it causes in its human host. It should provide new insights for the diagnosis, treatment, and prevention of V. parahaemolyticus infection.

Development of a functional antibody by using a green fluorescent protein frame as the template.

Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.

A prenatal missed diagnosed case of submicroscopic chromosomal abnormalities by karyotyping: the clinical utility of array-based CGH in prenatal diagnostics.

BACKGROUND: Array-based comparative genomic hybridization possesses a number of significant advantages over conventional cytogenetic and other molecular cytogenetic techniques, providing a sensitive and comprehensive detection platform for unexpected imbalances in the genome wide. CASE PRESENTATION: The newborn proband, demonstrated with craniofacial dysmorphism and multiple malformations, was born to a family with spontaneous abortions. This pregnancy was uneventful, except the prenatal ultrasound examination showed an increased nuchal translucency at 12(+) weeks of gestation. Cytogenetics revealed an apparently normal karyotype, and the couple decided to continue the pregnancy. Array-based CGH analysis was applied to the affected infant, identified a combination of 18p deletion and 7q duplication. Further study indicates that the unbalanced translocation was inherited from a balanced translocation carrier parent. CONCLUSIONS: In review of the case, several overlooked points leading to the missed diagnosis should be discussed and certain quality control strategies should be adopted to avoid similar problems in the future. Array-based CGH and karyotyping techniques are complemented by diverse detection spectrum and resolutions, and a combination of these methods could help providing optimal genetic diagnosis. Given that the array-CGH analysis will not introduce additional risk to patients, it is reasonable to recommend those already undergoing invasive testing should take array-based CGH as an adjunct to conventional cytogenetic tests and other molecular cytogenetic analysis.

Reference values of fetal serum ß2-microglobulin in the Chinese: evaluation of its clinical usefulness.

BACKGROUND: The level of ß2-microglobulin was generally used to evaluate the renal function in adults. Elevated levels of ß2-microglobulin were also applied to assess the perinatal situations in neonates and fetuses. The aim of our study was to establish and determine the reference values of fetal serum ß2-microglobulin in the Chinese and to assess its clinical benefits in abnormal fetuses. METHODS: Data from 308 normal cord blood samples were obtained to calculate the normal reference values of fetal serum ß2-microglobulin. According to the equations we obtained, we analyzed the level of ß2-microglobulin in four case groups: renal malformation, hydrops, cytomegalovirus (CMV) infection and rubella virus (RV) infection. RESULTS: In the normal group, the concentration of ß2-microglobulin decreased with the gestational age, with a mean value of 4.35±0.59 mg/L. The upper limit of ß2-microglobulin was calculated as 7.55-0.074×gestational age in weeks. The levels of ß2-microglobulin were significantly higher in the four case groups than the normal group. For the four groups, the sensitivity is 72.7%, 69.6%, 86.7% and 100%, respectively. CONCLUSIONS: Fetal serum ß2-microglobulin may be used as a predictor to evaluate the situations of fetal diseases.

[A de novo mutation of P gene causes oculocutaneous albinism type 2 with prenatal diagnosis].

OBJECTIVE: To determine the genotype of a family affected with oculocutaneous albinism (OCA) and to provide genetic counseling and prenatal diagnosis. METHODS: To determine the genotypes and mutational sites through PCR and sequencing for all exons and exon-intron junctions of 4 OCA genes in the proband and the P gene of her parents. Prenatal genotyping of the fetus was carried out using amniocentesis sample. RESULTS: The patient was diagnosed with OCA2 based on a genotype of c.1327G>A/c.2360C>T. Her father was heterozygous for c.2360C> T, whilst her mother has none of the two mutations. c.1327G>A is therefore a maternal de novo mutation. Neither of the mutations was found in the fetus. CONCLUSION: A maternally inherited de novo mutation c.1327G>A has been identified in the patient. In order to detect de novo mutations, full sequence analysis is necessary.

[Rapid prenatal genetic diagnosis of a fetus with a high risk for Morquio A syndrome].

OBJECTIVE: To provide rapid and accurate prenatal genetic diagnosis for a fetus with high risk of Morquio A syndrome. METHODS: Based on ascertained etiology of the proband and genotypes of the parents, particular mutations of the GALNS gene were screened at 10th gestational week with amplification refractory mutation system (ARMS), denaturing high performance liquid chromatography (DHPLC), and direct DNA sequencing. RESULTS: DHPLC screening has identified abnormal double peaks in the PCR products of exons 1 and 10, whilst only a single peak was detected in normal controls. Amplification of ARMS specific primers derived a specific product for the fetus's gene, whilst no similar product was detected in normal controls. Sequencing of PCR products confirmed that exons 1 and 10 of the GALNS gene from the fetus contained a heterozygous paternal c.106-111 del (p.L36-L37 del) deletion and a heterozygous maternal c.1097 T>C (p.L366P) missense mutation, which resulted in a compound heterozygote status. CONCLUSION: The fetus was diagnosed with Morquio A syndrome and a genotype similar to the proband. Termination of the pregnancy was recommended. Combined ARMS, DHPLC and DNA sequencing are effective for rapid and accurate prenatal diagnosis for fetus with a high risk for Morquio A syndrome. Such methods are particularly suitable for early diagnosis when pathogenesis is clear. Furthermore, combined ARMS and DHPLC are suitable for rapid processing of large numbers of samples for the identification of new mutations.

Novel rare alleles of ABCA1 are exclusively associated with extreme high-density lipoprotein-cholesterol levels among the Han Chinese.

BACKGROUND: High-density lipoprotein (HDL) is a major plasma lipoprotein directly associated with cholesterol metabolism. The ATP binding cassette transporter 1 gene (ABCA1) is one of the major genes modulating plasma levels of HDL-cholesterol (HDL-C). Rare alleles of ABCA1 associated with extreme HDL-C concentrations have not been previously investigated in the Chinese. METHODS: Blood samples were collected from 470 subjects whose HDL-C concentrations were within the top 5% of the distribution, 335 subjects in the lowest 5%, and 220 within the range 5%-95%. First, we sequenced all exons of the ABCA1 gene from 50 subjects from the group with extremely high HDL-C, and 50 from the group with extremely low HDL-C concentrations. Next, in the remaining subjects, we genotyped the non-synonymous variants identified exclusively with either extreme group. RESULTS: Four novel non-synonymous alleles were identified; all were rare. Alleles c.3029C>T (p.Ala1010Val) and c.5399A>G (p.Asn1800Ser) were found exclusively in the low group, c.2031C>A (p.Asp677Glu) and c.2660G>T (p.Cys887Phe) exclusively in the high group. CONCLUSIONS: Our results show that some rare alleles of ABCA1 are associated with marked phenotypes, supporting the "rare-variant common-disease" hypothesis. Certain alleles also provide tools for identifying individuals at high risk of dyslipidaemia, allowing for early therapeutic intervention.

[Studies on safety of cordocentesis guided by transabdominal ultrasound for prenatal diagnosis].

OBJECTIVE: To assess the safety and efficacy of diagnostic cordocentesis during pregnancy. METHODS: During March 1990 to June 2003, 2403 consecutive cordocenteses were performed under transabdominal ultrasound guidance at Guangzhou Women and Children's Hospital. The results of each procedure was prospectively collected and subsequently analysed in terms of operational complications and pregnancy outcomes. RESULTS: Success rate of cordocentesis: totally 2368 procedures (98.5%) were done successfully at the first attempt, and 35(1.5%) required repeated cordocentesis, 16 of which were performed successfully at second attempt. Duration of cordocentesis: In 75.5% cases, the procedure was completed in less than 5 min, and in 93.0% cases in less than 10 min. COMPLICATIONS: Transient bleeding at puncture site was observed in 315 cases (13.1%), transient fetal bradycardia in 125 cases (5.2%), and chorioamnionitis in 2 cases (0.1%). Pregnancy outcomes: The total fetal loss rate was 0.8% (18 cases of abortions). The rate of premature birth after cordocentesis was 0.2% (4 cases). CONCLUSION: Cordocentesis during pregnancy is a useful, relatively safe, and effective procedure for prenatal diagnosis.