X.Y. Meng

A rapid immunomagnetic beads-based immunoassay for the detection of β-casein in bovine milk

An immunomagnetic beads-based enzyme-linked immunosorbent assay (IMBs-ELISA) was developed for the detection of β-casein in bovine milk. Immunomagnetic beads (IMBs) were employed as the solid phase. The anti-β-casein monoclonal antibody (McAb) bound to IMBs was used as capture probe and an anti-β-casein polyclonal antibody (PcAb), labelled with horseradish peroxidase (HRP), was employed as detector probe. Three reaction and two washing steps were needed. Each reaction needed 10 min or less, which significantly shortened detection compared with classic sandwich ELISA. β-Casein in bovine milk was detected across a linear range (2-128 μg mL(-1)). Application results were in accordance with the Kjejdahl method, which suggests the IMBs-ELISA is rapid and reliable for the detection of β-casein in bovine milk.

Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples.

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample.

Real-time immuno-PCR for ultrasensitive detection of pyrene and other homologous PAHs

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental pollutant that can lead to cancer and endocrine system disrupting. Here we developed a real-time immuno-PCR (RT-IPCR) assay based on a biotinylated reporter DNA system for ultrasensitive detection of pyrene (PYR) and homologous PAHs in water. The PAHs in sample compete with PYR-OVA coated on PCR plate to bind with monoclonal antibody (McAb). The biotinylated goat anti-mouse IgG (Bio-IgG) can be captured by the McAb bound with PYR-OVA. Then streptavidin is bound with biotin on Bio-IgG. Finally biotinylated reporter DNA is captured by the streptavidin and quantified by real-time PCR using FastStart universal SYBR Green Master (ROX) kit. The linear range of the assay was from 500 fmol L(-1) to 5 nmol L(-)) with a detection limit of 450 fmol L(-1). The average recoveries of PYR and homologous PAHs from lake water, tap water and commercial mineral water were 96.8%, 101.4% and 99.6% respectively, indicating that water samples had little interfere with the assay. The results demonstrated that the developed RT-IPCR might be a potential method for ultrasensitive detection of PYR and homologous PAHs in drinking and environment water sample.

Gold nanoparticle aggregation-based colorimetric assay for β-casein detection in bovine milk samples

Traditional Kjeldahl method, used for quality evaluation of bovine milk, has intrinsic defects of time-consuming sample preparation and two analyses to determine the difference between non-protein nitrogen content and total protein nitrogen content. Herein, based upon antibody functionalized gold nanoparticles (AuNPs), we described a colorimetric method for β-casein (β-CN) detection in bovine milk samples. The linear dynamic range and the LOD were 0.08-250 μg mL(-1), and 0.03 μg mL(-1) respectively. In addition, the real content of β-CN in bovine milk was measured by using the developed assay. The results are closely correlated with those from Kjeldahl method. The advantages of β-CN triggered AuNP aggregation-based colorimetric assay are simple signal generation, the high sensitivity and specificity as well as no need of complicated sample preparation, which make it for on-site detection of β-CN in bovine milk samples.

A monoclonal antibody based enzyme-linked immunosorbent assay for detection of phenylethanolamine A in tissue of swine

Phenylethanolamine A (PEA) is a phenethanolamine member of the family of β-adrenergic agonists compounds illegally used as feed additives for growth promotion. In this study, PEA was covalently linked to carrier protein cationized bovine serum albumin (cBSA) and egg albumin (OVA). A monoclonal antibody specific for PEA was produced and characteristics of monoclonal antibody (McAb) were studied. Based on the McAb, an enzyme-linked immunosorbent assay (ELISA) for detection of PEA was developed, which showed an IC50 value of 6.25 ng mL(-1) and a detection limit of 0.19 ng mL(-1). The average recovery of PEA from spiked samples was 103.4%, which demonstrated that the matrices of meat where PEA may be found do not interfere with the assay.

An enzyme-linked immunosorbent assay for detection of pyrene and related polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) can form DNA-binding compounds that show genotoxicity and carcinogenicity. Pyrene, as a PAH, was covalently linked to carrier protein bovine serum albumin and ovalbumin. A monoclonal antibody (McAb) was produced that showed high cross-reactivity values with chrysene (169.73%), benzo[a]pyrene (693.34%), benzo[a]anthracene (16.36%), and indeno[1,2,3-cd]pyrene (40.96%) and showed no significant cross-reactivity values with other homologues (<0.1%). A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of pyrene and some homologues in water samples. The detection limit of the assay was 65.08 pg ml(-1). The average recoveries of PAHs from tap water, lake water, and mineral water were 99.13, 99.74, and 99.19%, respectively, indicating that matrices of water samples do not interfere with the assay. The results demonstrated that the developed ELISA seems to be a potential method for monitoring of pyrene and some homologous PAHs in water samples.

Production of a monoclonal antibody and development of an immunoassay for detection of Cr(III) in water samples

In this study we report the production of a monoclonal antibody (Mab) specific for Cr(III)-chelate and the development of a competitive immunoassay for detection of Cr(III) in water samples. In the assay, the complete antigen (Cr(III)-ITCBE-BSA) was used as coating antigen, and Cr(III)-ITCBE as competitor competes with coating antigen to bind with Mab. Using this approach, the spiked water samples with Cr(III) were detected. The linear range of the detection was 0.7-12.4 ng mL(-1). The limit of the detection (LOD) was 0.51 ng mL(-1). The spiked results were also confirmed by ICP-MS, which showed a good correlation (R(2)=0.997) between the two methods. The results indicated that the developed assay was reliable and suitable for the detection of Cr(III) in water samples.

Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of kappa-casein in bovine milk samples

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample.

An improved RT-IPCR for detection of pyrene and related polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous homogeneous chemicals which are well known by carcinogens, mutagens and endocrine disorder. Here, an improved real-time immuno-PCR (RT-IPCR) was developed for detection of pyrene and its homologs in water samples. The PAHs in sample compete with pyrene-modified DNA to bind with monoclonal antibody (McAb) coated on PCR plate. The reporter DNA was exponentially amplified by real-time PCR instrument using Fast Start universal SYBR Green Master (ROX) kit. Only two reaction steps were needed to accomplish the detection. The assay had a good linear range from 5 pmol L(-1) to 5 nmol L(-1) with a detection limit of 3.5 pmol L(-1). For application assay, the average recoveries from tap water, lake water and mineral water were 98.4%, 98.2% and 99.7%, respectively which showed a good correlation (R(2)=0.9906) with those from GC-MS. The results indicated that the improved RT-IPCR seems to be a potential method for simple and ultrasensitive detection of pyrene and some homologues in environment water samples.

A novel immunotoxin – rCCK8PE38 targeting of CCK-R overexpressed colon cancers

Abstract Background: Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, such as pancreatic, colon and gastric cancers. Previous studies have shown that the specific receptor-binding property of CCK for CCK receptors (CCKRs) can be exploited to produce immunotoxins (ITs) that target cancer cells overexpressing CCK receptors. Purpose: Construct a new IT-targeting CCKR-overexpressing colon cancers. Methods: To construct the CCKR-targeted IT, a reverse CCK8 peptide was fused with a modified 38-kDa truncated form of the Pseudomonas exotoxin (PE38KDEL). An efficient immunoaffinity purification procedure was used to produce a PE38-based IT. Several analyses, including CCK8 competition and indirect immunofluorescence assays, were performed to confirm the interaction between rCCK8 and CCKR. After cytotoxic assays on several cell lines, the anti-tumor activity of the new IT was detected in nude mice. Results: The rCCK8PE38 IT showed specific cytotoxicity for two colon cancer cell lines and one gastric cancer cell line. After purification, 18–26 mg of pure rCCK8PE38 per 1 L of culture was obtained. Purified rCCK8PE38 showed high cytotoxicity in colon cancer cell lines with IC50 values of 0.8–3.5 ng/mL. The results of the CCK8 competition and indirect immunofluorescence assays showed that rCCK8 had a specific interaction with CCKR. Nude mice inoculated with HCT-8 tumor xenografts were treated with rCCK8PE38, which efficiently decreased the tumor size in those mice. Conclusions and discussion: All of these data suggest that rCCK8PE38 has potential as a new immunotherapy agent. Furthermore, the results of this study further support the high value of the immunoaffinity method for IT purification procedures.