cDNA Display-Mediated Immuno-PCR (cD-IPCR): An Ultrasensitive Immunoassay for Biomolecular Detection.

Immuno-PCR (IPCR) is a sensitive antigen detection by means of specific antibody-DNA conjugates. To ensure the successful conjugation of a protein (an antibody) with a reporter DNA, immuno-PCR method based on cDNA display (cD-IPCR) has been introduced. The cDNA display molecule is a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level, which is directly used for antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

Critical review on conventional spectroscopic α-amylase activity detection methods: merits, demerits, and future prospects.

α-Amylase is an endoenzyme that catalyses the hydrolysis of internal α-l,4 glycosidic linkages in polysaccharides to produce maltose, maltotriose, and α-limit dextrins. It is widely used in the laboratorial and industrial workflow for several applications. There are several methods utilizing different techniques and substrates to assess α-amylase activity, among which the spectroscopic methods have found widespread applicability due to their ease of use and cost-effectiveness. Depending upon the reaction principle, these assays are classified into four groups: reducing sugar, enzymatic, chromogenic, and amyloclastic methods. Despite the presence of numerous methods, there is no general reliable method to assess α-amylase activity. Each method is shown to have its own merits and demerits. Many improvements have been made to make the available methods more accurate, reliable, and easy. This communication briefly discusses the basic reaction mechanisms and critically reviews the advantages and shortcomings associated with each method. Further recommendations are made for future development. © 2020 Society of Chemical Industry.

In vitro selection of anti-gliadin single-domain antibodies from a naïve library for cDNA-display mediated immuno-PCR.

Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. In this study, cDNA display method was used to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naïve VHH library. The cDNA display method is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. Three candidate VHHs were selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. In addition, a novel cDNA display mediated immuno-PCR method (cD-IPCR) was successfully applied to detect gliadin in food. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner.

A novel immuno-PCR method using cDNA display.

Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level. Several other in vitro display technologies (e.g., ribosome display, mRNA display) have similar simple nucleic acid-peptide linkage. However, they should be unsuitable for diagnostic applications because of their lability against heat and RNase. The newly developed system here, termed cDNA display mediated immuno-PCR (cD-IPCR), proved to work in direct- and sandwich-type detection of target proteins. Detection of a target in serum was also possible, using a VHH (variable domain of the heavy chain of a heavy chain antibody) antibody as a binding molecule. Although further improvement on sensitivity and quantitativity is necessary before the method becomes useful, we believe this work demonstrated a potential of cD-IPCR as an alternative novel format of IPCR.

Applicability and reliability of the glucose oxidase method in assessing α-amylase activity.

Glucose oxidase (GOD) is an enzyme widely used in glucose monitoring systems owing to its high specificity towards glucose. However, in our previous work maltose was found to show significant interaction with GOD and based on this observation, a novel microplate-based method was developed to assess α-amylase inhibitory activity (GOD method). Concerns regarding the interaction of GOD with maltose has limited the widespread use of the GOD method in assessing α-amylase activity. The present paper provides answers to concerns regarding the interaction of GOD with maltose using HPLC studies and application of the GOD method in assessing α-amylase activity. According to the results, the newly developed GOD method can be considered as a well-suited method for the determination of α-amylase activity and as an easy method to do kinetic studies compared to other available methods.

cDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method.

Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level. The cDNA display molecule for IPCR is generated first by transcribing the DNA that encodes the detection antibody into an mRNA by in vitro transcription. A puromycin DNA linker is then ligated to the mRNA and then in vitro translation and reverse-transcription are performed to generate the cDNA display molecule. The molecule is then directly used in antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

Boiled, sprouted, and raw cowpea-incorporated diets modulate high-fat diet-induced hypercholesterolemia in rats.

This study was carried out to investigate the effect of processed (boiled and sprouted) cowpea-incorporated experimental diets on serum cholesterol and serum antioxidant capacity in high-fat diet (HFD)-fed Wistar rats. Seven weeks old male Wistar rats were fed 20% fat as a control (CD), for comparison with 20% fat-enriched diets containing 20% whole raw cowpea diets (Bombay Raw Diet; BRD and MI35 Raw Diet; MRD), boiled cowpea diets (Bombay Boiled Diet; BBD and MI35 Boiled Diet; MBD) and sprouted cowpea diet (Bombay Sprouted Diet; BSD) for 6 weeks. The increase in serum total cholesterol as a result of high-fat diet was significantly countered by boiled and raw cowpea-incorporated diet-fed rats. Increased serum non-HDL-C level caused by HFD was significantly (p < 0.05) countered by raw, boiled, and sprouted cowpeas, while HDL-C was increased by raw MI and boiled Bombay incorporated diets. Boiling has improved the hypocholesterolemic ability of Bombay cowpea and BBD has significantly (p < 0.05) modulated serum HDL-C level and liver weight in rats. These findings were supported significantly high soluble fiber content in processed cowpea powder than that in raw cowpea powder. The decrease in serum antioxidant activity as a result of HFD was significantly countered by BRD. Processing has reduced the antioxidant activity in cowpeas and serum antioxidant activity in rats. Cecal lactobacilli population was significantly high in all cowpea diet-fed groups compared to control. Modulated serum cholesterol level in cowpea diet-fed rats was accompanied by dietary fiber composition, antioxidant activity in cowpeas and fecal weight, cecal weight and cecal lactobacilli population in rats compared to control. Both processed and raw cowpea-incorporated diets have modulated HFD-induced hypercholesterolemia by modulating serum antioxidative capacity, cholesterol metabolism, and cecal fermentation.

Cowpea: an overview on its nutritional facts and health benefits.

Cowpea (Vigna unguiculata) is a legume consumed as a high-quality plant protein source in many parts of the world. High protein and carbohydrate contents with a relatively low fat content and a complementary amino acid pattern to that of cereal grains make cowpea an important nutritional food in the human diet. Cowpea has gained more attention recently from consumers and researchers worldwide as a result of its exerted health beneficial properties, including anti-diabetic, anti-cancer, anti-hyperlipidemic, anti-inflammatory and anti-hypertensive properties. Among the mechanisms that have been proposed in the prevention of chronic diseases, the most proven are attributed to the presence of compounds such as soluble and insoluble dietary fiber, phytochemicals, and proteins and peptides in cowpea. However, studies on the anti-cancer and anti-inflammatory properties of cowpea have produced conflicting results. Some studies support a protective effect of cowpea on the progression of cancer and inflammation, whereas others did not reveal any. Because there are only a few studies addressing health-related effects of cowpea consumption, further studies in this area are suggested. In addition, despite the reported favorable effects of cowpea on diabetes, hyperlipidemia and hypertension, a long-term epidemiological study investigating the association between cowpea consumption and diabetes, cardiovascular disease and cancer is also recommended. © 2018 Society of Chemical Industry.

Comparative Analysis of Nutritional and Bioactive Properties of Aerial Parts of Snake Gourd (Trichosanthes cucumerina Linn.).

The present investigation was carried out to determine the nutritional and functional properties of T. cucumerina. Water extracts of freeze dried flowers, fruits, and leaves of T. cucumerina were evaluated for their total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, α-amylase inhibitory activity, and fiber and mineral contents. Antioxidant activity, TPC, and TFC were significantly higher (P ≤ 0.05) in leaves than in flowers and fruits. A significant linear correlation was observed between the TPC, TFC, and antioxidant activities of plant extracts. Although, leaves and flower samples showed a significantly higher (P ≤ 0.05) amylase inhibitory activity than the fruit samples, the overall amylase inhibition was low in all three parts of T. cucumerina. Soluble and insoluble dietary fiber contents were significantly higher (P ≤ 0.05) in fruits than in flowers and leaves. Ca and K contents were significantly higher (P ≤ 0.05) in leaf followed by fruit and flower and Mg, Fe, and Zn contents were significantly higher (P ≤ 0.05) in leaves followed by flowers and fruits. In conclusion, T. cucumerina can be considered as a nourishing food commodity which possesses high nutritional and functional benefits for human health.

Health-beneficial properties of potato and compounds of interest.

Potatoes have shown promising health-promoting properties in human cell culture, experimental animal and human clinical studies, including antioxidant, hypocholesterolemic, anti-inflammatory, antiobesity, anticancer and antidiabetic effects. Compounds present such as phenolics, fiber, starch and proteins as well as compounds considered antinutritional such as glycoalkaloids, lectins and proteinase inhibitors are believed to contribute to the health benefits of potatoes. However, epidemiological studies exploring the role of potatoes in human health have been inconclusive. Some studies support a protective effect of potato consumption in weight management and diabetes, while other studies demonstrate no effect and a few suggest a negative effect. As there are many biological activities attributed to the compounds present in potato, some of which could be beneficial or detrimental depending on specific circumstances, a long-term study investigating the association between potato consumption and diabetes, obesity, cardiovascular disease and cancer while controlling for fat intake is needed. © 2016 Society of Chemical Industry.

A simple microplate-based method for the determination of α-amylase activity using the glucose assay kit (GOD method).

For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of α-amylase inhibitory activity using the glucose assay kit was developed. The new method facilitates rapid screening of a large number of samples, reduces labor, time and reagents and is also suitable for kinetic studies. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The test is done in microtitre plates with a total volume of 260µL and an assay time of 40min including the pre-incubation steps. The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining α-amylase activity.

Banana Blossom (Musa acuminate Colla) Incorporated Experimental Diets Modulate Serum Cholesterol and Serum Glucose Level in Wistar Rats Fed with Cholesterol.

Hypocholesterolaemic and hypoglycaemic effect of banana blossom were studied in high-cholesterol fed rats. Experimental groups were fed for 4 weeks, with casein as the basal diet (CN), in comparison with two diets containing 0.5% cholesterol (CD) and 0.5% cholesterol + 21% banana blossom powder (CDB). Serum total cholesterol, non-HDL-cholesterol level, and serum glucose concentrations were lower in CDB fed group compared with CD fed group. Lower serum cholesterol and glucose level (P < 0.05) in CDB fed group were followed by higher faecal weight, caecal weight, caecal Lactobacilli, and Bifidobacteria population in CDB fed group compared to CD diet fed group. Lower serum AST level in banana blossom fed rats showed the reduction in oxidative stress induced by high cholesterol diet. Based on these data, it could be speculated that banana blossom incorporated experimental diets may modulate the hypocholesterolaemic and hypoglycaemic responses in Wistar rats.