Chemical extraction of indigo from Indigofera tinctoria while attaining biological integrity

Indigofera tinctoria was permeabilized with 20% methanol (v/v) at 25 °C and released 8±2 μg indigo g −1 dry plant material (excluding roots). This is equivalent to 42±11% of the total indican within the cells. The plants began to recover after 2 weeks.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42486/1/10542_2004_Article_239463.pd

Chemical permeabilization and in situ removal of daidzein from biologically viable soybean (Glycine max) seeds

After 24 h of chemical permeabilization with 20% (v/v) methanol at 25 °C, the amount of daidzein released from soybean seeds is 15 to 20% of the amount (0.0423 ± 0.0045 mg/g seed dry wt) obtained by physical grinding. With this chemical permeabilization condition, 70% of the permeabilized seeds are still able to germinate. The release of daidzein is enhanced to 33% with the addition of XAD-4 to 20% (v/v) methanol without affecting seed viability. © Rapid Science Ltd. 1998Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42485/1/10542_2004_Article_176543.pd

Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

AbstractAflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC50) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- (n = 6) and inter- (n = 12) variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R2 of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples

Light -directed parallel synthesis of oligopeptide microarrays using in situ photogenerated acid.

Presently, there are strong demands for libraries of small peptides containing sequences of eight residues or less for applications such as screening antibodies and specific binding proteins. Light-directed synthesis on microarray platform developed by Affymetrix offers a mean to synthesize thousands of predetermined sequence oligopeptides at defined sites. In this technique, solid-phase peptide synthesis and photolithography approach are combined to construct the peptide microarray. Light illumination through photolithographic masks is used to control the pattern of the localized photodeprotection in different areas. As a result, the simultaneous synthesis of different peptide analogs at different locations can be achieved. However, many photolithographic masks, which are very expensive to make, are usually required for the synthesis. Consequently, establishment of the method as routine is simply not possible. Therefore, the objective of this research is to develop an inexpensive method to make the peptide microarray with high efficiency and accuracy. The development of the new technique stems from two main concepts. First, an acid compound generated from triarylsulfonium hexafluoroantimonate (SSb) by light activation is used in the deprotection step of the peptide synthesis. Second, laser scanner is used to replace the photolithographic mask in order to control the locations of the illumination by laser diode to simultaneously synthesize different oligopeptides on the microwell array platform. The comparative oligopeptide synthesis using the conventional acid, trifluoroacetic acid (TFA) and the photogenerated acid reveals that SSb can replace TFA in the pentapeptide synthesis with high efficiency (93 +/- 4%). The developed technique is used to synthesize two microarray models of human protein p53 (residue 20--25) and lead binding tetrapeptide (Glu-Cys-Glu-Glu). The oligopeptide models and their analogs are parallel synthesized in the predetermined locations of the microarrays. The specific binding of the oligopeptide models is detected at the correct synthesis locations as designed and can be distinguished from their analogs. The cost of making the peptide microarray using the developed technique and the photolithographic mask technique are estimated and compared. The comparison shows that the cost to operate the deprotection of 20 amino acids using the laser scanner is less expensive than that using the photomask. The technique developed in this research can be used to make the oligopeptide microarray with high efficiency, accuracy and is cost effective. Importantly, this technique is convenient to set up in any regular research laboratories.Ph.D.Applied SciencesChemical engineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129789/2/3042100.pd

Development and validation of point-of-care testing of albuminuria for early screening of chronic kidney disease.

IntroductionChronic kidney disease (CKD) is a significant global health issue. As the prevalence of renal replacement therapy (RRT) in Thailand is increasing, early detection and management of CKD is the most important step to prevent CKD progression and the need for RRT. Current diagnostic tests for CKD are non-specific and expensive. We aimed to develop and validate antibody-based-albumin point-of-care testing (POCT) to detect patients with impaired kidney function at early stage.MethodsThe prototype strip test was developed under the concept of competitive lateral flow immunochromatography assay, or strip test. Monoclonal antibodies (MAbs) to human serum albumin (HSA) were harvested from the hybridomas of spleen cells from immunized mice and mouse myeloma cells. Presence of MAbs was detected by enzyme-linked immunosorbent assay (ELISA). Spot urine was obtained from patients with kidney disease, type I, or type II Diabetes Mellitus upon their visit at King Chulalongkorn Memorial Hospital during 2018-2019. All samples were analyzed for urine albumin with our POCT (CU microalbumin) and the other two commercial POCTs (Microalbu PHAN and MICRAL). The results were validated against standard method for urine microalbumin measurement. A urine microalbumin concentration of less than 20 ug/ml was defined as normal. The sensitivity, specificity, and predictive values were calculated in comparison with the standard laboratory method.ResultA total of 100 adult patients were included. CU microalbumin had a sensitivity of 86%, a specificity of 94%, and a positive predictive value of 96%. Our POCT showed good correlation with the laboratory results.ConclusionCU microalbumin correlated well with the standard method for quantitative measurement of urine albumin. Therefore, it has the potential for early screening of CKD, especially in primary health care facilities in resource limited settings