Development of Methyl Ester Antibody-Based Competitive Indirect ELISA for Quantitative Detection of Mitragynine in Human Urine.

Mitragynine is the main psychoactive compound of Mitragyna speciosa Korth. (kratom). This alkaloid could render psychotropic effects and is often misused as a substitute for commercial drugs. Nowadays, the increasing popularity of kratom has led to the development of a rapid and effective detection method. The detection of mitragynine in a biological sample such as urine requires a highly sensitive and specific method due to the complex nature of mitragynine in urine. Enzyme-linked immunosorbent assay (ELISA) is well known as a rapid screening method for biological samples. In this study, a competitive indirect ELISA was successfully developed using MG-22-OCH3 IgG as a detection antibody for mitragynine in human urine. The mitragynine immunoassay showed a limit of detection and a limit of quantification of 0.412 and 1.25 µg/mL, respectively. The measurement range was between 0.01 and 100.0 µg/mL, with a minimal inhibition (IC50) value of 0.152 µg/mL. The developed ELISA was validated using a gold method such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). The percentage of recovery and the coefficient of variation (CV) for the ELISA and LCMS/MS analyses were 84.0-95.70%, 99.20-112.0%, 7.69-9.78%, and 2.86-6.62%, respectively. This indicates that the developed ELISA is a reliable method that can be used as a rapid approach for quantifying mitragynine content in biological samples.

Cross-reactivity analysis of milk proteins from different goat breeds with cow's milk allergens using a proteomic approach.

INTRODUCTION: Goat's milk thought to be a good substitute for cow's milk protein allergic (CMPA) individuals. However, there is growing evidence that their proteins have cross-reactivities with cow's milk allergens. This study aimed to profile and compare milk proteins from different goat breeds that have cross-reactivity to cow's milk allergens. METHODOLOGY: Proteomics was used to compare protein extracts of skim milk from Saanen, Jamnapari, and Toggenburg. Cow's milk was used as a control. IgE-immunoblotting and mass spectrometry were used to compare and identify proteins that cross-reacted with serum IgE from CMPA patients (n = 10). RESULTS: The analysis of IgE-reactive proteins revealed that the protein spots identified with high confidence were proteins homologous to common cow's milk allergens such as α-S1-casein (αS1-CN), ß-casein (ß-CN), κ-casein (κ-CN), and beta-lactoglobulin (ß-LG). Jamnapari's milk proteins were found to cross-react with four major milk allergens: α-S1-CN, ß-CN, κ-CN, and ß-LG. Saanen goat's milk proteins, on the other hand, cross-reacted with two major milk allergens, α-S1-CN and ß-LG, whereas Toggenburg goat's milk proteins only react with one of the major milk allergens, κ-CN. CONCLUSION: These findings may help in the development of hypoallergenic goat milk through cross-breeding strategies of goat breeds with lower allergenic milk protein contents.

A highly selective two-way purification method using liquid chromatography for isolating αS2-casein from goat milk of five different breeds.

The main challenges in the purification of αS2-casein are due to the low quantity in milk and high homology with other casein subunits, i.e., αS1-casein, ß-casein, and κ-casein. To overcome these challenges, the aim of this study was to develop a two-step purification to isolate native αS2-casein in goat milk from five different breeds; British Alpine, Jamnapari, Saanen, Shami, and Toggenburg. The first step of the purification was executed by anion-exchange chromatography under optimal elution conditions followed by size exclusion chromatography. Tryptic peptides from in-gel digestion of purified αS2-casein were sequenced and analyzed by LC-ESI-MS/MS. From 1.05 g of whole casein, the highest yield of αS2-casein (6.7 mg/mL) was obtained from Jamnapari and the lowest yield (2.2 mg/mL) was from Saanen. A single band of pure αS2-casein was observed on SDS-PAGE for all breeds. The αS2-casein showed coverage percentage of amino acid sequence from 76.68 to 92.83%. The two-step purification process developed herein was successfully applied for isolating native αS2-casein from goat milk with high purity, which will allow for future in vitro studies to be conducted on this protein.

Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein.

The chemical, technological and allergy properties of goat's milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10-3 µM compared to 9.046 × 10-3 µM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.