Immunological Separation of Bioactive Natural Compounds from Crude Drug Extract and Its Application for Cell-Based Studies

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray–Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

The The Effect of Elution Volume for Immunoprecipitation on m6A-Seq Analysis

Objective: To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation. Method: Two volumes (125 ?L for Group C and 100 ?L for Group T) of elution buffer were used to explore whether smaller volume could save testing time. Result: Time consumption of elution in Group T was significantly shorter than that in Group C, while the efficiency of eluted m6A-containing fragments and the performance of m6A-Seq as indicated by m6A peak distributions showed no difference between the two groups. Conclusion: A smaller volume of elution buffer was an economical way to reduce time consumption in immunoprecipitation.

Melanin, a potential new matrix material for recombinant His-tag protein purification

Nickel-Sepharose (Ni-Sepharose) has been being applied as the most common matrix in purifying His-tag proteins based on the affinity interaction between histidine residues and Ni2+ ion. However, Sepharose still comes at high cost for this purification purpose, especially in developing countries as Vietnam. Here, we show for the first time that melanin from ink sacs of squids which is considered as biowaste in the food industry, can be used as a new potential matrix material instead of Sepharose. We utilized either melanin or melanin charged with metal ions as the stationary phase of affinity purification of His-tag proteins. The results showed that a recombinant His-tag protein VP28 in a protein pool was captured by melanin and Ni2+/Fe3+/Zn2+ chelated melanin. Experiments for releasing VP28 were performed only on the melanin and Ni2+-melanin matrices. The result showed that VP28 was quite selectively eluted when applying elution buffer of 250 mM imidazole overnight. The relative efficiency in releasing VP28 of melanin and Ni-melanin matrices roughly compared to Ni-Sepharose were about 38 and 18% respectively. Further optimization of this process may allow higher efficiency in the purification of His-tag proteins.

Purification of anti-glycoconjugate monoclonal antibodies using newly developed porous zirconia particles

Here, we describe porous zirconia particles (PZPs) optimized for the purification of immunoglobulins. PZPs, with a pore size of approximately 10 nm, were designed to specifically interact with antibodies via surface modification with a phosphate functional group. A simple PZP purification method based on precipitation enabled efficient purification of mouse anti-glycosphingolipid globoside/Gb4Cer monoclonal IgM (κ-light chains) from hybridoma culture supernatants. Over 99% of contaminating proteins were removed by the PZP purification process, and approximately 50% of the IgM was recovered in the purified fraction after eluting the PZP-adsorbed antibodies with 100 mM phosphate buffer. Other IgG3 and IgM monoclonal antibodies that react with Gb4Cer or α2,6-sialyl LacNAc-modified glycoproteins could also be purified using PZPs and elution buffer at concentrations of 100–500 mM. All of the purified antibodies retained their antigen reactivity and specificity, indicating that PZP purification does not affect antibody function. As PZP purification is also suitable for purification of IgM consisting of λ-light chains and IgG derived from other mammalian species, it is expected to be applied to the purification of a variety of antibodies, including anti-glycoconjugate IgMs.

Simple and Rapid Method for Determination of Abemaciclib in Human Serum using Supported Liquid Extraction Pretreatment and LC-MS/MS Analysis

We developed a simple and rapid method for the determination of abemaciclib in human serum using supported liquid extraction (SLE) method for pretreatment and LC-MS/MS. Abemaciclib was extracted using SLE method with methyltert-butyl ether (MTBE) as elution buffer, and analyzed by LC-QTOF MS system, LCMS9030 (Shimadzu). Abemaciclib and fluconazole (internal standard) were detected with ESI spray in positive ionization mode, and the transition were set at 507.3/393.1629 for abemaciclib and 307.1/220.0677 for fluconazole. The retention times of abemaciclib and fluconazole were 2.76 and 2.98 min, respectively, and good linearity was obtained from 20–1,000 ng/mL for abemaciclib. The regression equation (weight = 1/x2) describing the calibration curve in human serum was y = 0.0196 x – 0.056 (R2 = 0.999), where y is the peak area ratio of abemaciclib against the IS and x is the nominal concentration of abemaciclib. The intra- and inter-assay accuracy varied between -4.3–1.7%, and the precision varied between 0.90–6.19%. The mean recovery rate of abemaciclib was 87.7 ± 4.3%, and the mean matrix factor was 1.00 ± 0.083. Our method offers speed and simplicity of sample preparation, which is one of great advantages in the analysis of clinical specimens. We believe that the present method will contribute to establishing a methodology for determining the optimal dose of abemaciclib for individual patients. Key words: abemaciclib, SLE, supported liquid extraction, quantification

Improve sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane

In clinical applications of miRNAs, the purity and quality of the testing samples are very critical, especially the obtained tissue sample volume is limited. If the extracted miRNAs are contaminated or different in quality before analysis, it will increase the variance of the analysis result and make the medical information judgment incorrect and cannot be portable. Herein, we improved the commercially extraction kit by realizing the fundamental mechanism and hoped to serve finding optimal procedures for increasing the recovery of miRNAs extracted from cultured cells. In the adsorption process, the factors, like increasing the ethanol concentration or adding Ca2+, could influence the RNA adsorption were investigated. For the elution process, the effect caused by raising the elution temperature and raising the pH value of elution buffer was studied. Finally, the conditions for miRNA extraction are optimal modified by using a 65% (v/v) solution of ethanol in the adsorption process, and using TE buffer with the pH value of 8.0 and raising the temperature to 55 °C in the elution. According to the quantified results, the improved extraction kit can promote the recovery of endogenous miR-21 by about 6 times by using the optimal extraction conditions comparing with the miRNeasy Mini Kit.