IMMPACT:Initiative for Cell Mortality Programme Assessment.

Cell Death, Apoptosis, Oxidative stress, Caspase, BCL2

Proseek single-plex protein assay kit system to detect sAxl and Gas6 in serological material of brain tumor patients

The receptor tyrosine kinase (RTK) Axl and its ligand Gas6 are critically involved in the pathogenesis of high-grade glioma (HGG). Both proteins were found to be overexpressed e.g. in tumor cells, mediating cell proliferation and migration as well as tumor angiogenesis and neuroinflammation. The extracellular domain of Axl (sAxl) and Gas6 were found in the peri-tumoral edema and blood of animals as well as in human glioma tissue. Therefore, we monitored the level of sAxl and Gas6 in human blood samples.
To increase the sensitivity of protein detection beyond commonly used standard methods we preliminary tested the innovative Proseek Single-Plex Protein Assay Kit System from Olink Bioscience together with new antibodies against the soluble RTK sAxl and its ligand Gas6. We conclude that the Proseek method is a highly sensitive and fast procedure that can be used as a possible powerful tool compared to routinely used ELISA-methods.

Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions.
While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions.

Early dengue diagnosis by nonstructural protein 1 antigen detection: rapid immunochromotography versus two the enzyme-linked immunosorbent assay kits.

Dengue is known for its serious life-threatening complications. New rapid kits available recently in India target circulating non-structural protein (NS1) antigen from day one onwards. The sensitivity and specificity of a newly introduced rapid combo kit against two conventional ELISA kits is assessed. The performance of this kit is quite satisfactory since excellent agreement of 94.26% was observed with particular reference to NS1 antigen detection among all three kits namely Rapid SD Bioline dengue Duo (SD Korea), InBios DENV Detect NS1 ELISA, USA and dengue Early ELISA, Panbio, Australia. The false positivity of the rapid kit is very low since its specificity as for as NS1 antigen detection is concerned is 98.33%. The use of combination kit helps to detect additional cases of dengue, which are negative for NS1 antigen but positive for IgM and/or IgG antibodies, thus facilitating early diagnosis in remote areas and small laboratorie.

Effect of heat treatment on the quantitative detection of egg protein residues by commercial enzyme-linked immunosorbent assay test kits

This study examined the changes in the solubility of egg proteins as affected by different heat treatments and compared the performances of three commercial test kits for the quantitation of protein residues in heat-treated samples. National Institute of Standards and Technology (NIST) whole egg standard reference material #8415 and Henningsen spray-dried whole egg powder were subjected to heating in the presence of water at 60 and 100 degrees C, autoclaving for 5 or 10 min, or dry heating at 60-400 degrees C for 10 min. The amount of protein in the heated samples was assayed using the bicinchoninic acid total protein assay as well as egg-specific commercial enzyme-linked immunosorbent assay (ELISA) kits.
Elevated heat resulted in a lower level of proteins extracted. Neogen’s Veratox kit, which is reactive to multiple proteins in egg, greatly underestimated the amount of residual proteins in the boiled or autoclaved samples. Tepnel BioSystems’ Biokits assay, which employs antibodies specific to a heat-stable marker protein (ovomucoid), registered a higher level of protein in these samples. Both test kits substantially underestimated the amount of residual proteins in samples dry-heated at temperatures >176 degrees C. The Morinaga test, using an improved extraction buffer, registered the highest level of protein in the heat-treated NIST samples but not the Henningsen samples. The underestimation by the commercial test kits was attributed to changes in the immunoreactivity of residual proteins after heat treatments and not the differences in the amount of protein extracted. These results suggest that thermal processing may affect the quantitative analysis of allergens and needs to be taken into account in the validation of commercial ELISA test kits.

Improvements in live cell analysis of G protein coupled receptors using second generation BD calcium assay kits

BD Calcium Assay Kits are designed for cell-based calcium mobilization high-throughput screening assays. The kits use a proprietary formulation including a non-fluorescent calcium indicator that becomes activated inside the cell and shows increased fluorescence upon calcium binding. The formulation includes a signal-enhancing reagent to maximize the signal over background in a homogeneous, no-wash assay format, based on a technology developed at BD. We have compared the next generation BD calcium assay kit product family to previous versions of the formulation, and to other commercially available homogeneous calcium assay kits. The improvements have enabled better performance on the cell lines and receptors that we have tested in all plate formats including 1536.