Capillary electrophoresis with laser induced fluorescence (CE-LIF): is a method for the direct quantitation of gene expression.
Detection and Quantification of Proteins Generally:
Gavin (US 2015/0093757) disclsoes capillary electrophoresis methods which include analysis and quantification of the amount of total protein in a sample. The method includes subjecting the sample to capillary electrophoresis in a separation matrix that includes a haloalkane compounds, exposing the separated sample to ultraviolet (UV) light for a sufficient period of time to react the haloalkane compound with tryptophan residues in the protein of the same to form fluroescent protein compoudns and detecting the fluroescence of the formed fluorescent compounds. In a further embodiment, the method includes detecting a specific target protein in a sample by performing capillary based immunoassay on the sample to detect the present or absence of the protein and detemining the results of the immunoassay to determine whether the protein is present or absent. The method includes icharged based (isoelectric focusing) and size (molecular weight based separation. The amount of protein is compared to other products in the sample to determine the relative amount of the protein.
Roach (US 8,940,232 (ProteinSimple) discloses the use of capillaries having a small internal dimension such as a microchannelsfor condcuting electrophoretic or isoelectric focusing sepration, immobilization and fluorescence detection under automated control. Following sepration of the biologcial molecuels in the fluid paths of the capillaries with theri ionic charge by isoelectric focusing, the seprated molecuels are immobilized in their focused positions in the capillaries with ultraviolet light.
Yang (US 9,400,277 (proteinSim;e) discloses methods for detecting analytes such as a protein in a fluid path which includes resolving asby isoelectric focusing (IEF), immobilizaiton as by photoimmobilzing one or more proteins in the capillary and detecting the protein(s) by contacting the photoimmobilized prtoeins with antibodies which can be labeled to form antibody-protein complex(es) in the capillary which are detected. In some embodiments, two or more detetion agents are used, one detection agetn for example a primary antibody that binds with one or mroe analytes and a second detection agetn, for example a secondary antibody, which can bind to the deteftion agent-analyte complex.
Voss (US 2009/0023156) disclsoes methods for quantifying analytes which includes the steps of loading a microfluidic device, seperating the standard and the analyte by electrophoresis, immobilizing the standard and analyte, detecting the standard and anlyte with at elast one antiboy and comparing the signal of the standard to the signal of the analyte to determine the quantity of analyte in the sample.
Detection of RNA:
Goldsmith (US7939252) dicloses a method of measuring an amount of ribo-nucleic acid (RNA) comprising incubating an RNA smaple with a fluorescenlty labeled RNA probe complementary to a sequence in a target RNA under conditions where the probe can hybridize to the target RNA to form an RNA-fluorescently labeled probe hybrid, passing the RNA-fluorescently labeled probe hybrid through a capillary electrophoresis system, detecting and recoridng changes in fluorescence as a peak as the RNA-fluorescenlty labeled probe passes through a detection window and determining an area under the peak, whereby the area under the peak indicates the amount of target RNA.
Detection of Activatable Antibodies (Probodies): (see also Common Analytical Types under “Analystics and Characterization”)
Carman (US 2019/0117789) discloses activatable antibodies that bind CD166. The activatable antibody (AA) includes an antibody that binds to CD166 including a heavy chain and light change, a masking moeity (MM) coupled to the Ab wherein the MM inhibits the binding of the AB to the CD166 when the AA is in an uncleaved state and a cleavable moeity (CM) coupled to the Ab, wehrein the CM is a substrate that functions for a protease. In some embdoiments an agent such as maytansinoid is conjugated to the AA. The anti-CD166 conjugated activatable antibody was activated with matriptase or MMP14 for 2 hours at 37C and mixed with intact conjugated activatable antibody. The mixture was then analysed by the The WES Capillary electrophoresis system using anti-human IgG (American Qualex Catalog #A110UK). Carman showed the ability to separate matriptase-activated or MMP14 activated conjugated activatable antibodies form intact conjugated activatable antibodies.
Desnoyers (US Patent Application No: 16/632265) discloses a method of quantitating a level of activation of an activatable antibody which includes the steps of 1) loading at least one capillary with a stacking matrix and a separation matrix. the “stacking matrix” refers to a highly porous (relative to the separation matrix) material that functions to concentrate proteins present in the sample and “stack” them at the interface with the separation matrix so that the proteins start migration under electrophoresis conditions form the same physical starting point. Suitable stacking matrices include those used to prepare stacking gels for Western blotting such as acrylamide, SDS. Capillaries pre-loaded with stacking matrix and separation matrix are available commercially as Wes Separation Module. Electrophoresis causes the compounds in the sample to migrate through the separation gel at differential rates according to molecular size/weight. 2) the HMW and LMW compounds are immobilzied within each capilalry as by using UV light. 3) Each capilarry is immunoprobed with a first reagent such as an idiotypic antibody that is specific for the activatable antibody. 3) Detection of the first reagent can be accomplised for example with a second reagent that binds to the first reagent. Exemplary detectably labeled second r agents include HRP-conjugate anti-mouse secondary antibody.
Vasiljeva (US 2021/0025877; see also WO 2019/018828) disclsoes a method of quantitating a level of activation of an activatable antibody which includes constacting a loaded capillary with a biological sample which includes an antivatable antibody, an activated activatable antibody and combinations of both wherein the loaded capillary is preloaded with a stacking matrix (a highly porous material that fand a separation matrix , separating one or more HMW components from LMW components within each capillary, immobilizing HMW componetns and LMW components within each capillary, immunoprobing each cunctions to concetnrate proteins present in the sample and stack them at the itnerface with the separation matrix so that the proteins start migration under electrophoresis conditions form the same physical starting point. Suitable stacking matrices may be prpared form teh same materials used to prepare stacking gels for Western bloting such as acrylamide). apillary with at elast a first reagent that is specific for at least one activatable antibody and detecting and quantitating a level of the first reagent in each capillary.