Methods to reduce evaporation during elevated temperature
Inventors
YAMANISHI, Douglas T. • HOM, Clifford • Shah, Amit D.
Assignees
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Publication Number
US-10006084-B2
Publication Date
2018-06-26
Expiration Date
Abstract
A method including contacting a tissue or cellular sample with a reagent including an evaporation reducing agent(s) having the general formula X1—R—X2, wherein R is an alkyl, alkenyl, alkynyl or an aromatic moiety of 1 or more carbon atoms that may be substituted with an oxygen, nitrogen or sulfur and X1 and X2 are independently selected to be moiety that is susceptible to hydrogen bonding and processing the tissue or cellular sample. A reagent used in the processing of a tissue or cellular sample with the reagent containing an evaporation reducing agent.
Core Innovation
The document discloses an assay reagent and a method of use that reduce evaporation of aqueous processing reagents during elevated-temperature tissue or cellular assays. A tissue or cellular sample is contacted with a hybridization solution or a primary antibody diluent solution that includes a detergent and an evaporation reducing agent, and the tissue or cellular sample is processed.
The evaporation reducing agent is defined by a general formula X1—R—X2. R is an alkyl moiety, an alkenyl moiety, an alkynyl moiety, or an aromatic moiety that may be substituted or interrupted with an oxygen, sulfur, or nitrogen atom, while X1 and X2 are independently selected to be moieties that comprise an electronegative atom and/or are susceptible to hydrogen bonding.
The reagent includes a hybridization solution or a primary antibody diluent solution containing the detergent and the evaporation reducing agent above specified volume thresholds. The disclosed examples and results describe reduced volume loss during elevated-temperature steps associated with oligonucleotide hybridization, denaturation, and antibody incubation, while maintaining similar assay performance.
Claims Coverage
The provided portion includes two independent claims covering both a method and a corresponding reagent for processing a tissue or cellular sample. The independent claims include two key inventive features: contacting the sample with a hybridization solution or a primary antibody diluent containing a detergent and an evaporation reducing agent, and specifying the evaporation reducing agent by the X1—R—X2 general formula together with volume thresholds that depend on glycol-containing versus non-glycol-containing agents.
Contacting a tissue or cellular sample with a hybridization or primary antibody diluent containing detergent and an evaporation reducing agent
A method comprising contacting a tissue or cellular sample with a composition of a hybridization solution or a primary antibody diluent solution, wherein the hybridization solution or the primary antibody diluent solution comprises a detergent and at least one evaporation reducing agent, and processing the tissue or cellular sample.
Using an evaporation reducing agent defined by X1—R—X2 with glycol/non-glycol volume thresholds
The composition includes at least one evaporation reducing agent having the general formula X1—R—X2, where R is an alkyl, alkenyl, alkynyl or aromatic moiety optionally substituted or interrupted with an oxygen, sulfur or nitrogen atom, and where X1 and X2 are independently selected to be moieties comprising an electronegative atom and/or susceptible to hydrogen bonding, wherein the at least one evaporation reducing agent is present in an amount greater than 20 percent by volume when the evaporation reducing agent is a non-glycol-containing agent or a combination of more than one evaporation reducing agent, or an amount greater than 30 percent by volume when the evaporation reducing agent is a glycol-containing agent.
Providing a reagent formulation of hybridization solution or primary antibody diluent with detergent and threshold evaporation reducing agent
A reagent operable for use in processing of a tissue or cellular sample, the reagent comprising a composition of a hybridization solution or a primary antibody diluent solution, wherein the hybridization solution or the primary antibody diluent solution comprises a detergent and an evaporation reducing agent present in an amount greater than 20 percent by volume when the evaporation reducing agent is a non-glycol-containing agent or a combination of more than one evaporation reducing agent or an amount greater than 30 percent by volume when the evaporation reducing agent is a glycol-containing agent.
Across the independent claims, the coverage centers on contacting tissue or cellular samples with a hybridization solution or a primary antibody diluent that includes a detergent and an evaporation reducing agent, and employing an evaporation reducing agent defined by the X1—R—X2 general formula with specified volume thresholds that depend on whether the agent is glycol-containing or non-glycol-containing, and on whether one or multiple agents are combined.
Stated Advantages
Reduced evaporation (reduced volume loss) of aqueous processing reagents during elevated-temperature tissue or cellular assays.
Maintains similar assay performance while reducing evaporation-related volume loss.
Documented Applications
Oligonucleotide hybridization at elevated temperature, including denaturation and wash steps in situ hybridization processing.
Immunohistochemistry or immunocytochemistry processing, including antibody incubation and antigen retrieval.
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