As used herein, the term “antibodies” includes intact immunoglobulins as well as когда появилась вспышка коронавируса в китае a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments когда появилась вспышка коронавируса в китае are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment когда появилась вспышка коронавируса в китае of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab?, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab? fragments are obtained per antibody molecule; (3) (Fab?) 2 , the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab?) 2 , a dimer of two Fab? fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment когда появилась вспышка коронавируса в китае containing the variable region of the light chain and the variable region of когда появилась вспышка коронавируса в китае the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region когда появилась вспышка коронавируса в китае of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual , CSHL, New York, 1999). Antibodies for use in the methods and devices of this disclosure can be monoclonal or polyclonal.
Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein ( Nature 256:495–97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual , CSHL, New York, 1999. Antigen: A когда появилась вспышка коронавируса в китае compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. In one embodiment, an antigen is a coronavirus antigen. Binding or Stable Binding: An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the target:oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including functional or physical binding assays. Binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like. Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, Southern blotting, dot blotting, and light absorption detection procedures. For example, a method which is widely used, because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target dissociate or melt. The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T m ) at which 50% of the oligomer is melted from its target. A higher T m means когда появилась вспышка коронавируса в китае a stronger or more stable complex relative to a complex with a lower T m . cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) когда появилась вспышка коронавируса в китае and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Electrophoresis: Electrophoresis refers to the migration of charged solutes or particles in a liquid medium under the influence of когда появилась вспышка коронавируса в китае an electric field. Electrophoretic separations are widely used for analysis of macromolecules. Of particular importance is the identification of proteins and nucleic acid sequences. Such separations can be based on differences in size and/or charge. Nucleotide sequences have a uniform charge когда появилась вспышка коронавируса в китае and are therefore separated based on differences in size. Electrophoresis can be performed когда появилась вспышка коронавируса в китае in an unsupported liquid medium (for example, capillary electrophoresis), but more commonly the liquid когда появилась вспышка коронавируса в китае medium travels through a solid supporting medium. The most widely used supporting media are gels, for example, polyacrylamide and agarose gels.
Sieving gels (for example, agarose) impede the flow of molecules. The pore size of the gel determines the size of a molecule that can flow freely through the gel. The amount of time to travel through the gel increases as the size of the molecule increases.
As когда появилась вспышка коронавируса в китае a result, small molecules travel through the gel more quickly than large molecules and thus progress further from the sample application area than larger molecules, in когда появилась вспышка коронавируса в китае a given time period.
Such gels are used for size-based separations of nucleotide sequences.
Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log 10 of their molecular weight. By using gels with different concentrations of agarose, different sizes of DNA fragments can be resolved. Higher concentrations of agarose facilitate separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which когда появилась вспышка коронавируса в китае includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic когда появилась вспышка коронавируса в китае acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as “base pairing.” More specifically, A will hydrogen bond to T or U, and G will bond to C. “Complementary” refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence. “Specifically hybridizable” and “specifically complementary” когда появилась вспышка коронавируса в китае are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its когда появилась вспышка коронавируса в китае target sequence to be specifically hybridizable. An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there когда появилась вспышка коронавируса в китае is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding когда появилась вспышка коронавируса в китае is referred to as specific hybridization. Hybridization conditions resulting in particular degrees of когда появилась вспышка коронавируса в китае stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences.
Generally, the temperature of hybridization and the ionic strength (especially the Na + and/or Mg ++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2 nd ed., vol. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Ausubel et al. Short Protocols in Molecular Biology, 4 th ed., John Wiley & Sons, Inc., 1999. For purposes of the present disclosure, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence.
