Rapid protein identification using antibody mixtures

Details Rapid protein identification using antibody mixtures Rapid protein identification using antibody mixtures

1999-11-19

2004-01-20

Chin, Christopher L. (Department: 1641)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

C436S528000, C436S529000, C436S530000, C436S532000, C436S535000, C436S541000, C436S824000, C435S007100, C435S004000, C435S007920, C435S287300, C435S288600, C422S051000, C422S063000, C422S068100, C422S070000, C204S450000, C204S451000, C204S452000, C204S455000, C204S456000, C204S546000, C356S344000

Reexamination Certificate

active

06680208

ABSTRACT:

FIELD
This invention relates to a method for identifying proteins in a protein mixture using electrophoresis.
BACKGROUND
Immunoassay systems have long been used to analyze proteins. Such methods include traditional Western blotting, in which a sample is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which resolves the individual proteins present in the sample by their molecular weights. The proteins (antigens) in the gel are subsequently transferred to a membrane, which is exposed to an antibody-containing solution. The antibody recognizes a specific protein, which allows the protein of interest to be identified. To detect this interaction, a secondary antibody containing a detectable marker is added. Typically, this procedure is used to detect only one protein on a membrane.
With conventional techniques, protein blotting is not well suited to handle large numbers of proteins. Traditionally, to analyze several different proteins simultaneously, individual lanes of the sample are run on a gel and the proteins are transferred to a membrane which is cut into a series of strips (each strip containing the samples). Each of the individual membrane strips are incubated with a different antibody-containing solution. This requires that each strip be handled separately, which is a very laborious and tedious task.
The detection of multiple proteins on a single gel using sequential probes has been described. In these methods, samples are resolved using SDS-PAGE, transferred to membranes, and exposed to primary and color-conjugated secondary antibodies. The resulting bands of differently colored products allow for the identification of several antigens on a single lane of a blot. In one variation of the method (Krajewski et al., 1996
, Anal. Biochem
. 236:221-8; Lin and Pagano, 1986
, J. Virol
. 59:522-4; and Theisen et al., 1986
, Anal. Biochem
. 152:211-4), the membrane is exposed to one primary antibody, which is detected with a colored-secondary antibody, producing a colored reaction. The blot is subsequently reprobed with different primary and colored-secondary antibodies, producing a colored reaction different from the first.
In another variation of this method, Lee et al. (
J. Immunol. Meth
. 106:27-30, 1988) describes the simultaneous probing of different proteins. This method requires the use of primary antibodies generated in different species, for example one antibody made in rabbits (i.e. polyclonal), the other in mice (i.e. monoclonal), which are added to the membrane at the same time. The differently color-conjugated secondary antibodies (anti-rabbit or anti-mouse), will bind to only one of the primary antibodies. This allows for the detection of at least two different proteins simultaneously.
The use of two-dimensional (2D) gel electrophoresis to detect multiple proteins on a single blot has been reported. Sanchez et al. (
Electrophoresis
. 18:638-41, 1997) discloses the use of a monoclonal antibody mixture to detect nine individual proteins which are sufficiently different in both pI and molecular weight to avoid ambiguities in their identification. Faleiro et al. (
EMBO J
. 16:2271-81, 1997) teaches the use of 2D gel electrophoresis to detect multiple caspases.
Three U.S. Patents to Levin (U.S. Pat. Nos. 4,713,349, 4,834,946, and 4,978,507) describe an apparatus that can be used to detect multiple antigens on a single protein-containing membrane. The apparatus includes an upper and lower plate, between which a protein-containing membrane is placed. The upper plate contains an array of channels, into which an antibody-containing liquid is introduced.
SUMMARY
The present invention provides an improved method for detecting and quantitating proteins present in a sample. The present invention allows several proteins to be identified and/or quantitated in the same sample, overcoming many of the limitations of current immunoassay systems. In certain particular examples, multiple proteins in the sample can be identified simultaneously.
The present invention provides a method for identifying different proteins in a protein mixture sample by electrophoretically separating proteins by molecular weight from at least one sample into at least a two-dimensional molecular weight gradient pattern in a direction of separation on a gel. The molecular weight gradient pattern is exposed simultaneously or sequentially to a plurality of adjacent, elongated specific binding agent applicators that extend in the direction of separation. Each applicator applies a plurality of different sets of specific binding agents in different lanes, wherein each set recognizes proteins of sufficiently different molecular weights to distinguish different proteins from one another along the direction of separation. Proteins in the mixture that have similar molecular weights are recognized in different lanes, such that otherwise partially or completely overlapping protein bands are detected in different lanes.
The proteins may be electrophoretically separated by introducing the protein mixture into an elongated sample chamber that extends transverse to the direction of separation, and performing electrophoresis to separate the proteins in the direction of separation by molecular weight. There can be at least three (for example at least ten) applicators, each of which applies at least 2, 5, 10, or 50 different specific binding agents. In one embodiment, the applicators are substantially parallel. The applicators may be slots or elongated channels having slots through which the specific binding agents are applied. Alternatively, each applicator may include multiple, closely spaced nozzles or holes in place of the elongated slot. In a further embodiment, the ratio of different specific binding agents applied to the number of elongated applicators is at least 1 or 3 different specific binding agents to each applicator (for example at least 5 or more different specific binding agents).
In a particular embodiment, the specific binding agent is an antibody, such as a monoclonal antibody. The specific binding agents may recognize signal transduction proteins. In another embodiment, the protein mixture sample includes a cell lysate.
In disclosed embodiments, the pattern is transferred from the gel to a transfer member, and the different sets of specific binding agents are applied to the transfer member. The method may also include detecting a location of binding of specific binding agents, and correlating each location with a particular specific binding agent that identifies a protein of interest. In a further embodiment, the method includes quantitating the protein of interest, for example by measuring intensity or luminescence, after detecting the protein of interest.
The cell lysate sample can be introduced into an elongated sample trough in an electrophoresis gel. Electrophoresis is performed on the sample to separate the cell lysate from the elongated sample chamber into a molecular weight gradient pattern in a direction of separation that extends transverse to the sample chamber. The pattern is transferred to a transfer member, and subsequently exposed simultaneously to a plurality of adjacent, elongated antibody applicator channels having slots that extend in the direction of separation, substantially transverse to the sample chamber. The plurality of different applicators apply different sets of specific binding agents to the transfer member in side-by-side lanes. The specific binding agents within each slot recognize proteins of distinguishable molecular weights, and the specific binding agents in different slots are sufficiently different to recognize a variety of different proteins of interest. Proteins that are of similar molecular weights, and would otherwise partially or completely overlap in a single lane, are detected in separate lanes. Each location where a protein is detected may then be correlated with a particular specific binding agent that identifies a protein of interest, by comparing a detected location of binding with an expected location of binding for each applicator. In a partic

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