Blotting Technique for Membrane Assay

The invention relates to laboratory techniques for detecting the positions of or presence/absence of substances which are putatively contained in membrane interstices. More particularly, it concerns a blotting technique for applying reagents and wash solutions to accomplish this detection.

There are a number of instances in which it is desirable to detect the presence and/or location of substances embedded in a membrane.

For instance, such membranes are used to obtain replicas of developed gel electrophoresis supports. The use of gel electrophoresis is currently a ubiquitous technique for the separation of biological materials. (Nonbiological materials can be separated using gels or other chromatographic supports as well, but the scope of effort with regard to biologicals is very great.) Typical applications include separation of nucleic acid fragments of various sizes either in the context of sequence determination; in the detection of polymorphisms; or verification of sizes in other contexts. Also frequently conducted are separations of proteins and glycoproteins and application of gel separations as verification of homogeneity or purity. In all of these procedures samples are applied to gels and the components are separated by application of an electric field across the gel. Regardless of the manner in which the gel is developed, the resulting pattern of migration of the substances contained in the sample must be visualized in some manner.

To conduct this visualization, typically the gel support is contacted with a membrane to which the substances will be transferred in the same pattern in which they appeared on the gel. The "spots" are then visualized by, at a minimum when the substances are labeled, washing the membrane to remove background contaminants. In most cases detection is by adding to the membrane detecting reagents which react with the substances to be detected and then washing the excess away.

The present procedures, for applying and removing these reagents and for washing away contaminants involve substantial volumes of fluid and are difficult to conduct. The typical procedure for applying reagent or wash is to immerse the membrane in a liquid and agitate the liquid bath. The membrane is removed from the bath and excess liquid drained off. Alternatively, the wash solutions are withdrawn using a vacuum. This procedure is disclosed, for example, in U.S. Pat. No. 4,427,415. This procedure takes a good deal of time and requires large volumes of solution. Furthermore, the vacuum application tends not to result in an evenly applied pressure gradient so that large surface areas do not behave well under this protocol. The result is that the design of systems for vacuum application has been limited to small areas such as those associated with spot testing on, for example, a microtiter plate, e.g., the Dot-Blot system marketed by Bio-Rad. This type of spot test is another instance in which visualization of substances embedded in membranes is required, as explained below.

A recent effort to overcome the problems of large volumes and uneven vacuum pressure is exemplified in the Hybrid-Ease.TM. hybridization chamber marketed by Hoefer Scientific Instruments. This chamber comprises two grids between which the membrane is sandwiched. The grid plates are snapped into position surrounding the membrane, and syringes fitted into the open space created by the grids. One syringe is used to apply reagents and wash, and the other to withdraw excess. This system still requires large volumes of liquid and is cumbersome to employ.

Bibulous materials have been used in other contexts to draw fluids through membranes where only the presence, absence or amount of a material at a known location is to be determined. For example, immunoassay procedures, such as those described in Syva U.S. Pat. No. 4,366,241 and Hybritech U.S. Pat. No. 4,632,901, use the capillary action of an absorbent material to draw fluids through a reaction-supporting membrane.

It is clear that a more efficient method for detection of the migration patterns resulting from electrophoresis on supporting gels or for general assay procedures is required. The present invention permits a more efficient elucidation of the migration pattern as replicated on a transfer membrane, and more effective detection of results of specific binding reactions on solid supports.

A rapid, efficient and convenient method to detect the pattern, nature or amount of substances on a membrane is provided. The invention method involves a simple positive-pressure squeeze-blotting procedure which effectively supplies and removes reagents and permits washing of the contaminants from substances embedded in the membrane to be detected using very low volumes of liquid.

Thus, in one aspect, the invention is directed to a method to passage liquid, such as a reagent solution or wash, through a membrane in which substances to be detected are embedded. The membrane may, for example, correspond to the migration pattern of a sample subjected to separation on a solid support. The membrane may also be a specific-binding assay solid support. The method comprises positioning a donor bibulous matrix containing the liquid to be supplied onto one surface of the membrane, and an acceptor bibulous matrix on the opposite surface of the membrane. The resulting "sandwich" is then squeezed by application of even positive pressure over the surface on both sides, and the liquid is forced from the donor matrix through the membrane onto the acceptor matrix. This method is particularly useful for membranes that are obtained by blotting of a gel support which has been used for electrophoretic separation of materials contained in a sample or verification of purity.

In another aspect, the invention is directed to an apparatus useful in conducting the method of the invention.

FIGS. 1A-1C show a perspective view of the apparatus of the invention absent and present the application of pressure, respectively.

FIG. 2 shows results of successive washings using Dot-Blots of IgG and KLH.

FIGS. 3A-3B shows the results of three washes using the technique of the invention on Western Blots of IgG.

The invention relates generally to the technique of passing liquids through membranes by means of a sandwich arrangement wherein the membrane has positioned on one of its surfaces a donor bibulous matrix which contains the liquid, and on the other, opposing, surface an acceptor bibulous matrix which contains substantially no fluid. The resulting sandwich, when compressed using positive pressure results in the transfer of the liquid from the donor matrix through the membrane to the acceptor matrix. The sandwich can be held in place for any desired period, e.g., 30 minutes to 24 hours, resulting in a corresponding period of incubation of the membrane contents with the liquid and the solutes it contains. Simple washing can be conducted in this manner as well; in this case no substantial holding period is required.

In a less preferred embodiment, the donor matrix may first be positively pressed onto the membrane without the acceptor matrix. This step may be followed, optionally, by pressing the acceptor matrix against the other side of the membrane, or by removal of the donor matrix followed optionally by compression against the acceptor matrix placed against either side of the membrane. This embodiment is less preferred because the membrane itself often has no holding capacity, and thus washing, for example, by this protocol is not very effective.

The membrane contains, in its interstices, one or more substances to be detected. Generally these substances are present in the interstices either by virtue of having been blotted from a solid support for electrophoresis or chromatography or by direct application, usually to detect the presence, absence, or amount of a particular type of material such as an antibody or specific protein--i.e., a Dot-Blot type assay. The definition of the membrane is not limited, however, to these instances, but applies to any case wherein a membrane contains in its interstices one or more substances to be detected. Included in the types of membranes envisioned are paper chromatographic strips, membranes commonly used to blot electrophoresis gels such as nitrocellulose; nylon; membranes of proprietary composition such as that marketed under the trademark Immunodyne.TM.; or various other polymeric membranes, such as polyvinyl difluoride, marketed under the trademark Immobilon.TM.. A variety of materials can be used to replicate the results of electrophoresis gels performed on various samples as is understood in the art. Most commonly, the samples contain biological substances such as individual proteins, antibodies, nucleic acids, oligonucleotides, complex carbohydrates, and the like, but the application of the technique is not limited to membranes containing these substances. The invention technique is applicable to any membrane containing within its interstices a substance to be detected regardless of the chemical composition of the membrane or of the target substances.

When membranes which represent replicas of electrophoretic results are employed, the transfer of the substances to be detected from the gel to the membrane can be conducted by utilizing membranes containing transfer buffer, by electroelution, or by dry blotting of the gels. Techniques for these transfers are well understood in the art, and do not constitute part of the invention herein.

Another major type of membrane utilized in current work is the Dot-Blot type membrane which permits the impregnation of multiple samples on the same experimental surface. Typical configurations of dot blots include those which approximate microtiter plates.

Other suitable membranes include those which are directly used themselves as chromatographic supports.

The donor and acceptor bibulous matrices can be made of a variety of materials as well. Because the transfer of liquid from donor matrix to membrane and from membrane to acceptor matrix is conducted under positive pressure, restriction on the relative pore sizes of the membrane and matrices is not of great importance. The donor matrix must be bibulous in the sense that it is able to retain, absent the application of pressure, or absent contact with a matrix of smaller pore size, sufficient liquid to conduct the procedure described for detection of substances in the membrane. Similarly, the acceptor matrix must be sufficiently bibulous to absorb the liquid from the membrane under applied pressure. Suitable matrices of the invention include cellulosic filter paper, as well as other woven or spongelike materials. (Typically, bibulous materials are capable of retaining fluid volumes of at least 0.1 ml/cm.sup.2, but the "bibulousness" required varies with the application.) Generally the donor and acceptor matrices are sufficiently planar to be considered two dimensional, but thicker "block" forms of these materials can also be used.

The liquid to be supplied may contain detecting reagent or may simply be provided as a wash. The nature of the detecting reagent depends, of course, on the substance to be detected. Typically, proteins are detected by immunological reactions between antigen and antibody or immunoreactive portions thereof; typically the presence of nucleic acid fragments is detected by suitable oligonucleotide probes. The detecting substances responsible for the immediate or specific reaction with the substance to be detected may be further supplemented, if needed, with label and a multiplicity of applications of the detecting reagents may be needed--e.g., a protocol may include detection of an antigen by supplying an antibody labeled with an enzyme, e.g., commonly, horseradish peroxidase, and then this binding is detected by means of supplying substrate for this enzyme. In application of reagent, it is possible, though not preferred, to use only a positively pressed donor matrix to expose this component of the membrane for a defined period.

The membrane is typically washed after the application of each detecting reagent; the wash solutions are applied using the method of the invention, wherein both donor and acceptor membranes are compressed in a "sandwich," as it is desirable for the wash to passage through the membrane. The wash technique of the invention requires only surprisingly small volumes of wash liquid to be effective.

It is most convenient to conduct the method of the invention at room temperature, but elevated and lower temperatures can also be used. These can be effected by thermostatting the apparatus, or by preheating or cooling the means for applying pressure.

The apparatus in which the method of the invention is conducted must contain a means for positioning each of the matrices at the surface of the membrane and a means for applying even pressure to the membrane in contact with the matrices. One preferred embodiment is shown in FIGS. 1A-1C. As shown in FIG. 1A, a cradle 10 contains a delineated surface for positioning of the matrix and membrane. The positioning means 11, which in this case is simply a line indication of where to place the matrix/membrane sandwich, is covered first with the acceptor matrix 12, then with the membrane 13, and then with the donor matrix 14. The pressure plate 15 is available to mediate even the application of positive pressure. Pressure providing means, 16 and 17, are in the open position. These pressure application means are standard pressure clamps which are commercially available.

FIG. 1B shows pressure plate 15 in position, prior to application of pressure.

FIG. 1C shows this apparatus when pressure is being applied. The pressure mediating plate 15 is placed on top of the matrix/membrane/matrix sandwich and the pressure providing devices 16 and 17 are readjusted to supply pressure. Within only a few seconds, the transfer of liquid contained in the donor matrix to acceptor matrix is complete.

The temperature of the blotting can be regulated by applying or subtracting heat from the cradle 10 and/or the pressure mediating plate 15.

It should be noted that the foregoing is simply an illustrative design. The positioning means is arbitrary; it can be a raised frame, a delineated surface, or, in some cases, the judgment of the operator. The pressure application means may include, instead of pressure plate 15, rollers which traverse the surface, either individually in counterpart to a supporting surface on the other side of the matrix/membrane composite, or in a laundry wringer type arrangement. Any means of applying even, positive pressure may be used.

The following examples are intended to illustrate but not to limit the invention.

Dot-Blots were obtained by spotting labeled IgG or labeled KLH at several dilutions from 4 ug/dot to 0.062 ug/dot using Immobilon.TM. membranes. FIG. 2, rows (1)-(4), show the results of successive washing conducted with the apparatus of FIG. 1 using filter paper matrices saturated with wash buffer.

A. SDS-polyacrylamide gel electrophoresis of a sample mouse IgG was conducted following standard procedures.

The resulting gel was blotted onto nitrocellulose in a transverse electric field using commercially available tanks. After blotting, the membrane was incubated in a 1% solution of casein to block excess protein-binding sites. This "Western" blot was then exposed to a small volume of HRP-labeled goat antimouse serum. After 3 washes using the apparatus of FIGS. 1A-1C, using filter paper matrices saturated with buffer, the membrane was exposed to HRP substrate. Specific detection of IgG was accomplished, as shown if FIG. 3, where lane A is the un-washed membrane and lane B is a corresponding washed membrane.

B. Using the procedure of paragraph A, SDS-polyacrylamide gel electrophoresis was performed using various antibodies and antigens, including anti-KLH and anti-fluorescein, alone or mixed, and yeast whole cell lysate. The resulting patterns were transferred to various membranes, i.e., Immunodyn.TM., Immobilon.TM., and nitrocellulose. Detection and washing were conducted as in paragraph A, with comparable results.