JP2002542487A - Polypeptide microarray - Google Patents

Abstract

  (57) [Summary] A microarray of polypeptides on a solid support is provided. The composition of the microarray is used for duplicate detection and quantification of ligands, eg, antigens or antibodies, in a miniaturized format. The substrate is used to detect binding of the ligand to the plurality of polypeptides for screening and diagnostic purposes.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to methods and devices for making microarrays of biological samples, and uses thereof.

BACKGROUND OF THE INVENTION [0002] The life and development of all organisms are determined by intermolecular interactions, such as, for example, DNA and proteins, proteins and proteins, or proteins and small molecules. Among these, protein-protein interactions play a particularly important role, for example, the interaction between antibodies and antigens, receptors and peptide or protein hormones, enzymes and substrates or inhibitors. Many of the best-selling drugs either target proteins or are themselves proteins. Many of the disease molecular markers that underlie the diagnosis are also proteins.

[0003] The development of high throughput protein analysis techniques and reagents has been of great interest. In particular, interest in protein expression analysis has increased due to an increase in knowledge of the DNA sequence of the target organism. There is a growing awareness of the importance of proteomics in understanding and organizing the human genome. Information on the total amount of proteins present in cells is key to accelerating the discovery of medically important proteins and the genes from which they are derived.

[0004] Genetics establishes a relationship between gene activity and certain diseases. However, most of the course of the disease occurs at the protein level, not at the genetic level. Often there is little correlation between the levels of various gene activities and the relative amounts of the corresponding proteins. Also, proteins and post-translational modifications of proteins are not directly encoded by the same gene, so the complete structure of an individual protein cannot be determined with reference to the gene alone.

[0005] Assays focused on protein binding can be used to quantify protein expression; determine specific interactions; determine the presence of protein ligands, and the like. Methods for quantifying proteins in a sample by determining binding to the antibody of interest are well known in the art.

For example, solid-phase radioimmunoassay of antigens or antibodies in serum samples
(RIA) is well known. Cat et al., Plastic tubes (U.S. Pat. No. 3,646,346) and plastic discs (J. Lab. & Clin. Med., 70: 820).
(1967)) reported such a technique on the surface. In such a technique, an excess amount of a specific antibody is first adsorbed on the surface of a support. The sample to be assayed is then immunologically reacted with the surface in a sandwich or competitive binding technique. In the competitive binding technique described in U.S. Pat. No. 3,555,143, a concentration-determining antigen and a known amount of a radiolabeled antigen are immunologically reacted with the antibody-adsorbed surface. Thereafter, the amount of labeled antigen bound to the antibody on the surface is quantified to indirectly determine the total amount of antigen in the original sample. In the sandwich technique, serum containing an unknown concentration of antigen is immunologically reacted with a surface containing antibodies. In the next step, the bound antigen is incubated with the labeled antibody and the amount of immunologically bound labeled antibody is measured.

[0007] The development of high-throughput parallel protein analysis systems is of great interest, especially when they can be used to analyze small amounts of material. Preferably, such a system can be used for complex molecules with high binding affinity for ligands, such as antibodies, protein receptors and the like.

References The documents of interest include the following:

SUMMARY OF THE INVENTION A method is provided for making a microarray of analyte assay regions on a solid support, wherein each region of the array comprises a known amount of a selected analyte-specific reagent. The method comprises: (i) formed by spaced, coextensive, elongated components; and (ii)
A) a selected analyte in a reagent dispenser having an elongated capillary channel adapted to hold an amount of the reagent solution, and (iii) having a tip region such that the aqueous solution in the channel forms a meniscus; First loading a solution of the specific reagent. Preferably, the channel is formed by a pair of spaced tapered components. Microarray compositions can be used for a variety of miniaturized detection and quantification of ligands, such as, for example, antigens or antibodies.

In another aspect, the invention includes a substrate having a microarray of at least 10 ³ different polynucleotide or polypeptide biopolymers in a surface area of less than about 1 cm ² . Each different biopolymer is located at a separate defined location in the array, has a length of at least 50 subunits, and is in a defined amount between about 0.1 femtomolar and 100 nanomolar.

The substrate can be used to detect binding of a ligand to a plurality of arrays of immobilized biopolymers. In one aspect, the substrate comprises a glass support, a coat of a polycationic polymer, such as polylysine, on such a support surface, and an array of different biopolymers non-covalently and electrostatically attached to the coat. And each different biopolymer is located at a separate defined location in the surface array.

DETAILED DESCRIPTION OF THE INVENTION Methods and compositions are provided for making microarrays of polypeptide regions on a solid support, wherein each region of the array comprises a known amount of a selected polypeptide. Robotic printers are used to deposit minute amounts of protein solutions on a derivatized two-dimensional surface substrate that binds to a polypeptide such as polylysine.
Substrates with a microarray on the surface are spotted at a high density of typically at least 10 ³ different polypeptides on a surface area of less than about 1 cm ² . Each different polypeptide is present in a defined amount between about 0.1 femtomolar and 100 nanomolar. Generally, the polypeptide will be at least 50 amino acids long, but any polypeptide can be used.

[0013] Microarrays are widely used in quantification and analysis methods for the detection and quantification of proteins or compounds that interact with proteins, such as polynucleotides, hormones, vitamins, and other cofactors. . Normally,
A sample containing a ligand believed to bind to the polypeptide immobilized on the microarray is added to the microarray under conditions that permit specific binding between the polypeptide and the ligand. Unbound sample is washed off the microarray and the bound ligand is detected by, for example, a detectable label present on the ligand, or a suitable method provided in the second detection step. Because the present invention is highly parallel and miniaturized, it consumes much less sample than conventional immunoassays. In order to quantify many components in parallel, the physiological and phenotypic characteristics of a sample can be diagnosed and recognized based on a multidimensional pattern of expression, rather than just some parameters.

[0014] In one embodiment of the invention, comparative fluorescence is used to monitor the presence of ligand bound to the microarray. The use of comparative fluorescence measurements allows more precise measurements over a wide range of ligand concentrations and binding affinities as compared to methods that measure the absolute amount of bound ligand.

In one embodiment of the invention, the biopolymer is a polypeptide whose functional binding properties are obtained by the three-dimensional structure of the polypeptide, such as antigens, antibodies, receptors, etc., wherein the structure comprises cysteine It often relies on contacts between non-adjacent amino acid residues, such as disulfide bonds between residues, hydrophobic pockets, and the like. Such binding properties include various protein and polypeptide molecules that provide specific interactions in biological systems, including protein receptors and one or more naturally occurring ligands, e.g., cytokines. Specific binding between cytokine receptors, hormones and hormone receptors, chemokines and chemokine receptors and the like are included.
DNA binding proteins such as nuclear hormone receptors, transcription factors, etc.
The ability to specifically define NA motifs can be provided on microarrays carried by proteins. Of particular interest are microarrays that retain the binding properties of antigen-specific immunoreceptors, including receptors, T cell antigen receptors, and major histocompatibility complex proteins.

[0016] These and other objects and features of the invention will become more apparent from the following detailed description of the invention when read in conjunction with the accompanying drawings.

Definitions Unless defined otherwise, the terms defined below have the following meanings.

“Ligand” means one member of a ligand / antiligand binding pair. For example, the ligand may be one of the nucleic acid strands of a complementary hybridized double-stranded nucleic acid binding pair; an effector molecule in an effector / receptor binding pair; or an antigen in an antigen / antibody or antigen / antibody fragment binding pair. There may be.

“Antiligand” means the opposite element of a ligand / antiligand binding pair.
The anti-ligand is the remaining one of the nucleic acid strands of a complementary hybridized double-stranded nucleic acid binding pair, respectively; a receptor molecule in an effector / receptor binding pair; or an antigen / antibody or antigen / antibody fragment. It may be an antibody or an antibody fragment in the binding pair.

“Analyte” or “analyte molecule” refers to a molecule, usually a macromolecule, such as a polynucleotide or polypeptide, whose presence, quantity, and / or properties are to be determined. The analyte is one member of the ligand / antiligand pair.

“Analyte-specific assay reagent” means a molecule that is capable of binding specifically to an analyte molecule. The reagent is the opposite element of the ligand / antiligand binding pair.

An “array of regions on a solid support” is a linear or two-dimensional array of preferably discontinuous regions, each having a defined region formed on the surface of the solid support. is there.

A “microarray” is an array of regions having a density of discontinuous regions of at least about 100 / cm ² , and preferably at least about 1000 / cm ² . The regions in the microarray typically have dimensions, for example, ranging between about 10-250 μm in diameter, and are separated from other regions on the array by about the same distance.

A support surface is “hydrophobic” if the droplets of aqueous medium added to the surface do not extend substantially beyond the size of the added droplets. That is, the surface prevents the droplets added to the surface from spreading due to hydrophobic interactions with the droplets.

By “meniscus” is meant a concave or convex surface formed at the bottom of a liquid in a channel as a result of the surface tension of the liquid.

“Different biopolymers” as used for biopolymers forming a microarray refers to different biopolymer sequences and / or different concentrations of the same or different biopolymers and / or at different or different concentrations. By different mixtures of biopolymers is meant an array element that is different from other array elements. Thus, "different polynucleotides"
The array of elements comprises (i) different polynucleotides, each of which may be present in a certain defined amount, for each element, (ii) different graded concentrations of a polynucleotide of a given sequence, and / or (iii) An array comprises a mixture of two or more different polynucleotides of different composition.

By “cell type” is meant a cell from a particular source, such as, for example, a tissue or organ or a cell of a particular differentiation state, or a cell with a particular pathology or genetic makeup.

Methods for Making Microarrays This section describes how to make microarrays of analyte assay areas on a solid support or substrate, where each area on the array contains a known amount of a selected analyte-specific reagent. I do.

FIG. 1 shows a partial schematic diagram of a reagent dispensing device 10 useful for performing the present method. Generally, the device comprises a reagent dispenser 12 having an elongated open capillary channel 14 adapted to hold an amount of a reagent solution as indicated at 16 as described below.
including. The capillary channels are formed by a pair of spaced, coextensive elongated elements 12a, 12b that taper toward each other and converge at the distal end or distal region 18 of the channel. More generally, the open channel is adapted to hold an amount of reagent solution and is tipped into the aqueous solution in the channel forming a meniscus, such as a concave meniscus as depicted at 20 in FIG.2A. It is formed by at least two spaced elongated elements having an area. The advantages of the dispenser having an open channel configuration are discussed below.

Still referring to FIG. 1, the dispenser device may be used to dispose a known amount of solution in the dispenser on a support, as described below with respect to FIGS. Includes structures for quickly moving the dispenser away. In the embodiment shown, the structure includes a solenoid 22 that can quickly pull the solenoid piston 24 downward and activate, for example, with the bias of a spring, to release the piston to a normally raised position as shown. included. The dispenser is carried on the piston by the connecting element 26 as shown. The moving structure described above is also referred to herein as dispensing means for moving a dispenser and engaging a solid support to dispense a known amount of liquid onto the support.

The dispenser device described above is carried by an arm 28 that can move in a line or in the xy plane to place the dispenser at a selected location as described below.

FIGS. 2A-2C illustrate a method of disposing a known amount of a reagent solution in a dispenser as described above on a surface of a solid support, such as the support shown at 30. The support is a polymer, glass, or other solid material support having the surface shown at 31.

In one general aspect, the surface is a surface that is relatively hydrophilic, ie, wettable, such as a surface with charged, naturally or bound covalently bound groups. One such surface described below is a glass surface having an adsorbed layer of a polycationic polymer such as poly-L-lysine.

In another aspect, the surface has or is made to have relatively hydrophobic properties. That is, the aqueous medium disposed on the surface becomes a ball. Various known hydrophobic polymers, such as polystyrene, polypropylene, or polyethylene, have desirable hydrophobic properties, and various lubricants or other hydrophobic films that can be applied to the support surface, and glass also have such properties. Have.

First, the dispenser is loaded with the selected analyte-specific reagent solution, such as by washing the tip of the dispenser, immersing it in the reagent solution, and filling the dispenser channel by capillary flow. Next, the dispenser is moved to a position selected with respect to the support surface, and the tip of the dispenser is positioned directly above the support surface position where the reagent is to be placed. This movement is performed with the tip of the dispenser in the raised position, as shown in FIG. 2A, typically the tip is at least 1-5 mm above the surface of the substrate.

The position of the dispenser is thus determined, and the solenoid 22 is activated to cause the tip of the dispenser to move quickly toward and away from the substrate surface, making instant contact with the surface, resulting in a dispenser. Lightly tap the surface of the support. The movement of the tip lightly tapping the surface destroys the liquid meniscus at the channel tip and causes the tip liquid to contact the support surface. This causes the liquid to flow into the capillary space between the tip and the surface,
Liquid is withdrawn from the dispenser channel as shown in 2B.

FIG. 2C shows the flow of liquid from the tip to the support surface, which in this case is a hydrophobic surface. This figure shows that the liquid continues to flow from the dispenser to the
Is formed. At a particular ball size, or volume, the tendency of the liquid to flow to the surface is such that the hydrophobic surface interaction between the support surface and the ball, which acts to limit the total area of the ball on the surface, and the Balances the surface tension of the droplet towards the curvature.
At this point, if a particular ball volume is formed and the dispenser tip continues to contact the ball as the dispenser is withdrawn, there is little or no effect on the ball volume.

In the arrangement of the liquid on the more hydrophilic surface, the liquid has a weak tendency to form beads,
The volume to be placed is susceptible to the total time the dispenser tip is immersed in a proximate portion of the support surface, as shown, for example, in FIGS.

The desired placement volume, ie the volume of the ball, formed in this way is preferably 2 pl
Volumes range from (picoliters) to 2 nl (nanoliters), but volumes of 100 nl or more can be placed. The selected placement volume depends on (i) the area of the dispenser tip, i.e. the size of the area where the tip expands, (ii) the hydrophobicity of the support surface, and (iii) the contact time with the support surface and the rate at which the tip pulls down. It turns out that it depends. In addition, increasing the viscosity of the medium and consequently reducing the time for the liquid to flow from the dispenser to the support surface reduces the size of the ball. By arranging the droplets in a hydrophilic area surrounded by a hydrophobic grid pattern on the support surface, the size of the ball can be further limited.

In an exemplary embodiment, the tip of the dispenser quickly taps the support surface,
The dwell time in contact with the support is less than about 1 millisecond, and the speed of movement upward from the surface is about 10 cm / sec.

Assuming that the ball formed in contact with the surface is a hemisphere having a diameter approximately equal to the width of the dispenser tip, as shown in FIG. 2C, the width of the dispenser tip (d)
The volume of the ball formed with respect to is as shown in Table 1 below. As shown in the table, as the width increases from about 20 to 200 μm, the ball volume goes from 2 pl to 2 nl.

[0042]

[Table 1]

In one tip size, the volume of the ball is increased by increasing the hydrophobicity of the surface, decreasing the time the tip contacts the surface, increasing the speed at which the tip separates from the surface, and / or increasing the viscosity of the medium. It can be reduced while controlling. Once these parameters are fixed, the placement of the selected volume in the desired pl to nl range can be performed reproducibly.

After placing the ball at one selected position on the support, the tip usually moves to a corresponding position on the second support, where the droplet is placed and a plurality of supports are placed. This process is repeated until a droplet of reagent is placed at the selected location in each of the.

Thereafter, the tip is washed to remove the reagent solution and filled with another reagent solution, and the reagent will be placed at another array position on each support. In one embodiment, the tip comprises: (i) immersing the capillary channel of the device in a washing solution; (ii) removing the washing solution drawn into the capillary channel; and (iii) immersing the capillary channel in a fresh reagent solution. Washed and refilled by stages.

From the above, it can be seen that the tip of an open capillary dispenser like tweezers can be quickly and efficiently washed and dried before loading a new reagent into the tip because (i) the tip is an open channel, (ii) Passive capillarity can be used to load samples directly from a standard microwell plate, while sufficient samples for printing a large number of arrays can be retained in an open capillary container; (iii) Open It can be seen that capillaries have the advantage of being less clogged than closed capillaries, and that (iv) open capillaries do not require a perfectly finished bottom surface for the delivery of liquid.

The microarray formed on the surface 38 of the solid support 40 according to the method described above
A portion of 36 is shown in FIG. The array is comprised of a plurality of analyte-specific reagent regions, such as region 42, each of which may contain a different analyte-specific reagent. As mentioned above, the diameter of each region is preferably about 20-200 μm
Range. The spacing between each region and the nearest (not oblique) neighboring region is preferably in the range of about 20-400 μm (shown at 44) as measured from center to center. Thus, for example, for an array with a center-to-center spacing of about 250 μm, 40 areas / cm or 1,600 areas
/ cm ² is included. After formation of the array, the support is treated by evaporating the droplets of liquid that form each area, leaving a desired array of dry, relatively flat areas. This drying can be performed under heating or under reduced pressure.

In some cases, it may be desirable to reconstitute the water in the droplets containing the analyte reagents first with water to increase the adsorption time on the solid support. The spotting of the analyte reagents in a humid environment may be performed to ensure that the droplets do not dry until array preparation is complete.

Automated Instruments for Array Formation In another aspect, the invention includes automated instruments for producing an array of analyte assay areas on a solid support, wherein each area in the array has a known amount. Of selected analyte-specific reagents.

The device is shown in FIG. 4 as a two-dimensional partial schematic. The instrument dispenser device 72 has a basic structure as described above with respect to FIG. 1 and includes a dispenser 74 having an open capillary channel terminating at a tip substantially as shown in FIGS. 1 and 2A-2C. Including.

The dispenser is attached to a device for moving in a direction toward and away from the placement position, in which the tip of the dispenser taps the surface of the support, and a volume of the reagent solution selected as described above. Place. This movement is performed by the solenoid 76 as described above. Solenoid 76 is a control unit
It is under the control of 77 and its function is described below. A solenoid is also referred to herein as a dispensing means that moves the dispenser into engagement with the support when the device is at a defined array position relative to the support.

The dispenser device is carried by an arm 74 which is threaded onto a worm screw 80 which is driven (rotated) in the desired direction by a stepper motor 82 which is also under the control of the unit 77. The left end of the illustrated screw 80 is housed in a sleeve 84 for rotation with respect to the screw shaft. The opposite end of the screw is attached to the drive shaft of a stepper motor, which is housed in a sleeve 86. Dispenser device, worm screw, two sleeves mounting the worm screw, and x (
The stepper motor used to move the device in the (horizontal) direction forms what is collectively referred to herein as transposition assembly 86.

The transposition assembly is made for precise and minute movement along the direction of the screw, ie, the x-axis in the figure. In one mode, the assembly is 5 to
It functions to move in the x-axis direction at a selected distance in the range of 25 μm. In another mode, the dispenser unit can move precisely in the x-axis a distance of a few microns or more to place the dispenser at an associated location on an adjacent support, as described below. it can.

The transposition assembly has been mounted in the direction of the “y” (vertical) axis of the figure to position the dispenser at the selected y-axis position. The structure mounting this assembly has a fixed rod 88 fixedly mounted between a pair of frame bars 90, 92 and a rotatable mount between a pair of frame bars 96, 98. A worm screw 94 is included. This worm screw is driven by a stepper motor 100 operating under the control of the unit 77 (
Rotate. The motor is mounted on bar 96 as shown.

The above-described structure including the worm screw 94 and the motor 100 is made to make a minute movement in the direction of the screw, that is, the y-axis in the figure. As described above, in one mode, the structure functions to move in the y-axis direction at a selected distance in the range of 5-250 μm, and in a second mode, the structure moves to a relevant position on an adjacent support. It can be moved precisely in the y-axis direction at a distance of a few microns or more, like installing a dispenser.

As used herein, the transposition assembly and the structure that moves the assembly in the y-axis direction are collectively referred to as positioning means for positioning the dispenser device at a selected array position with respect to the support.

The holder 102 in the instrument can hold a plurality of supports, such as the support 104, on which the instrument is forming a microarray of reagent areas. The holder is provided with a number of indented slots, such as slot 106, which receive the support and are in a selected precise position with respect to the frame bar on which the dispenser means of movement is mounted. Install the support.

As described above, in a series of procedures for automatically operating an instrument for forming a selected microarray of reagent areas on each of a plurality of supports, the control unit of the apparatus comprises two It functions to operate a stepper motor and a dispenser solenoid.

The control unit is constructed to provide the appropriate signals to each solenoid and each stepper motor in a fixed timed sequence and at appropriate signal times, according to conventional microprocessor control principles. The making of the unit and the settings selected by the user to obtain the desired array pattern will be understood from the following description of the operation of a typical instrument.

First, one or more supports are placed in one or more slots of the holder. Then, a well containing the first reagent solution placed on the support (not shown)
Move dispenser to a position directly above. The dispenser solenoid is now activated to lower the tip of the dispenser into this well and fill the capillary channel of the dispenser. The motors 82, 100 are now activated to move the dispenser to the selected array position on the first support. Solenoid actuation of the dispenser places a selected volume droplet of the reagent at this location.
As mentioned above, this operation preferably places a selected volume of the reagent solution between 2 pl and 2 nl.

The dispenser is now moved to the corresponding position on the adjacent support where a similar amount of solution is placed. This process is repeated until the reagent is located at the corresponding preselected location on each support.

If it is desired to place a single reagent at more than one array location on a single support, the dispenser may be moved to a different array location for each support before moving the dispenser to a new support. After moving or placing the solution at individual locations on each support at one selected location, the cycle can be repeated for each new array location.

To place the next reagent, move the dispenser over the wash solution (not shown) and move the dispenser tip into and out of this solution until the reagent solution is substantially flushed from the tip. I do. After each immersion, decompress the solution, spray with compressed air,
The solution can be removed with a sponge or the like.

Next, the tip of the dispenser is dipped into the second reagent well and the filled tip is moved to the second selected array position on the first support. Thereafter, as described above, a process of placing a reagent at each of the corresponding second array locations is performed. This process is repeated until an entire microarray of reagent solutions on each support has been made.

Microarray Substrates This section describes an embodiment of a substrate having a microarray of biopolymers on the substrate surface, and in particular describes a microarray of different polypeptides bound on a glass slide coated with a polycationic polymer. I do.

The substrate is made according to another aspect of the present invention and is intended for use in detecting the binding of a labeled ligand to one or more of a plurality of different biopolymers. In one embodiment, the substrate comprises a glass substrate having a surface coated with a polycationic polymer, preferably a cationic polypeptide such as polylysine or polyarginine. On the polycationic coat, different biopolymers are made, each located at a known selected area.

By placing a uniform thickness film of a polycationic polymer, eg, poly-L-lysine, on the surface of the slide and drying the film to produce a dry coat,
You can coat slides. The amount of the polycationic polymer added is at least an amount sufficient to form a monolayer of the polymer on the glass surface. The polymer film attaches to the surface by electrostatic bonding between negative silyl-OH groups on the glass surface and charged amine groups on the polymer. Glass slides coated with poly-L-lysine are commercially available, for example, from Sigma Chemical Co. (St. Louis, Mo.).

Suitable microarray substrates can also be made by chemical derivatization of glass.
A silane compound having a suitable leaving group on the terminal Si is covalently bonded to the glass surface.
The derivatizing molecules can be designed to impart the desired chemistry to the surface of the glass substrate. An example of such a bivalent reagent is aminopropyl-tri (ethoxy) silane, which reacts with the glass surface at the tri (ethoxy) silane portion of the molecule, while the amino portion of the molecule remains free. Like a slide coated with polylysine,
Surfaces with terminal amino groups are suitable for biopolymer adsorption. The body of the terminal surface group can be modified by further chemical reactions. For example, a terminal aldehyde group is generated by the reaction between the terminal amine and glutaraldehyde in the above example. Prior to spotting on the microarray, silyna
ted) Further modifications can be made, such as applying Protein A or Protein G to the glass. Other surfaces that bind to polypeptides are nitrocellulose-coated glass slides available from Schleicherand Schuell, and protein-binding plastics such as polystyrene.

[0069] The spotted polypeptides may adhere by adsorption or covalent bonding. Adsorption occurs by electrostatic, hydrophobic, van der Waals forces, or hydrogen bonding interactions between the spotted polypeptide and the array substrate.
Simply applying the polypeptide solution to the surface in an aqueous environment is sufficient for polypeptide adsorption. Covalent bonding occurs when a functional group of the polypeptide reacts with a chemically activated surface. For example, if the surface has been activated by a highly reactive electrophilic group such as an aldehyde group or a succinimide group, the unmodified polypeptide will react with an amine group such as at a lysine residue or terminal amine. , A covalent bond is formed.

To make microarrays, defined amounts of different biopolymers are placed on polymer-coated slides, as described in Section II. When an aqueous sample is added to a substrate under conditions that allow the labeled ligand in the sample to bind to the relevant binding partner in the substrate array, the biopolymer disposed according to the important properties of the substrate is coated. Non-covalently bound to the slide surface.

In a preferred embodiment, each microarray comprises at least 10 ³ different polynucleotide or polypeptide biopolymers per surface area of about 1 cm ² or less. In one embodiment, the microarray comprises 400 regions in about 16 mm ² , or 2.5 × 10 ³ regions / cm ² . Also, in certain preferred embodiments, the biopolymer in each microarray region is present in a defined amount (for polynucleotides) between about 0.1 femtomolar and 100 nanomolar. As mentioned above, the ability to make such high-density arrays, where each region is made of a well-defined amount of disposed material, can be obtained according to the microarray formation method described in Section II.

Also, in certain preferred embodiments, the biopolymer has a length of at least about 50 units, eg, amino acids, nucleotides, etc., ie, substantially longer than the high density arrays formed by various in situ syntheses. It is a polymer.

The polypeptide biopolymer can include polypeptides from any source. Polypeptides of interest include polypeptides isolated from cells or other biological sources, synthetic polypeptides including synthetic peptides and peptides selected from combinatorial libraries, and polypeptides synthesized from recombinant nucleic acids, and the like. included. In one embodiment, the polypeptide is isolated from a phage display library or clone (Huse et al., (1989) Science. 1989).
246 (4935): 1275-81; Winter et al., (1994) Annu Rev Immunol. 12: 433-55; Clacks.
on et al. (1991), Nature 352 (6336); 624-8). Usually, the polypeptides in each distinct region of the array are substantially pure.

Use of Microarrays Whole cells, peptides, enzymes, antibodies, antigens, receptors, ligands, phospholipids, polymers, pharmaceutical preparations, or arrays of chemicals can be used in medical applications by the methods described in the present invention. Can be made for large-scale screening assays in diagnostics, drug discovery, molecular biology, immunology and toxicology.

The microarray of immobilized polypeptides prepared according to the present invention can be used in a number of diagnostic and screening large-scale binding assays. The ability to measure quantitative variations in the levels of multiple proteins in multiples allows recognition of patterns determined by several to many proteins. Many physiological parameters and disease-specific patterns can be evaluated simultaneously.

One embodiment of the present invention includes separating, identifying, and analyzing proteins present in a biological sample. For example, comparing disease and control samples allows for the identification of “disease-specific proteins”. These proteins are
It can be used as a target for drug development or as a molecular marker for disease.

[0077] Polypeptide arrays can be used to monitor the expression levels of proteins in a sample, including biopsies of the tissue of interest, cultured cells, microbial cells, and blood, It may include plasma, lymph, synovial fluid, cerebrospinal fluid, cell lysates, culture supernatants, body fluids including amniotic fluid, and the like, and their derivatives. Of particular interest are clinical samples of bodily fluids including blood and its derivatives, cerebrospinal fluid, urine, saliva, lymph, synovial fluid, and the like. Measurements can be quantitative, semi-quantitative, or qualitative. Where the assay is quantitative or semi-quantitative, it preferably includes a competitive format, for example, between labeled and unlabeled samples or between differently labeled samples.

Assays for detecting the presence of the ligand of the immobilized polypeptide can be performed as follows, but need not be the methods described herein.

The sample, fractions or portions thereof, are added to a microarray containing the bound polypeptide. The sample may include various body fluids or extracts as described above. Preferably, a series of standards containing known concentrations of the control ligand are also assayed in parallel with the sample or a portion thereof as a control. The incubation time is sufficient for the ligand molecule to bind to the polypeptide. Generally, about 0.1 to 3 hours is sufficient, usually 1 hour is sufficient.

After incubation, unbound components are washed away from the insoluble support.
Generally, a diluted nonionic surfactant, usually at an appropriate pH of 7 to 8, is used as the washing medium. Washing may be performed 1 to 6 times using an amount sufficient to completely wash away proteins that are not specifically bound present in the sample.

Various methods can be used to detect the presence of bound ligand. These fall into three general groups. The ligand itself can be labeled with a detectable label and the bound label can be measured directly. Alternatively, a labeled sample can be mixed with a different labeled or unlabeled sample in a competitive assay. In yet another embodiment, the sample itself is unlabeled and to quantify the amount of ligand present,
Add the second stage labeling reagent.

Examples of labels for which ligand binding can be measured directly include radiolabels such as ³ H or ¹²⁵ I, fluorescent agents, dyes, beads, chemiluminescent agents, colloid particles, and the like. Suitable fluorescent dyes are well known in the art and include fluorescein isothiocyanate (FITC)
Rhodamine and rhodamine derivatives; Texas red; phycoerythrin; allophycocyanin; 6-carboxyfluorescein (6-FAM); 2 ', 7'-dimethoxy-4
', 5'-dichloro-6-carboxyfluorescein (JOE); 6-6 carboxy-X-rhodamine (ROX); 6-carboxy-2', 4 ', 7', 4,7-hexachlorofluorescein (HEX); 5-carboxyfluorescein (5-FAM); N, N, N ', N'-tetramethyl-6-carboxyrhodamine (TAMRA); sulfonated rhodamine; Cy3; Preferably, the compound to be labeled is mixed with an activating dye that reacts with groups present on the ligand, such as amine groups, thiol groups, aldehyde groups, and the like.

In particular, when performing a second stage of detection, such as by the addition of a labeled antibody that recognizes a ligand, the label may be a covalently linked enzyme capable of providing a detectable product signal after the addition of a suitable substrate. It can be. Examples of suitable enzymes that can be used in the conjugate include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, and the like. If not commercially available, such antibody-enzyme conjugates can be readily made by techniques well known in the art. The second stage binding reagent may be any compound that binds the ligand with sufficient specificity and is distinguishable from other components present. In a preferred embodiment, the second stage binding reagent is a ligand-specific antibody, such as a monoclonal or polyclonal serum, such as a mouse anti-human antibody.

To increase the signal, the ligand can be labeled with an agent such as biotin, digoxigenin, etc., with the second stage reagent being avidin, streptavidin, anti-digoxigenin antibody, etc. compatible with that label .

To detect binding of a ligand, for example, a scanning laser microscope, a fluorimeter,
The microarray can be scanned using an improved ELISA plate reader or the like. For example, with a scanning laser microscope, separate scans can be made using appropriate excitation radiation for each phosphor used. The digital images obtained from the scan are combined for subsequent analysis. For any array element, the ratio of the fluorescent signal of one label can be compared to the fluorescent signal of the other labeled DNA to determine the relative amount.

[0086] Microarray and ligand detection methods can be used for several screening, research, and diagnostic assays. In one application, for research or diagnosis,
The total protein of the organism is bound to an array of antibodies and protein expression is monitored. When the total protein of normal cells is labeled with one color of fluorophore, the total protein of diseased cells is labeled with another color of fluorophore, and two samples are simultaneously bound to the same array, different protein expression can result in two It can be measured as a ratio of the intensity of the phosphor. This two-color experiment can be used to monitor expression in response to different types of tissues, disease states, drugs, or environmental factors.

For example, screening assays to determine whether one or more proteins are involved in a disease pathway or correlate with a disease-specific phenotype can be measured from cultured cells. Such cells can be manipulated experimentally by adding pharmaceutically active agents that act on the target or pathway of interest. This use is important for elucidating biological functions or finding therapeutic targets.

For many diagnostics or studies, it is useful to determine the level of a ligand, such as a protein ligand, in blood or serum. This use is important for the discovery and diagnosis of clinically useful markers that correlate with a particular diagnosis or prognosis. For example, by monitoring the specificity of a series of antibodies or T cell receptors in parallel, the levels and kinetics of the antibodies in the course of autoimmune disease, infection, and graft rejection can be determined. Alternatively, comparison of normal and diseased blood samples, or comparison of clinical samples at different stages of the disease, may lead to the development of new protein markers associated with the disease of interest.

In another aspect of the invention, polypeptide arrays can be used to detect post-translational modifications of proteins, which are important for studying signaling pathways and cell regulation. Post-translational modifications can be detected using antibodies specific for a particular state of the protein, such as phosphorylation, glycosylation, famesylated, and the like.

The detection of these interactions between the ligand and the polypeptide leads to a medical diagnosis. For example, by linking an unknown pathogen sample to an array containing various antibodies specific for known pathogen antigens, pathogenic microorganisms can be unambiguously identified.

EXPERIMENTAL The present invention relates to the specific methods, protocols, cell lines, animal species or genera described,
And it is not limited to a reagent, but is various. Also, the terms used in the present specification are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, and the scope of the present invention is defined by the appended claims. It is to be understood that only limited.

As used in this specification and the appended claims, the singular forms “a”, “a”,
"And" and "the" also include plural referents unless the context clearly indicates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The following examples are intended to fully disclose and explain to those skilled in the art how to make and use the invention, and are not intended to limit the scope of what is considered to be the invention. Absent. Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperatures, concentrations, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, amounts are parts by weight, molecular weight is average molecular weight; temperature is in degrees Celsius; and pressure is at or near atmospheric.

Example 1 Using a different ligand / antiligand pair on the antibody and antigen microarray 115, a set of antibody / antigen pairs was collected which allows highly controlled experiments to be performed.

Method Array Preparation: Antibody solutions were prepared at 100-200 μg / mL in PBS / 0.02% sodium azide buffer without glycerol. Antibodies were spotted on glass slides treated with poly-L-lysine. Slides were derived by the following procedure. Place the slide on the slide rack and place the rack in the chamber. Dissolve 70 g NaOH in 280 mL ddH2O, and add 420 mL 95% ethanol to prepare a washing solution. The total volume will be 700 mL (= 2 X 350 mL). Stir until thoroughly mixed. Pour the solution into the chamber containing the slides; cover the chamber with a glass lid. Mix for 2 hours on a rotary shaker. Quickly transfer the rack to a new chamber filled with ddH2O. Raise and lower the rack and rinse well. Each time, repeat the rinse four times with fresh ddH2O. Prepare polylysine solution: 70 mL poly L lysine + 70 mL tissue culture PBS in 560 mL water. Transfer slides to polylysine solution and shake for 15 minutes to 1 hour. Transfer the rack to a new chamber filled with ddH2O. Shake up and down 5 times to rinse. Spin the slides on a microtiter plate carrier for 5 minutes at 500 rpm. Dry slide rack in vacuum oven at 45 ° C for 10 minutes.

Antibodies and antigens were prepared in 384-well microtiter plates, each containing at least 3 wells of 110 different antibodies or antigens. A 16-chip printhead on the arrayer spots the plate three times, 9-12 for each antibody or antigen
A total of 1152 spots were created as overlapping spots. The spacing between spots was 375 micrometers. The array was sealed in a closed container. Short-term storage (
11 month) if stored at 4 ℃, if stored longer than that can be stored frozen.

Inscribe the position of the spot on the back of the slide with diamonds (di
amond scribe) or marked with an indelible marker. To remove unbound proteins, arrays were soaked several times in PBS / 3% skim milk / 0.1% Tween-20, immediately transferred to a PBS / 3% skim milk solution and blocked overnight at 4 ° C. The skim milk solution was first centrifuged (10,000 xg for 10 minutes) to remove particulates.

After blocking, the slides were soaked in 0.2 × PBS three times for 1 minute at room temperature and shaken to remove unbound milk protein. The array was soaked in the last wash until the protein mixture was added.

Sample preparation: The protein solution was prepared at a concentration such that the final protein concentration after mixing with the dye solution (see below) was 0.2-2 mg / mL, with a maximum of ~ 15 μg of protein for each array (each (When using 25 μL of array) and prepared in 0.1 carbonate or phosphate buffer at pH 8.0.

Cy dye activated with NHS-ester (Amersham, catalog number PA23001 (Cy3)
And PA25001 (Cy5)) were dissolved in 0.1 M pH 8.0 carbonate buffer so that the final concentration of the dye after mixing with the protein solution was 100-300 μM. (Each vial of dye contains 200 nmol). The dye solution and the protein solution were mixed and reacted at room temperature in the dark for 45 minutes. The standard protein solution was mixed with the Cy3 dye solution, and the test protein solution was mixed with the Cy5 dye solution. The reaction was quenched by the addition of sufficient 1 M each of pH 8 Tris or glycine to obtain at least a 200-fold quencher: dye concentration.

Each reaction was applied to a microconcentrator with the appropriate molecular weight cutoff. At a cutoff of 3000 D, most of the protein was obtained while removing the dye. If low molecular weight proteins are not important, a cutoff value of 10,000 D is faster. The reaction solution was centrifuged according to the method of using the microconcentrator. Centrifugation at 10,000 xg at room temperature usually takes 20 minutes at 10,000 D and 80 minutes at 3000 D. After centrifugation, Cy5 or Cy3
3% milk blocker was added to any of the labeled protein solutions. (To remove microparticles, the milk must first be centrifuged: 10,000 xg for 10 minutes.) Add 25 μL of milk to each array made from the protein solution. Add PBS to each microcon so that the concentration becomes 500 μL, and centrifuge again. The concentrated sample was taken in a small volume (（5 μL) of PBS to prevent drying and precipitation.

The Cy3-labeled standard protein solution is partitioned into the appropriate Cy5-labeled test protein solution,
PBS was added to each mixture to make 25 μL of each array. Microparticles or precipitates were either 1) passed through a 0.45 μm spin filter or 2) centrifuged at 14000 xg for 10 minutes and the supernatant was removed by pipetting.

Detection: Each array was individually removed from the PBS wash. To prevent the array from drying, 25 μL of the dye-labeled protein solution was placed on the spot (within the boundaries described) and a coverslip was placed on the protein solution. The coverslip is at least 1/4 inch longer than the dimensions of the array. A layer of PBS was placed under the array and the array was placed in a sealed humidified chamber and incubated at 4 ° C. for about 2 hours. Gently immerse each array in PBS to remove protein solution and coverslips, and immediately
Transferred to slide rack in .1% Tween-20 solution. Once all racks were in the PBS / Tween solution, they were washed on a rotary shaker for ~ 20 minutes at room temperature. Array was transferred to a new rack in PBS solution (to suppress the residual Tween), gently 5-10 minutes shaking, PBS, transferred to H ₂ O, H ₂ O wash solution and gently rocked for 5 minutes each . The arrays were then spin dried and scanned.

Analysis: The fluorescence intensity of each spot reflects its level of binding to a particular protein. The relative concentration between proteins in the pools labeled with different dyes can be determined by comparing the fluorescence intensity between the color channels of each spot. Relative concentrations were determined using the following method.

The location of each analyte spot on the array was plotted using “grid” software such as GenePix or ScanAlyze, which bounded each spot on the array.

The fluorescence signal of each spot was determined as the mean or median pixel intensity within the boundaries drawn using grid software. Each color channel was processed separately. Optionally, a statistical technique was used to reject bad pixels in each circle, ie, pixels with intensities that deviated significantly from the average pixel intensity.

[0107] Background was subtracted from the signal. The background is 1) the median or average pixel intensity of the local part around each spot, or 2)
It can be determined as the median or average pixel intensity within a particular spot or area that is determined to be a non-combined background area. Statistical techniques can be used to reject bad pixels in the background.

At each spot, the relative binding between the proteins in the separately labeled pool corresponds to the ratio of the fluorescence intensities of the two color channels. For this ratio to reflect the true relative concentration, the signal minus one background of the color channel is
It is necessary to multiply by the standardization factor. The normalization factor can be determined by selecting spots of known true density and calculating the factor that most accurately gives the true color ratio. Alternatively, if control spots are not used, the average binding to all spots on the array can be assumed to be approximately equal for the two protein pools. Then, for all spots on the array, a standardization factor that gives the average color ratio is calculated.

Once all arrays have been normalized and the color ratios calculated, the changes in protein concentration between the arrays are compared. Interpretation is easiest if the same standard pool is used for each experiment.

Results In order to examine the specificity, quantification, and detection limit of the protein array, six types of mixed solutions of antigens, each having a different protein concentration, were prepared. For example, one protein changed from a high concentration to a low concentration, another protein changed from a low concentration to a high concentration, and another changed from a low concentration to a high concentration and then to a low concentration. For the entire set, the concentration varied by three orders of magnitude. This set of six mixtures was detected at different concentrations and at different levels of fetal calf serum (FCS) background. The ability to reconstruct the actual concentration change from the data was indicative of the performance level of the microarray.

A microarray was generated containing 6-9 overlapping spots for each antibody. FIG. 5 compares these arrays of arrays made from six different protein mixtures (labeled with the red fluorescent dye Cy5) with a standard mixture (labeled with the green fluorescent dye Cy3) containing equal amounts of each protein. Things. The red / green ratio was calculated for each spot on the array and plotted as a function of dilution. FIG. 6 plots the log of the ratio of red to green (R / G) versus dilution for the eight antigens. The ideal slope, calculated as the log of the protein concentration ratio, is shown as a straight line falling from 1.5 to -1.5. Other lines on the graph represent overlapping spots on the array.
The slope of this experimental data is very similar to the ideal slope over the six concentrations examined, indicating that these antibodies specifically and quantitatively detect the relevant antigen. Deviations from the ideal slope occur systematically between overlapping spots,
This suggests that the largest error in quantification occurs in pipetting or data conversion rather than random fluctuations in the system.

The detection of specific proteins is limited not only by the concentration, but also by the background protein concentration. When the protein background is high,
Different sets of protein mixtures were added to different amounts of FCS before dye labeling to determine how much specific protein could be detected. FCS concentrations of 10 and 100 times the concentration of the antigen mixture were used. FIG. 7 shows the effect of protein background on the quantification of proteins IgG and flags. In the absence of serum background, accurate quantification was observed for all proteins over the entire concentration range of 120 ng / mL to 120 pg / mL. At a serum concentration of 10x, the flag protein still shows accurate quantification, but the IgG deviates slightly from the ideal slope at the upper and lower limits. At a serum concentration of 100x, both proteins show significant deviations from the ideal slope. In experiments with 100x serum, partial concentrations (
The antigen concentration divided by the total protein concentration) ranged from 4 x 10 ^-5 to 4 x 10 ^-8 . Therefore, the detection limit of the partial concentration when using these antibodies was x2 × 10 ⁻⁶ for the flag and 22 × 10 ⁻⁷ for the IgG. These partial concentrations are the physiological range of many clinically interesting serum proteins. The results of this type of analysis for each antigen tested are shown in the table below. Antibodies have been classified (++) in the presence of accurate quantification in the entire concentration range of all low background experiments and at least in some of the high background tests. If the correct quantification was shown for most of the low background experiments, it was classified as (+). Many of the antibodies showed either no signal or a non-specific signal.

In a second detection format, antigen was spotted on the array to detect labeled antibodies.
FIG. 7 is an example of specific detection of antibodies in four different mixtures. A combinatorial labeling scheme was used that allowed the identification of specific antigen / antibody binding. An analysis similar to that described above was performed to classify the binding specificity of the antigen on the microarray. The results of this analysis are shown in the table below, together with the results of the antibody array. According to this analysis, protein arrays spotted with antigen perform at least as well or better than protein arrays spotted with antibodies.

[0114]

[0115] From the experimental data and description above, it is clear that the present method provides a useful method for the production of microarrays containing immobilized polypeptides. Polypeptides retain their binding specificity and are useful for detecting and quantifying ligands that bind to polypeptides, peptides, nucleic acids, factors, cofactors, etc., including proteins and fragments thereof.

[0116] All articles and patent applications cited herein are, as noted, that each article or patent application is specifically and individually incorporated by reference herein.
Which is incorporated herein by reference.

The foregoing invention has been described in detail by way of drawings and examples for a clearer understanding, but without departing from the spirit or scope of the appended claims in light of the present disclosure. It will be apparent to those skilled in the art that certain changes and modifications can be made.

[Brief description of the drawings]

FIG. 1 is a side view of a reagent dispensing device with an open capillary dispensing head made for use in one embodiment of the present invention.

2A-2C illustrate the steps of delivering a fixed amount of droplets to a hydrophobic surface using the dispensing head of FIG. 1, as described in one embodiment of the method of the present invention.

FIG. 3 shows a portion of a two-dimensional array of analyte assay areas made according to the methods of the present invention.

FIG. 4 is a two-dimensional view showing the components of an automatic device for producing an array according to the invention.

FIG. 5 shows the concentration profile of 110 antigens on a microarray.

FIG. 6 shows detection of protein as a ratio of signal from two fluorescent dyes to dilution of the protein sample.

FIG. 7 shows a quantitative graph of the protein after serum dilution.

FIG. 8 shows multiple antibody combination detection.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Brown Patrick United States of America Stanford Peter Kouts Circle 76 (72) Inventor Herb Bryan United States of America Berkeley Grant # 1 1922

Claims (12)

[Claims] Contacting a sample with a microarray of polypeptides comprising 100 or more discrete regions of different polypeptide chains per cm ^{2 of a} two-dimensional solid support; Washing the unabsorbed sample; and detecting the presence of bound ligand. A method for simultaneously detecting the presence of a plurality of protein-bound ligands in a sample. 2. The method of claim 1, wherein the ligand present in the sample is labeled with a detectable label. 3. The method of claim 2, wherein the detectable label is a fluorescent dye. 4. The method of claim 2, further comprising contacting a second sample comprising a ligand labeled with a second detectable label with the microarray. 5. The method of claim 4, wherein the second detectable label is a fluorescent dye. 6. The method of claim 1, wherein the sample is a clinical sample of a bodily fluid. 7. The method of claim 6, wherein the bodily fluid is blood or a derivative thereof. 8. The method of claim 1, wherein the sample is a cell culture supernatant. 9. The method of claim 1, wherein the sample is a cell lysate. 10. The method according to claim 1, wherein said polypeptide is an antibody. 11. The method according to claim 1, wherein the polypeptide is an antigen. 12. The method of claim 1, wherein the polypeptide is at least 50 amino acids in length.