METHOD AND APPARATUS FOR SUBSTRATE HANDLING AND PRINTING

Abstract

The present invention relates to a method and device for manufacturing microarrays, wherein a microarray comprises a plurality of spots, for testing the interaction of biomolecules. Disclosed herein is a method for enhancing efficiency of overlay printing of spot positions on multiple slides or plates arranged in an array wherein a slide or plate order is provided by rows and columns.

Claims

1. A method for enhancing efficiency of overlay printing of spot positions on multiple slides arranged in an array comprising the steps: printing at least a one spot of a first test material comprising a first type of first potential binding partner pair onto a first row (r1) of slides in an array of n columns in a printing order to provide the first test material on at least slide r1n1 and a replicate slide r1n2 printing at least one spot of a second test material comprising a second type of first potential binding partner pair onto a second row (r2) of slides in an array of n columns in a printing order to provide a second test material on at least slide r2n1 and a replicate slide r2n2, reordering the slides, printing spots of at least a first overlay material comprising a first type of second potential binding partner pair to overlay the spots of the at least first test material and/or the spots of the at least second test material wherein when the overlay material is printed, a slide is provided at the same position in the array at which the test material was applied and the overlay material is provided without requiring movement of a printhead between rows when overlaying the first overlay material.

2. The method of claim 1 wherein when at least two different overlay materials are printed the slide is provided at the same position in the array at which the first test and material was applied.

3. The method of claim 1, wherein the test material comprises an analyte, in particular a cell, cell-derived product, cell lysate, a protein, a protein fragment, a receptor provided on an intact cell, a receptor provided in a cell lysate, a fusion protein, a nucleic acid sequence or the like.

4. The method of claim 1, wherein a second potential binding partner (provided in the overlay material) is a specific binding molecule selected from a group comprising an antibody or a fragment thereof, a small molecule, an aptamer, or a nucleic acid molecule.

5. The method of claim 1, wherein a subgroup of a number of slides (Sr)′ may be printed with a first test material (t1) to provide replicate slides with each replicate slide providing a first position of a column in a row (for example: r1n(Sr)′t1, r1n(Sr)′t1, r1n(Sr)′t1, r1n(Sr)′t1, r1n(Sr)′t1).

6. A method of claim 5 wherein the subgroup is 5 slides, such that in the method, in each row, five replicate slides are printed with a first test material (t1) with each replicate plate providing a first position of a column in a row: r1n1t1, r1n2t1, r1n3t1, r1n4t1, r1n5t1.

7. The method of claim 6 wherein the overlay material is printed following reordering of the slides determined based on the following input Binding partner Print Run (overlay Hybridoma Print Run Number (H)) Slide Number (S) Tray Number (T) Slides per subrun (Sr)=5 Runs/cycle (Rc)=20 Slides per total run (St)=100 Slides per tray=25
Binding partner Offsset (e.g. HybridomaOffsset)=MOD(Rc*(Sr−(H−1)), St); Which provides (based on above):
Binding Partner Offsset (e.g. HybridomaOffsset)=Ho=MOD(20*(5−(H−1)), 100);
SlidePosition=Sp=MOD(S−1, 25)+1;
SlideOffset=So=MOD((SlidePosition−1)*20, 100);
SubrunOffset=Sro=QUOTIENT(Sp'11,5);
TrayOffset=To=(T−1)*5;
Test Material (e.g. Lysate)=L=MOD(Ho+So+Sro+To, 100); where MOD is the module operation (remainder after division of one number by another).

8. The method of claim 1, for detection of analyte to a specific binding molecule comprising: incubating the printed test material and overlay material to allow binding between an analyte and specific binding member provided in the test material and overlay material to occur, detecting any binding between an analyte and specific binding member provided in the test material and overlay material.

9. The method of claim 8 wherein the detecting step is an immunohistochemical detecting step, optionally wherein the detecting comprises detecting fluorescence, colorimetry, quantum dots, biotin/avidin, surface plasmon resonance to identify binding.

10. The method of claim 1, wherein there is provided a blocking step prior to the printing of overlay material.

11. A method of diagnosing a disorder, the method comprising determining the presence of an analyte in a test material sample using a method of claim 1, wherein when binding of a specific binding molecule provided in an overlay material and analyte in the test material is detected it is indicative of the disorder.

12. A system to provide the method of claim 1, wherein the system comprises a printer adapted to print a test material onto a substrate, to provide a first row (r1) of slides in an array of n columns in a printing order to provide the first test material on at least slide r1n1 and a replicate slide r1n2, to print at least one spot of a second test material onto a second row (r2) of slides in an array of n columns in a printing order to provide a second test material on at least slide r2n1 and a replicate slide r2n2, to print spots of at least a first overlay material to overlay the spots of the at least first test material and/or the spots of the at least second test material wherein when the overlay material is printed, a slide is provided at the same position in the array at which the test material was applied and the overlay material is provided without requiring movement of a printhead between rows when overlaying the first overlay material.

13. The system of claim 11 wherein the system comprises an environmentally controlled module around the printer.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0136] There will now be described, by way of example only, various embodiments of the invention with reference to the following drawings, of which:

[0137] FIG. 1 illustrates a functionalised glass slide printed with analyte/antigen (e.g. cell lysate) library;

[0138] FIG. 2 illustrates an analyte/antigen binding agent (e.g. hybridoma) library printed directly on top of antigen library;

[0139] FIG. 3 illustrates non-binding analyte/antigen binding agents after a washing step being washed away;

[0140] FIG. 4 illustrates specific binding of analyte/antigen being detected with a labelled secondary affinity reagent (e.g. antibody);

[0141] FIG. 5 illustrates a high resolution scan of an analyte/antigen binding agent library printed directly onto a printed lysate library

[0142] FIG. 6 illustrates the data generated from a spot on spot printing example in which four different antibodies were screened at four different concentrations against sixteen cell lysates. In parallel, two RPPA experiments were performed comparing the reactivity of two control antibodies (EGFR and 2C8) at a single concentration (0.1 μg/mL) against fourteen cell lysates (A431, A549 and SKMEL28) which were included in the spot on spot printing example. In the spot on spot array image the box labels highlight sets of four spots per antibody+lysate combination and the data on the graphs represent 0.1 μg/mL antibody (on the image this is the third spot from the top of each box);

[0143] FIG. 7 illustrates a spot on spot study wherein four antibodies and supernatants at four concentrations were screened against 16 cell lysates;

[0144] FIG. 8 illustrates the data generated from a comparison study of the reactivity of two control antibodies (EGFR and 2C8) against three cell lysates (A431, A549 and SKMEL28);

[0145] FIG. 9 illustrates a RPPA experiment wherein antibody 2C8 was provided at 0.1 μg/mL vs cell lysates A431, A549 and SKMEL28;

[0146] FIG. 10 illustrates a RPPA experiment wherein EGFR antibody is provided at 0.1 μg/mL vs cell lysates A431, A549 and SKMEL28;

[0147] FIG. 11 illustrates the data generated from a comparison of the interactions of four lysates (A431, A549, SKMEL28 and HT29) with antibody 2C8 (0.1 μg.Math.ml) and shows a >10-fold higher in signal using spot-on-spot (B) than RPPA (A).

[0148] FIG. 12 is a plan view of apparatus for printing.

[0149] FIG. 13 A illustrates a print run of test materials on a configuration of substrates in horizontal arrangement of subgroups and figure B with vertical arrangement of subgroups

[0150] FIG. 14 illustrates the selective reordering of the substrates of FIG. 13

[0151] FIG. 15 illustrates the printing of overlay materials on the reordered substrates illustrated in FIG. 14.

[0152] Alternative FIG. 16 illustrates a print run of test materials on a configuration of substrates, the selective reordering of the substrates and two printing strategies for overlay materials

[0153] FIG. 17 illustrates tray arrangement of test materials on plates

[0154] FIG. 18 illustrates a printing pattern for the first layer of printing of test materials for 20 different test materials

[0155] FIG. 19 illustrates a printing pattern for an overlay material.

[0156] FIG. 20 illustrates use of module operation using parameters specified herein to illustrate movement of slides during printing.

[0157] FIG. 21 illustrates in A that all the slides in position 1 are designated as S1 and in B that slides in the same tray or row are designated by second positional indicator T1, T2, T3 or T4 with lysates in this example in batches of 5.

[0158] FIG. 22 illustrates printing of second binding member (hybridoma) and retaining position of first printed binding member (lysate) in reordered slides.

[0159] FIG. 23 A, B, C and D illustrates FIG. 13 B in expanded view

[0160] FIG. 24 illustrates FIG. 14 in expanded view, and

[0161] FIG. 25 illustrates FIG. 15 in expanded view

[0162] Definitions

[0163] Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0164] All numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.”

[0165] It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise.

[0166] As used herein, unless the context demands otherwise, the terms “substrate”, “slide” and “plate” are to be seen as interchangeable synonyms.

DETAILED DESCRIPTION OF THE INVENTION

[0167] A method of printing as described herein could utilise printing apparatus as discussed in patent applications WO 2004/028683 as would be known in the art. Accordingly, suitably the printing apparatus may include a platen, four cages, and a linear rail. Each cage may be a rectangular metal frame having a series of vertically stacked substrate (plate or slide) tray supports in the form of inwardly protruding ledges. The cages may be shaped to receive a number of substrate trays and each tray holding a linear array of slides to be printed. Each slide tray may be oriented lengthways across the width of the platen, and the length of the trays can be greater than the width of the platen.

[0168] In specific embodiments discussed herein, the arrangement for the slides within a tray is a linear array of, say, twenty-five slides.

[0169] The operation of the apparatus to print a first spot will now be described. The present invention provides an efficient means of printing a large number of substrates, by providing rapid means of storing, retrieving, printing and re-ordering of the slides.

[0170] The arrangement reduces the requirement for reloading the printhead with different liquids. This is particularly appropriate where the liquids are valuable and available in small quantities only. As discussed above, the loading of liquids into the printhead is inevitably wasteful in that only a proportion of each liquid is usefully printed. Inkjet printheads produce very small drops, so once liquids are introduced into the printhead, it can print a very large number of spots.

[0171] An additional advantage of this approach is that larger numbers of trays can be printed than those which fit on the platen: one or more cages can be used to store the trays when they are not being printed, to feed them onto the platen for printing and to remove them afterwards. A number of trays can be stored above each other on shelves in each cage; one or more cages can be moved vertically downwards so as to deposit the trays in turn onto the platen in preparation for printing. One or more other cages, moving vertically upwards, can remove them afterwards. The cages can perform these functions while the platen is stationary, during the printing stroke of the printhead. Thus the loading of trays onto the platen and unloading from it need not add to the time taken to print the overall number of slides in the machine; this scheme is equivalent in speed to a system using an impracticably large platen to hold all the trays. This is discussed, for example in WO 2004/028683.

[0172] A further advantage of this approach is that the cages and their motion need not be precise: if the trays are equipped with location features which engage with matching features on the platen, the act of loading each tray onto the platen ensures its accurate positioning with respect to the platen. The only parameters that need to be accurate are the printhead mounting, its motion, the platen's location features and its motion. Both of these motions are one-dimensional.

[0173] The inventors have determined that the spot on spot method described herein can provide for improved sensitivity (higher signal to noise ratio) of detection of binding between an analyte and specific binding molecule. Additionally, it is considered the spot on spot method may provide for improved discrimination between positive and negative samples (hybridoma supernatants), improved throughput and lower background.

[0174] It is considered the method can have advantages over alternative screening techniques such as RPPA (reverse phase protein array), mass spectroscopy, western blotting, or ELISA as it allows the binding of large libraries of unknown antibodies to large libraries in unknown analytes/antigens to be specifically detected in a high-throughput manner. By way of reference, it is considered that a single operator would be limited to generating an estimated 2000 data points (i.e. a specific test of the binding ability of one antigen binding agent to one analyte) in a working week using ELISA or Western Blotting which would allow the same sensitivity as the present test, whereas the present method may provide, for example 2 million data points in the same period.

[0175] It is further considered that the spot on spot method described herein can advantageously provide for a reduction in the quantity of specific binding molecule—for example antibody utilised, for example four orders of magnitude less antibody than RPPA for an equivalent test (e.g. RPPA would use ˜100 μl primary antibody to interrogate 100 lysates, whereas the present method may use ˜0.01 μl). It is considered the present method only requires 100 pL of ˜1 mg/ml of lysate and 100 pL of antibody per test. It is considered that significantly increased volumes of lysate and/or antibody would be required to test for binding using ELISA, western blot or mass spectrometry.

[0176] It is further considered the present method is advantageous as it allows a library of lysates to be screened against a library of antibodies in a single experiment, for example as different antibodies can be printed at spot positions of cell lysate, whereas RPPA would typically only allow a single antibody to be screened against a library of lysates. For example, the method of the present invention may allow 250 lysates to be screened against 250 hybridomas on a single portion of substrate (slide) containing 62,500 features, whereas RPPA would require 250 slides.

[0177] The present method can advantageously allow identification of a positive interaction between a specific binding member and an analyte. Whilst mass spectroscopy could provide increased granularity of what protein or proteins may be present in a sample, resolving these is an inherent challenge with mass spectroscopy, because the most abundant proteins (e.g., actin) will compete for detection with less abundant (but more interesting) proteins such as cytokines. Mass spectroscopy is also resource-intensive from an informatics standpoint. As noted above the method of the present invention may allow a library of lysates to be screened against a multitude of antibodies in a single experiment. Mass spectroscopy typically only allows a single antibody to be screened against a single analyte.

EXAMPLES

Example 1

[0178] A lysate library was normalised to 2.5 mg/mL before diluting 1:1 in 2× Protein Printing Buffer C (Arrayjet Ltd, UK) (PPBC×2). Negative control samples (BSA in PBS) were normalised to 1 mg/mL before diluting 1:1 in PPBC×2. Positive control samples (IgG in PBS) were prepared at 2 μg/mL before diluting 1:1 in PPBC×2.

[0179] 200 pL of lysate library and control samples were printed onto PATH nitrocellulose slides (Grace Bio, Inc., Oregon, USA) with an Arrayjet Marathon series inkjet bioprinter (Arrayjet Ltd, UK) at 4° C. and 80% relative humidity (% RH). The slides were incubated overnight at 4° C. and 80%RH. The slides were then incubated at 30° C. for one hour. The slides were then blocked with 5% BSA in PBS-T (IgG-free) for 30 mins. Excess blocking reagent was washed off with three sequential 30 minute washes of PBS-T, PBS and distilled water. The slides were dried. 200 pL of specific binding agents (antibodies) in printing buffer were printed onto each of the lysate library ensuring that the specific binding agents are printed directly on top of the printed lysate spots. Printing conditions of 4° C. and 80% RH were used.

[0180] Excess specific binding agent was washed off with three sequential 30 minute washes of PBS-T, PBS and distilled water. The slides were incubated with labelled secondary antibody diluted 1/1000 in BSA (1% in PBS-T) (protected from light for 90 minutes) at room temperature. Excess secondary antibody was washed off with nine sequential 5 minute washes of PBS-T (×3), PBS (×3) and distilled water (×3). The slides were dried. Using a confocal laser microarray scanner (Innoscan 710 AL, Innopsys, France) the slides were scanned and data was extracted for analysis. Specific binding between the specific binding agent to the analyte is indicated by elevated fluorescence levels when compared to the negative control samples.

Example 2

[0181] When testing the same set of lysates, the spot-on-spot method was more sensitive than RPPA (FIG. 11), indicating a higher true positive rate for the spot-on-spot technique. This was achieved by increasing the signal and reducing the background, giving an improved signal to noise ratio by approximately ten-fold. One of the key steps to achieving this is by printing the antigen binding member onto the lysate; this means that the antigen binding member is only bound to the analyte and does not bind to the entire surface of the slide, which is the case in the RPPA method.

[0182] Subsequently, when the labelled secondary antibody is applied it is less likely to create background signal.

Example 3

[0183] An example assay was provided comprising approximately 10,000 hybridoma samples against 100 different lysate. Suitably assaying of about 1,000,000 tests a week is discussed.

[0184] Each test is carried out in duplicate. i.e. a total of up to 2,000,000 tests a week

[0185] To allow for lysate printing, five print runs of one hundred slides were utilised. This therefore provides for printing of 500 slides consisting of 5 identical slides for each lysate, i.e. 5 slides×100 lysates=500 slides.

[0186] As illustrated in FIG. 13, this means that, Print run 1 will print lysate 1 to lysate 20; Print run 2 will print lysate 21 to lysate 40; Print run 3 will print lysate 41 to lysate 60; Print run 4 will print lysate 61 to lysate 80; Print run 5 will print lysate 81 to lysate 100.

[0187] The total number of spots per lysate will be 20160. This is sufficient to print 10,000 hybridomas (second test material) in duplicate, there is space to add buffer spots if required.

[0188] The second test material to be printed (in this example hybridomas) are printed with different lysates such that up to 10,080 hybridomas in duplicate are provided. Suitably the hybridomas may be provided in 10% glycerol.

[0189] Suitably an algorithm may be provided to allow for slide loading for hybridoma printing. Suitably, the system provides for a left to right shift of lysate number such that the slide position and tray are always constant. In the embodiment described herein a rotation to the right of 5 slides creates a sequence such that the slides are in the correct position for the hybridoma print run. This is illustrated for example in FIGS. 15 and 16. A colour sequence is provided to more clearly illustrate the sequence such that the hybridoma printing of the slide is provided when the slide is in the same position as lysate printing.

Example 4

[0190] With reference to FIGS. 21 and 22, a first print run printed Lysates (L) in batches of 5, so the first five slides are all Lysate 1 L1, the slides 5 to 10 print Lysate L2, and so forth until 20 lysates are printed.

[0191] Hybridomas are then provided in overlay printing after the slides have been reordered such that the parameters S and T, are kept constant: so S1 T1 should be placed in slide 1 tray 1, etc. In this embodiment 100 different lysates are provided in one print run then were placed in same location for overlay printing where they were printed during the lysate print run, for example the first lysate 2 was printed at position 6 and thus requires to be provided in S6 T1 in the overlay printing.

[0192] Lysate 3 was printed in position 11, etc.

[0193] Because in this embodiment 20 lysates are printed in a first batch of concatenated print runs: ‘S’ and ‘T’ kept constant and the equation for ‘L’ is that the next lysate to lysate 1 should be 1+20

[0194] Within tray: in groups of 5:

[0195] Lx; L(x+20), L(x+40), L(x+60), L(x+80).

[0196] Inter-tray: The lysate should be as in the previous tray+5

[0197] Tray 1: L(x);

[0198] Tray 2: L(x+5)

[0199] Tray 3: L(x+10)

[0200] Tray 4: L (x+15)

[0201] For the following hybridomas run, a rotation between the 5 slides takes place: Lysate 1 is now in position 2, so the rotation system brings lysate 81 to position 1 (see FIG. 22 second print run)

[0202] Inter-tray is “+5” as before

[0203] For the next hybridoma run, the rotation continues and lysate 1 is now in position 3 (see FIG. 22 third print run).

[0204] As will be appreciated, a fourth print run would position lysate 1 at position 4 and the rotation system would bring

[0205] It will be evident to the skilled reader that various changes could be made to the above-described embodiments within the scope of the invention.

[0206] Various modifications and improvements can be made within the scope of the invention herein intended.