Multiplex ELISA Using Oligonucleotide Tethered Antibodies Protocol

Introduction of Multiplex ELISA Protocol

In this chapter we describe steps in preparing a quantitative multiplex immunoassay in a micro-plate format based upon the A² (A-squared) technology. Unlike conventional antibody microarrays in which antibodies are printed directly onto a substrate, the A² uses oligonucleotide tethering to attach antibodies to the surface. First, a polypropylene 96-well plate is surface treated for covalent attachment of capture oligonucleotides. Amine-terminated capture oligonucleotides are printed in a 7 × 6 array pattern in each well of the plate (A² Plate).

Next, the complementary oligonucleotides are conjugated to selected antibodies to form the oligo–antibody conjugate.

The conjugates are pooled and added to the plate under hybridization conditions for the creation of the antibody array.

Once prepared, standard immunoassays may be conducted in multiplex.

Figure 1. Close-up of the printing process.

What is the advantage of the A² approach? First, direct immobilization of antibodies often leads to random adsorption onto the surface and as pointed out by Cho et al., “random immobilization results in a low rate (e.g., 5–10 %) of the active antibody density, that is, those that can participate in the binding reaction, which decreases even further when the substance to be analyzed is large in molecular size.”. Thus, stochastic conditions may result in reduced capture efficiency due to steric hindrance or potential blocking of the antibody binding site. Furthermore, printing of proteins can lead to significant variation in spot morphology and protein density thereby reducing array quality. Alternatively, the printing of well characterized capture oligonucleotides offers several advantages. First, oligonucleotide arrays are highly reproducible to produce and extremely stable. Oligonucleotide array plates when properly stored have a shelf-life extending beyond 5 years. Because the formation of duplex DNA is thermodynamically controlled the density of the tethered oligo– antibody conjugates is relatively constant from spot to spot. As a result, the variation of immobilized antibody among spots is reduced which in turn aids in lowering assay imprecision.

The chapter is divided into three main methodologies: oligo-nucleotide–antibody conjugation, hybridization, and performing a multiplex immunoassay.

Methods of Multiplex ELISA Protocol

Conjugations

This protocol is for the preparation of oligonucleotide–antibody conjugates for use with the A^2® Plate Array. Approximately 2 OD_{260 nm} units of an activated oligonucleotide are reacted with 100 μg of purified antibody (IgG). The process involves adsorption of the immunoglobulin onto a solid-phase held within a chro matographic column (or large spin-column). Next, initiator is added to modify the antibody with reactive sulfhydryl (-SH) groups. Finally, the SMCC-oligonucleotide is added which couples to the thiolated antibody to form the oligo–antibody conjugate. The resulting conjugate still bound to the solid-support is rinsed free of reactants; and subsequently eluted in purified form from the column ready for use.

Protocol

Solid-Phase: bring to room temperature prior to use; gently mix slurry to dispense.

Spin Column: place bottom cap on column tip and insert capped spin column into a collection tube.

Prepare Spin Columns

1. Add 100 μL of a well-mixed solid-phase slurry to spin column ( see Note 8).
2. Spin: 2,000 rpm, 30 s.
3. Discard solution.
4. Add 600 μL DDI H₂O and mix bed by up and down with a pipette.
5. Spin and discard rinse.
6. Repeat steps #4, #5.
7. Add 600 μL binding buffer and mix bed by up and down with a pipette.
8. Spin and discard rinse.
9. Repeat steps #7, #8.
10. Cap bottom of spin column.
11. Cap top until ready for use.

Prepare Antibody Binding

1. Remove top cap from spin column.
2. Add 100 μL antibody solution directly to the bed and mix with pipette ( see Note 9).
3. Add 500 μL binding buffer to the column bed.
4. Mix bed by up and down by pipette action.
5. Securely cap top of the column.
6. Tumble: 10 min.
7. Remove bottom cap and spin 2,000 rpm, 30 s.
8. Measure OD 280 in effluent: if OD280 > 0.01 reapply effluent and repeat tumble.
9. If OD 280 < 0.01 then cap bottom of spin column, ready for next steps.

Prepare Protein Initiator

1. Weigh out ~1.9 mg initiator ( see Note 10).
2. Dissolve in 1 mL binding buffer.
3. For one spin column: combine 50 μL (step #2) with 750 μL binding buffer.
4. Add the entire volume from step #3 (800 μL) to the spin column and mix bed by pipetting.
5. Cap top and seal with Parafilm ( see Note 11).
6. Tumble: 1 h at room temperature.
7. Remove bottom cap (change collection tube).
8. Wash with 600 μL binding buffer.
9. Spin and discard rinse.
10. Repeat steps #8, #9- five (5) more times.
11. Cap bottom of spin column, ready for next steps.

Prepare Activated Oligo

1. Spin down tube containing activated oligo (2 OD₂₆₀) to pellet contents.
2. Reconstitute activated oligo in 800 μL binding buffer, mix well and spin tube again.
3. Add oligo solution to the spin column.
4. Mix column bed by up and down pipetting action.
5. Replace top cap and seal with Parafilm.
6. Tumble: overnight (15–20 h) at room temperature.

Prepare Oligo–Antibody Conjugate

1. Remove caps and spin 2,000 rpm, 30 s.
2. Replace collection tube to collect washes.
3. Wash with 600 μL binding buffer and check OD₂₆₀.
4. Repeat washes until OD₂₆₀ < 0.01.
5. Discard washes.
6. Change collection tubes.
7. Add 100 μL of elution buffer and mix well by pipetting action.
8. Spin: 2,000 rpm, 30 s.
9. Recover the eluate into a labeled 1 mL collection tube.
10. Repeat four more times with 100 μL elutions (500 μL total) collected into the same collection tube used in step #9 ( see Note 12).
11. Determine protein content of the collected eluate using the BCA method.
12. Store conjugates at 2–8 °C in preparation for hybridization to the A2 Plate.

Hybridization

This protocol is for the creation of the A2 Capture Antibody Array by hybridization of the previously prepared Oligonucleotide– Antibody Conjugates. The conjugates are pooled into a hybridization cocktail prepared by mixing Hybridization Buffers A and B; and then applied to the A2 Plate. The number of plates that can be prepared depends upon the conjugate yield and desired loading.

Protocol

1. Combine 6 μg of each of the conjugates together in a 15 mL centrifuge tube ( see Note 13).
2. Add 18 μL of the biotinylated reference oligonucleotide to the tube.
3. Add 1,500 μL HYB Buffer A.
4. Add HYB Buffer B to reach a final total volume of 6,000 μL.
5. Mix well.

Hybridization

1. Bring the A2 Plate to room temperature prior to removing from its pouch ( see Note 14).
2. Add 200 μL of Wash Buffer (1×) to each well of the A2 Plate.
3. Soak 5 min with shaking.
4. Remove the wash buffer but do not allow the plate to dry.
5. Tap the plate upside down onto several layers of clean towels to remove residual buffer.
6. Deliver 55 μL of the oligo–antibody pool to each well of the 96-well A2 Plate.
7. Cover the plate with a lid.
8. Incubate for 1 h at room temperature with shaking (100–300 rpm).
9. Remove the hybridization solution from the wells and discard.
10. Add 200 μL of Wash Buffer (1×) to each well and shake for 1 min.
11. Remove the wash buffer quickly by inverting over a sink.
12. Tap the plate upside down onto several layers of clean towels to remove residual buffer.
13. Repeat the wash ( steps #10–12) an additional 2 times.
14. Place the lid back onto the plate.
15. Return the plate to the aluminum pouch containing the drying packet and seal.
16. Store the plate at 2–8 °C until need for the assay.

Multiplex ELISA

This protocol is for the performance of an Enzyme Linked Immunosorbent Assay (ELISA), using A^2® Plate with immobilized oligo–antibody array. The previously prepared oligo–antibody conjugates are pooled and applied to the A2 Plate (A21002) under hybridization conditions, resulting in the self-assembly of a capture antibody array in each well of the 96-well plate. After rinsing, the plate is ready to perform the multiplex ELISA. An example of a quantitative multiplex ELISA using this protocol is provided.

1. Bring the A2 Plate to room temperature prior to removing from its pouch.
2. Rehydrate by delivering 200 μL of Wash Buffer (1×) to each well of the A2 Plate.
3. Soak for 5 min with shaking (250–300 rpm).
4. Remove the wash buffer but do not allow the plate to dry ( see Note 15).
5. Tap the plate upside down onto several layers of clean towels to remove residual buffer.
6. Deliver 55 μL of previously prepared standards or sample to each well in appropriate diluents.
7. Incubate for 1 h at room temperature with shaking.
8. Remove solution as previously described.
9. Rinse wells 3 times with 200 μL Wash Buffer (1×) with a 5-min soak between rinses.
10. Deliver 55 μL of biotinylated antibody (in biotin antibody diluent) to each well.
11. Incubate for 1 h at room temperature with shaking.
12. Remove solution and rinse wells 3 times with 200 μL Wash Buffer (1×) with 5-min soaks.
13. Deliver 55 μL of streptavidin-AP (1:20,000 v/v) to each well.
14. Incubate for 30 min at room temperature with shaking.
15. Remove solution and rinse wells 3 times with 200 μL Wash Buffer (1×) with 5-min soaks.
16. Deliver 55 μL of AP Enzyme Substrate to each well.
17. Cover plate with foil or place in dark.
18. Incubate for 30 min at room temperature without shaking ( see Note 16).
19. Carefully remove most of the solution from wells, but do not rinse or allow plate wells to dry out.
20. Wipe off the bottom of the plate with ethanol using a scratch free and lint free cloth.
21. Read the semi-wet plate on the A2 Reader (Channel B; 200 ms).

Multiplex Fluorescent Immunoassay

1. Perform assay steps #1–12.
2. Deliver 55 μL of a working stock solution of streptavidin P-1 to each well.
3. Incubate for 1 h at room temperature with shaking.
4. Remove solution and rinse wells 3 times with 200 μL Wash Buffer (1×) with 5-min soaks between rinses.
5. Fill wells with 200 μL of fresh Wash Buffer.
6. Wipe off the bottom of the plate with ethanol using a scratch free and lint free cloth.
7. Read the plate on the A2 Reader (Channel A; 200 ms).

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