How to couple antibodies to EYRAbeads

Looking to develop your very own EYRAplex assay with EYRAbeads? This protocol provides general guidelines for covalently coupling antibodies to Mabtech EYRAbeads for use in home-brewed multiplex bead-based immunoassays.

Coupling your own antibodies to EYRAbeads is a relatively straightforward process and can be done in as little as half a day. This means you could get an entire custom assay up and running and validated all in under a week! If the process below feels daunting, don't worry! Our field application scientists can jump on a call or join you at your lab to help develop your very own homebrew EYRAplex assay. There are a number of ways and different reagents one can use for coupling, but here we highlight one path forward to custom couplings.

I. Preparation of capture antibody

1. Buffer exchange the capture antibody into conjugation buffer using a desalting column.

• Note: Do not keep antibodies in conjugation buffer for longer than 2 hours before conjugation.

2. If needed, concentrate the mAb to achieve a concentration of 0.05 mg/ml (preferably use a high-concentration stock for buffer exchange).

3. After buffer exchange:

• Filter the mAb through a 0.22 µm sterile filter.
• Measure the antibody concentration (e.g., by absorbance at 280 nm).

4. Dilute the mAb in conjugation buffer to a concentration that results in 4 µg antibody/million beads in the specified volume of buffer-exchanged antibody in Table 1.

• For example, if conjugating 1 million beads, the buffer-exchanged antibody should be diluted to 13.3 µg/ml in 0.3 ml with a total conjugation volume of 0.6 ml. 

II. Preparation of EYRAbeads

1. Mix beads thoroughly by vortexing for 30 seconds, followed by sonication (if available) for 5-10 seconds before pipetting (within 30 seconds of vortexing).
2. Transfer the desired number of EYRAbeads to a suitable tube (see Table 1 above).
3. Add activation buffer to reach the recommended wash volume, according to Table 1.
4. Wash the EYRAbeads four times with the recommended wash volume of activation buffer, vortexing thoroughly after each wash.
5. Resuspend the EYRAbeads in the recommended resuspension volume of activation buffer, see Table 1.

III. Activation of EYRAbeads

Note: All steps should be performed in a chemical hood.

1. Dissolve sulfo-NHS and EDC freshly in activation buffer:
   1. Sulfo-NHS: dissolve to 50 mg/ml in activation buffer.
   2. EDC: dissolve to 50 mg/ml in activation buffer.
2. Add the recommended volume of sulfo-NHS and EDC (see Table 1) to the bead suspension prepared in Step II.
3. Incubate on a tube rotator, preferably in oscillating mode, at RT for 15 min.

IV. Conjugation of antibody to activated EYRAbeads

1. Remove the activation buffer using the magnetic rack.
2. Wash the EYRAbeads four times with conjugation buffer, vortexing thoroughly after each wash.
3. Resuspend the EYRAbeads in the recommended volume of conjugation buffer, according to Table 1.
4. Add the buffer-exchanged capture antibody in the same volume as the conjugation buffer to the bead suspension (1:1) and mix immediately by pipetting.
5. Incubate on a tube rotator, preferably in oscillating mode, at room temperature overnight.

V. Quenching of antibody-conjugated EYRAbeads

1. Wash the conjugated EYRAbeads with the magnet as described previously:
   1. Once with conjugation buffer
   2. Three times with quenching buffer (vortex thoroughly after each wash).
2. Resuspend the EYRAbeads in the recommended wash volume of quenching buffer.
3. Incubate on a tube rotator, preferably in oscillating mode, at RT for 60 min.

VI. Storage of conjugated EYRAbeads

1. Wash the quenched EYRAbeads three times with freshly filtered storage buffer, vortexing thoroughly after each wash.
2. Resuspend the EYRAbeads in a suitable final volume of storage buffer, e.g., 2 × 10⁶ beads/ ml.
3. Count the EYRAbeads using a cell/ bead counter or suitable flow cytometry instrument.
4. Aliquot into dark vials and store at 4-8°C.

SHOP EYRAbeads