Monoclonal antibody purification

2.9 Monoclonal antibody purification

Methods include ammonium sulphate precipitation, gel filtration and ion-exchange chromato- graphy, although a rapid and more convenient method is affinity chromatography. The use of protein G/protein A for mouse isotypes has previously been covered in Chapter 1. For IgM isotypes, alternative methods are now available (e.g. PROSEP®-Thiosorb-M©, Bioprocessing Ltd; rProtein L, Actigen Ltd), which can be followed according to manufacturer’s instructions. In general immunoglobulins are preferable as monoclonal reagents, since IgM antibodies are prone to degradation, particularly following freezing and thawing.

2.9.1 Purification of murine IgM using PROSEP®-Thiosorb-M© (Bioprocessing Ltd)

PROSEP ® -Thiosorb-M © is an affinity adsorbent which facilitates the purification for IgM by covalent attachment of a ligand with thiophilic properties, to PROSEP®. The matrix will bind IgM at high concentration of lyotropic (water-structuring) salt such as ammonium sulphate, or potassium sulphate, and release it at low concentration or in the absence of a lyotropic salt. Fur- thermore, standard chromatography columns are suitable for use with PROSEP®-Thiosorb-M©.

A simple protocol (from Emma Waldron, Konstantinos University, Greece, and Wolverhampton University, UK) is provided for the culture supernatant known to contain IgM.

MATERIALS PROSEP ® -Thiosorb-M © (Bioprocessing Ltd) Chromatography column, e.g. disposable syringe Culture supernatant containing monoclonal antibody B35B

20 m M HEPES, 0.5 M NaCl, 7.5% (NH 4 ) 2 SO 4 , pH 7.5 (wash buffer)

20 m M HEPES, 2 M NaCl, pH 7.5 (elution buffer 2) 60% ethylene glycol (elution buffer 3)

6 M urea (regeneration solution) BioLogic HR chromatography system (e.g. Bio-Rad) plus column

2.9MONOCLONAL ANTIBODY PURIFICATION

1.5-ml Eppendorf tubes UV spectrophotometer and quartz cuvette (particularly if performing purification without a flow

cell and chart recorder)

METHOD

1 Prepare a 50% slurry of the matrix in wash buffer (1 g swells to about 3.25 ml) and pour swelled slurry into column. Ensure the bed height is even and connect to a suitable chromatography system. (BioLogic HR, BioRad).

2 For a column containing 4–5 ml matrix, use 20 ml of wash buffer to flush out the tubing and wash the column. Check the absorbance of the flow-through.

3 Add 20 ml of culture supernatant (previously centrifuged at 1000 g for 5 min to remove any debris).

4 Add 20 ml of wash buffer (UV monitoring should ultimately indicate a zero base line).

5 Add 20 ml of elution buffer 2 and subsequently add 20 ml of elution buffer 3 to release IgM antibody. Check against UV monitor and test fractions for immunological reactivity. The absorbance of pooled fractions may also be analysed using a quartz cuvette and UV spectrophotometer set at 280 nm.

6 Aliquot antibody in Eppendorfs/dialyse against appropriate buffer/estimate protein concentration using extinction coefficient or bicinchroninic acid (BCA) system (see also Appendix B.5).

7 Regenerate column with 6 M urea, and then equilibrate with 10 column volumes of wash buffer.

2.9.2 Isotyping of murine monoclonal antibodies

Antibodies in hybridoma cell culture supernatant can be typed in a capture enzyme-linked immunosorbent assay (ELISA) using anti-isotype antibodies provided in a commercially avail- able kit, e.g. Sigma. The following protocol provides a means of qualitatively determining the isotype of murine monoclonal antibodies IgG1, IgG2a, IgG2b, IgG3, IgM or IgA. A 200 µl system is used.

MATERIALS AND EQUIPMENT Mouse monoclonal antibody isotyping reagents akit Sigma ISO-2 Washing buffer (phosphate-buffered saline (PBS)–0.05% Tween 20) PBS Horseradish peroxidase (HRP) conjugate diluted in washing buffer

0.1 M citrate buffer Hydrogen peroxide, 30% w/v solution 2,2′-azinobis(3′ethylbenzthiazoline sulphonic acid) (ABTS) tablets 0.1% sodium dodecyl sulphate Flat-bottomed 96-well ELISA plate Multichannel pipette plus tips 0.5–10-µl pipette 40–200-µl pipette 200–1000-µl pipette Universal tubes

60 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

1.5-ml Eppendorfs Petri dishes Plate reader and printer (Labsystems Multiskan MS or equivalent)

METHOD

1 Dilute the isotype-specific antibodies to 10 µg/ml in PBS, 1600 µl per plate required for each solution (see kit information leaflet for protein concentration).

2 Pipette 200 µl of IgG 1 into each well of column 2, do the same for IgG 2a in column 3 and so on for all the antibodies. Incubate plate at 37°C for 1 h, or at 4°C overnight.

3 Remove coating solution and wash plate three times with washing buffer.

4 Add 200 µl of hybridoma supernatants to be tested to appropriate row of wells, i.e. into wells A2–A7. Also add 200 µl of HT or HAT medium (according to the medium the cells were grown up in) to a row of wells as a control. Incubate plate at 37°C for 2 h.

Note: Supernatants of known isotype should also be included as controls and to ensure that the system is functioning.

5 Wash plate three times with washing buffer.

6 Dilute HRP conjugate 1 : 1000 (or an optimal dilution) with washing buffer (20 µl HRP in

20 ml buffer).

7 Add 200 µl HRP to all wells and incubate plate for 1 h at 37°C.

8 Wash plate as in step 5.

9 Dissolve one ABTS tablet in 20 ml 0.1 M citrate buffer. Immediately prior to substrate use, add 12 µl of hydrogen peroxide to solution and mix well.

10 Add 200 µl of above substrate solution to each well and allow reaction to proceed for

30 min. A green colour will develop in the appropriate wells to indicate the MAb isotype. If preferred, the reaction can be stopped by adding to each well 200 µl of 0.1% sodium dodecyl sulphate.

11 The absorbances produced by the antibodies should be read on a plate reader at 414 nm and a printed record made to aid identification of sample isotype.

TECHNICAL NOTES • Remove background noise by calculating the mean HT/HAT values and subtracting this from all

other values produced. • If more than one isotype is indicated, then the hybridoma may need recloning. • It is advisable for the laboratory to have a stock of immunoglobulin isotypes to be used as con-

trols in the experiments. • Check the isotype of the monoclonal antibody regularly when purified and after cryopreserva-

tion as there have been occasional incidences where IgM antibodies have switched to another isotype after cryopreservation.