Cunningham plaque assay

9.2 Cunningham plaque assay

9.2.1 Cunningham assay for plaque-forming cells

This assay was modified by Cunningham from the Jerne haemolytic plaque assay (Section 9.1) and can be used to enumerate antibody-forming cells against soluble antigens (haptens) conjugated to the surface of indicator erythrocytes, e.g. dinitrophenol (DNP) or trinitrophenol (TNP).

9.2.2 Preparation of DNP- or TNP-conjugated erythrocytes

There are three methods of sensitizing the indicator erythrocytes (e.g. sheep or horse RBC):

1 Chemical. TNP coupled directly to the erythrocytesaa more gentle reaction than dinitro- phenylation, and TNP cross-reacts strongly with DNP.

9.2CUNNINGHAM PLAQUE ASSAY

2 Dinitrophenylated, non-complement fixing antibodies. As chicken antibodies do not fix mam- malian complement, DNP–chicken anti-erythrocyte antibodies may be used to sensitize indicator cells at subagglutinating doses. Using this assay method for DNP plaques, fowl γ -globulin (FγG) cannot then be used as a carrier molecule.

3 Dinitrophenylated fragments of mammalian anti-erythrocyte antibodies. Addition of whole DNP– rabbit IgG anti-SRBC to the SRBC indicator cells would cause haemolysis, so the dinitrophenylated non-complement-fixing Fab fragment must be used to sensitize erythrocytes. DNP–Fab will still bind to the erythrocytes but cannot induce agglutination or fix complement.

9.2.3 Trinitrophenylation of erythrocytes

MATERIALS AND EQUIPMENT Erythrocytes, horse or sheep 2,4,6-trinitrobenzene sulphonic acid (TNP) Glycyl glycine Phosphate-buffered saline (PBS)

METHOD

1 Prepare a phosphate buffer solution by dissolving 5.62 g sodium dihydrogen phosphate dihydrate and 16.19 g disodium hydrogen phosphate in 1 l water. This solution is pH 7.2 and isotonic with sheep erythrocytes (SRBC) (289 mosmol) and so causes less SRBC lysis than the original method which used 0.28 M cacodylate buffer, pH 6.9.

2 Wash erythrocytes three times with PBS by centrifugation at 300 g for 10 min at room temperature.

3 Resuspend 4 ml of packed cells in 16 ml phosphate buffer, and react with trinitrobenzene sulphonic acid according to the Protocol.

4 Mix TNP solution with cells for 30 min on a magnetic stirrer at room temperature.

5 Add 50 ml of a solution of glycyl glycine (initial concentration of 2 mg/ml) in phosphate buffer to each aliquot to react the free TNP sulphonate.

6 Wash three times in PBS by centrifugation (300 g for 10 min) and store at 4°C. Determine the optimum conditions for sensitization according to Section 9.2.7.

Protocol.

Tube number

TNP in 2 ml buffer (mg)

20% suspension SRBC (ml)

C H A P T E R 9: Lymphocyte function

9.2.4 DNP–Fab′ anti-sheep erythrocyte (SRBC) sensitization

Preparation of Fab anti-SRBC The antiserum prepared in Section 3.11 is suitable for this purpose. This method described works

well but it is not the only route that may be used to prepare Fab or Fab′. MATERIALS AND EQUIPMENT

Rabbit anti-sheep erythrocyte (anti-SRBC) hyperimmune serum Other materials and equipment required asee the following sections: Sections 1.3, 1.7, 2.12, 3.11,

4.8, 7.5 and Appendix B.1.2.

METHOD

1 Isolate the IgG fraction of the antiserum by diethylaminoethyl (DEAE)–cellulose ion- exchange chromatography (see Section 1.3).

2 After concentration, dialyse the IgG anti-SRBC against 0.1 M sodium acetate and digest with

pepsin to obtain the F(ab′) 2 fragment (see Sections 1.7 and 2.12).

3 Apply the digest to a Sephacryl S-200 column equilibrated with PBS, recover the F(ab′) 2 peak, concentrate and dialyse against PBS (see Appendix B.1.2). Store a small sample at –20°C for testing later.

4 Reduce the F(ab′) 2 fragments with 0.02 M dithiothreitol for 30 min at 37°C.

5 Alkylate with 0.05 M iodoacetamide for 10 min at room temperature.

6 Dialyse Fab′ mixture overnight against PBS. Reduction and alkylation of the F(ab′) 2 is usually sufficient to prevent haemagglutination and so it is not necessary to fractionate the mixture any further.

7 Dinitrophenylate the Fab′ anti-SRBC, and determine the average number of DNP groups per Fab′ molecule (see Section 4.8).

8 Test each preparation by haemagglutination (see Section 3.11) and haemolysis (see Section 7.5) according to the Protocol (see below).

Protocol. Preparation

number Description

Haemagglutinin titre

Haemolysin titre

1 Rabbit anti-SRBC whole serum

2 IgG anti-SRBC

3 F(ab′) 2 anti-SRBC

4 Fab′ anti-SRBC

5 Fab′ anti-SRBC + goat or sheep anti-rabbit immunoglobulin

6 DNP–Fab′ anti-SRBC

7 DNP–Fab′ anti-SRBC + goat or sheep anti-rabbit immunoglobulin

The haemagglutination and haemolysis test should be carried out after each stage of the pro- cedure, and the procedure continued only if the results of the tests are satisfactory.

9.2CUNNINGHAM PLAQUE ASSAY

9.2.5 Sensitization of indicator cells

MATERIALS AND EQUIPMENT Sheep erythrocytes DNP–Fab′ anti-SRBC Phosphate-buffered saline (PBS)

METHOD

1 Wash SRBC three times in PBS by centrifugation (300 g for 10 min at room temperature) and adjust to a final concentration of 40% v/v.

2 Sensitize aliquots of SRBC according to the Protocol.

Protocol. Aliquot number

DNP–Fab′ anti-SRBC added (µl) (initial conc. 1 mg/ml)

1 5 10 20 40 100 SRBC 40% v/v suspension (ml)

3 Incubate at 37°C for 30 min, mixing occasionally.

4 Wash five times with PBS by centrifugation (300 g for 10 min at room temperature) to remove unbound protein.

5 Adjust cell concentration to 20% v/v and store at 4°C. Determine the optimum conditions for sensitization as described in Section 9.2.7.

TECHNICAL NOTES • Use 2–3-week-old SRBC, but fresh horse RBC. • The sensitized erythrocytes are stable for 1 week at 4°C. It is, however, advisable to wash the

cells each time before use.

9.2.6 Preparation of assay chambers

MATERIALS AND EQUIPMENT Glass microscope slides

Adhesive tape, double-sided Photographic roller

METHOD

1 Wash slides overnight in a strong solution of a free rinsing detergent and rinse thoroughly in distilled water. Soak overnight in absolute ethanol. Air dry the slides. Clean slides are absolutely essential to allow bubble-free filling of the assay chambers.

2 Place 20 slides in a line with their long edges adjacent and stick double-sided tape along each edge and along the centre of the row (see Fig. 9.1).

Continued

C H A P T E R 9: Lymphocyte function

Microscope

Double-

slides (layer 1

sided

with sticky

Fig. 9.1 Double layer of slides held

sticky

tape; layer 2

placed on Slides are then cut apart, giving two

together with double-sided tape.

tape

top of tape culture compartments for each slide

to form chamber)

pair.

3 Remove the backing from the tape and add a second row of slides to complete the sandwich.

4 Roll the slides firmly with a photographic roller to seal the chambers.

5 Separate adjacent slides.

9.2.7 Anti-DNP assay Direct plaque detection

MATERIALS AND EQUIPMENT Dinitrophenyl–fowl g-globulin (DNP–FgG)-, or DNP–keyhole limpet haemocyanin (DNP–KLH)-

primed mice Trinitrophenyl (TNP)-, or dinitrophenyl (DNP)-sensitized sheep erythrocytes (SRBC) Tissue culture medium Fetal bovine serum (FBS) Guinea-pig serum, as complement source (fresh or preserved serum) Assay chambers

50 : 50 mixture of paraffin wax and petroleum jelly on hot plate Micro-titre tray, U-shaped Dropping pipettes (18-gauge needle with end cut square attached to a 1-ml syringe barrel delivers

a constant but approximate 25 µl) or automatic pipette

METHOD

1 Remove the spleens from two or three mice and prepare a single-cell suspension as described in Section 6.3.

2 Adjust to 10 7 lymphocytes/ml.

3 Place 25 µl of medium into each well of the microtitre tray to be used.

4 Add 25 µl of 20% sensitized erythrocyte suspension.

5 Add 100 µl of spleen-cell suspension.

6 Add 25 µl of neat guinea-pig serum absorbed with SRBC (see Section 11.10.2, Technical notes).

7 Mix the suspension in each well of the tray and load into an assay chamber with a Pasteur pipette.

8 When both chambers are full, seal edges by dipping into paraffin wax–petroleum jelly mixture.

9 Incubate at 37°C and examine at 30, 45, 60 and 90 min.

Continued on p. 270

9.2CUNNINGHAM PLAQUE ASSAY

10 Remove all the assay chambers as soon as plaques are clearly visible to the naked eye.

11 Count the number of plaques per chamber using a low-power binocular microscope.

12 Calculate the number of plaques per total spleen for each group, and plot a graph of plaque-forming cells (PFCs) against volume or concentration of sensitizing agent, as shown in Fig. 9.2.

Use this optimum volume or concentration of sensitizing agent in all future assays.

10 5 Fig. 9.2 Relationship between

number of anti-DNP plaques and volume of DNP-Fab′′ anti-SRBC used

Direct plaques per spleen ( 0 to sensitize indicator cells. The

optimal volume for sensitization within Volume of DNP-Fab' anti-SRBC (µl)

the tinted area would be used in routine assays.

TECHNICAL NOTES • It is technically more convenient to remove clumps from the spleen suspensions by settling

them through 1 ml of fetal bovine serum instead of filtering through nylon wool. • It is not necessary to perform replicates of each assay point; the major source of experimental variation is the likely difference between mice receiving the same immunogen or similar num- bers of transferred cells. Immunization relies upon cell proliferation and so the experimental error arises exponentially.

• Use a 37°C incubator without forced air circulation: any vibration will prevent uniform settling of the erythrocytes. • Use a low angle of incidence for the light when counting the plaques under the microscope. The plaques will appear as dark holes in the birefringent layer of erythrocytes. In the assay described above, direct (mainly IgM) plaques were detected; indirect (mainly IgG) plaques may be detected by the addition of a developing serum, e.g. anti-mouse immunoglobu- lin, as described below.

Indirect plaque detection

MATERIALS AND EQUIPMENT Optimally sensitized erythrocytes (see Direct plaque detection above and Fig. 9.2)

Rabbit anti-mouse immunoglobulin Other materials and equipment as given for previous section

C H A P T E R 9: Lymphocyte function

METHOD

1 Dilute the anti-immunoglobulin serum with PBS as shown in the Protocol.

Protocol. Tube number

PBS (ml)

Antiserum (ml)

2 Add 25 µl of each dilution to corresponding wells of the microtitre tray.

3 Repeat the assay as in previous section, but omit the 25 µl of medium from each well of the tray; this has been replaced by the anti-immunoglobulin serum.

4 Determine the dilution of developing serum giving the maximum number of plaques. TECHNICAL NOTES

• The IgG plaques are taken to be the difference between the total number of developed plaques and the number of direct plaques. • Some anti-Ig sera inhibit IgM plaques while developing IgG plaques. If this is found to

be the case in your system, then no correction of the number of developed plaques is required. • Test for the inhibition of direct plaque formation as follows. Determine IgM (direct) plaques

with and without anti-Ig using either: (a) spleen cells from an animal 4 days after antigen prim- ing; or (b) spleen cells from an animal primed with a highly substituted carrier. This will prime for an IgM response but not allow IgG switching.

Although a great deal of time is spent standardizing reagents, application of the assay to an experi- mental situation is extremely rapid.