Enumeration of antibody-secreting cells in vitro

9.1 Enumeration of antibody-secreting cells in vitro

Before even contemplating biological assay it is essential to be aware that biological materials do not behave in an exactly similar manner between laboratories and so each system must be standardized for research purposes.

Plasma cells synthesize antibody for secretion and this can be used to quantify the number of antibody-producing cells in an organ. The basic assay developed by Jerne and Nordin can be used to detect cells producing antibody against erythrocyte antigens. Spleen cells from immune mice are incubated in an agar gel with the immunizing erythrocytes. After the addition of comple- ment, the erythrocytes in the locality of the plasma cells are lysed, producing macroscopic holes or plaques in the erythrocyte suspension. This relatively simple and robust assay has undergone several modifications to improve its convenience and application range.

9.1.1 Enumeration of total plasma cells by reverse Jerne plaque assay

This technique is useful for the enumeration of total immunoglobulin-secreting cells. Secreted immunoglobulin is captured by protein A coated onto indicator erythrocytes, which are then lysed by the addition of a developing anti-immunoglobulin serum plus complement.

Preparation in advanceacoating of sheep erythrocytes with protein A

MATERIALS AND EQUIPMENT Sheep red blood cells (SRBC) in Alsever’s solution

0.14 M sodium chloride in distilled water (saline) Protein A (2 mg/ml in saline)

9.1ENUMERATION OF ANTIBODY-SECRETING CELLS IN VITRO

Chromic chloride (0.1 mg/ml in saline) Phosphate-buffered saline (PBS)

METHOD

1 Wash SRBC six times in saline by centrifugation (300 g for 10 min at room temperature).

2 Add 1 ml of protein A solution (2 mg/ml initial concentration) to 1 ml of packed erythrocytes.

3 Add 6 ml of chromic chloride solution (0.1 mg/ml) dropwise. After mixing add 10 ml saline and incubate overnight at 4°C.

4 Wash the erythrocytes three times by centrifugation (300 g for 10 min at room temperature) and resuspend to 10% v/v in PBS.

Coated erythrocytes may be stored at 4°C and should be used within 1 week.

Assay

MATERIALS AND EQUIPMENT Cell suspension for assay, e.g. spleen from immunized mouse Protein A-coated erythrocytes (prepared as above) Phosphate-buffered saline (PBS) Agarose, 1.8% w/v in distilled water Tissue culture medium (three times concentrated) Bovine serum albumin (BSA) 7% w/v in PBS Anti-mouse immunoglobulin, developing serum Guinea-pig serum, as complement source Plastic Petri dishes, 50 mm

METHOD

1 Mix 14 ml agarose solution with 5.5 ml of tissue culture medium, which has been concentrated three times, and add 1.5 ml of BSA solution.

2 Dispense 0.7 ml volumes of agarose mixture into glass test tubes in a water bath at 45°C.

3 For each assay dish add 0.2 ml of lymphoid cell suspension, 0.1 ml of anti-immunoglobulin serum and 0.05 ml of protein A-coated sheep erythrocytes to each tube containing 0.7 ml agarose mixture. Mix thoroughly and pour into a plastic Petri dish. Swirl the dish to ensure an even covering of agarose–cell suspension.

4 Incubate for 1–1.5 h in a humid 37°C incubator gassed with 5% CO 2 in air.

5 Add 1 ml of diluted guinea-pig serum, as a complement source (the dilution must be predetermined empirically) and continue incubation at 37°C until haemolytic plaques are visible (within 30–60 min).

6 Count the number of haemolytic plaques and calculate the number of antibody-secreting cells in the original cell suspension.

TECHNICAL NOTES • The optimal dilution of anti-immunoglobulin serum and complement source must be determined. • Illuminate the assay dish with light at a low angle of incidence to aid visualization of the plaques.

They will appear as uniform dark holes in a light, birefringent layer of erythrocytes.

C H A P T E R 9: Lymphocyte function

• The cell suspension for assay should be used at a concentration to give not more than 100–200 plaques per assay dish.

9.1.2 Enumeration of antigen-specific plasma cells by Jerne plaque assay

The Jerne haemolytic plaque-forming cell technique uses the original immunogen, sheep eryth- rocytes (SRBC), in an antibody–antigen binding reaction to detect antibody-secreting plasma cells. In this case, the addition of complement results in the formation of a lytic halo associated only with SRBC antigen-secreting plasma cells.

Preparation in advance

• Immunize two mice with 2 × 10 8 SRBC given intraperitoneally 5 days before the experiment. • Agar underlay. Make up 1.4% Difco Bacto Agar in Hanks’ saline. Melt the agar in a microwave

oven and then directly over a bunsen flame to get rid of all of the lumps if necessary. Take care to swirl the agar gently to avoid charring. Add enough of the agar solution to a 5-cm plastic Petri dish to just cover its base. An underlay is used to ensure that the base of the assay dish is reasonably flat, so pouring must be done on a levelled surface.

• Agar overlay. Prepare 0.7% agar solution in Hanks’ saline containing 0.5 mg/ml of diethy-

laminoethyl (DEAE)–dextran (final concentration). DEAE–dextran is used to prevent anti- complementary activity of the agar. Alternatively, use the more expensive agarose as in the previous section.

MATERIALS AND EQUIPMENT SRBC-immunized mice 2–4-week-old sheep blood in Alsever’s solution Agar overlay Petri dishes containing agar underlay Hank’s saline, without phenol red indicator dye Water bath, 45°C Guinea-pig serum, as complement source

METHOD

1 Wash SRBC three times in Hank’s saline by centrifugation (300 g for 10 min at room temperature). Adjust to 20% v/v after resuspension in Hank’s saline.

2 Remove spleens from mice and prepare a single-cell suspension by teasing them apart with forceps into ice-cold saline. Discard the fibrous connective tissue that remains.

3 Suck suspension in and out of a Pasteur pipette to disperse the cells; it is not usually necessary to let the cells stand to settle out the small aggregates. (To avoid loss and damage to the plasma cells do not filter cells through nylon wool and do not use a syringe and needle for cell dispersion.)

4 Adjust to a total volume of 2.5 ml in Hank’s saline.

5 Dilute 1 ml of the spleen-cell suspension 1 : 10 and 1 : 100 with Hank’s saline.

6 Pipette out 0.8 ml of overlay into small test tubes in a 45°C water bath. Use a warm pipette.

7 Add 0.25 ml of the original or diluted spleen-cell suspensions to each assay dish according to the Protocol.

Continued on p. 264

9.1ENUMERATION OF ANTIBODY-SECRETING CELLS IN VITRO

Protocol.

Dish number

Spleen-cell suspension (ml)

(initial dilution)

Agar overlay (ml) ⎫

0.80 ml ⎫

SRBC (20%) (ml) ⎬ ⎯⎯⎯⎯⎯⎯→ ⎭

0.15 ml ⎭

Fraction of spleen assayed

8 Place dish on a levelled surface.

9 Add 0.15 ml SRBC suspension to each overlay tube just before use. Mix well by flicking the end of the tube.

10 Add overlay to dish and mix thoroughly with spleen cells.

11 Allow agar to set and add 1.0 ml of a 1 : 10 dilution of guinea-pig serum as a source of complement.

12 Incubate dishes at 37°C for 1–1.5 h. If the plaques are not clear when the dishes are

removed from the incubator, allow them to stand at room temperature for about 30 min before counting.

Plaques can be seen by holding the Petri dish up to the light (without its lid), but counting is eas- ier and more accurate if you use a low-power binocular microscope and draw lines on the bottom of the dish. Only direct plaques (mainly IgM antibody) are detected by the method described above because of the high haemolytic efficiency of this antibody class. In theory, a single molecule of IgM can initiate the complement cascade and cause SRBC lysis. To detect IgG plaques increase the number of molecules binding to any one site (two adjacent molecules of IgG are required to activate the complement sequence via the classical pathway); plaques must therefore

be developed with an antiserum against mouse immunoglobulin. This method of detecting so- called ‘indirect’ plaques can be used to assay each of the mouse IgG subclasses using appropriate subclass-specific antisera.

Indirect plaques

MATERIALS AND EQUIPMENT As preceding method, plus rabbit anti-mouse immunoglobulin (anti-Ig)

METHOD

1 Prepare a plaquing mixture as in the preceding method (steps 1–10) but instead of adding complement, add 0.1 ml of a 1 : 10 dilution of rabbit anti-mouse Ig and incubate at 37°C for 45 min.

2 Wash away the developing antiserum by flooding the plate twice with Hank’s saline.

3 Add l ml of 1 : 10 dilution of guinea-pig serum. Incubate for 45 min at 37°C.

C H A P T E R 9: Lymphocyte function

Plaques may be clearer if the plates are left at room temperature for 30 min before counting. Under experimental conditions it is necessary to titrate the developing antiserum until the maximum number of plaques are obtained. The direct, or ‘IgM’, plaques are then subtracted from the total to give the number of ‘IgG’ plaques. The technique as described can be used to enumerate antibody-producing cells in any species. In the case of chicken antibody-producing cells you must use an homologous serum as a source of complement. Chicken antibody does not fix the first component of mammalian complement (C1q). Alternatively, develop both IgM and IgG plaques using rabbit anti-chicken immunoglobulin class-specific antisera and guinea-pig complement.

TECHNICAL NOTES • If large numbers of SRBC are used for immunization (10 6 –10 9 per mouse) the peak of the direct plaque-forming-cell (PFC) response is 4 days; at lower doses (10 4 –10 5 per mouse) the peak of

the direct response is day 5. However, the numbers of indirect plaques peak at day 5–6 after immunization. Day 5 is usually an acceptable compromise for measuring both the direct and indirect PFC response to SRBC. Other antigens may show different kinetics.

• At doses of SRBC of 10 4 and below, the route of immunization is important: intravenous injec- tion gives more PFCs than intraperitoneal injection. Above 10 5 SRBC per mouse both routes of administration give approximately the same number of plaques. • Use a spleen dilution giving approximately 200–300 plaques per assay plate. As cell density increases plaque size decreases and PFC number is not linear. • Some anti-immunoglobulin developing sera suppress IgM direct plaques while revealing IgG

indirect plaques. Once the optimum dilution for plaque development has been determined, it is necessary to test for suppression of direct plaques by the serum, using PFC response, 3 days after SRBC immunization. The response is low but virtually all are IgM direct plaques.

The haemolytic plaque method can be extended using antigen coupled to erythrocyte-indicator cells. This, of course, allows more widespread application of the technique, especially in the tech- nically more convenient Cunningham modification.