Modification of cell-surface antigens

8.6 Modification of cell-surface antigens

Continuous flow cytometry is used to quantify antibody binding to cell surfaces after various manipulations designed to yield information about the nature and distribution of the target molecule. The technique relies on efficient detection of the maximum number of cell-surface epi- topes at all times. The antibody must be titrated for saturation binding (shown by plateau stain- ing as in Fig. 4.1) with minimum non-specific binding, both before and after each treatment. Few of the treatments are totally devoid of side reactions; for example, enzymes often contain other contaminating enzymes. For maximum reliability, link a degradation experiment with resynthe- sis in the presence of specific inhibitors. Resynthesis experiments are not limited to permanent cell lines grown in vitro; even normal cells are capable of extensive short-term resynthesis of mem- brane components in vitro. The connectivity pathways shown in the flow chart (Fig. 8.9) should give useful and related information; however, not all the assays are necessary for each mono- clonal antibody. The information to be gained from this general approach could be increased by

8.6MODIFICATION OF CELL-SURFACE ANTIGENS

Standardize monoclonal

Proportion positive cells

antibody binding by

and number sites per cell

flow cytometry

8.6.1 Assay

Unknown

Is epitope capped by

Culture for re-expression epitope?

Protein

Treat cells with

proteinases

in presence of inhibitors of protein synthesis

Culture for re-expression associated?

Mixed glycosidase

treatment

in tunicamycin

8.6.3 Assay

8.6.4 Assay

Mild periodate treatment of whole cells or immunoblot

8.6.5 Assay

Same determinant

Quantitate detected by existing

Determine saturation curve

binding of antibody?

for each antibody alone

by flow cytometry

Fluorochrome or hapten

Inhibition of labelling antibody

sandwich labelling of known

binding by unlabelled

Fig. 8.9 Determination of the chemical nature of unknown monoclonal antibody-defined epitope

using flow cytometry. The assay numbers refer to the technique in Section 8.6 for the measurement of antibody binding following the indicated treatment. The applicability of this general approach could be extended, where appropriate, by the inclusion of lipases and glycosidases or inhibition of antibody binding by mono- or oligosaccharides.

extending the range of useful reagents; for example, by inclusion of specific glycosidases, inhibi- tion of antibody binding by mono- or oligosaccharides, lipases, etc.

8.6.1 Binding site distribution

To determine the distribution of antibody-binding sites on the cell population of interest with respect to: (i) proportion of total cells carrying the epitope; and (ii) the relative distribution of

C H A P T E R 8: Lymphocyte structure

Channel number

Channel number

Fig. 8.10 Distribution of fluorescence intensity in lymphocytes stained with monoclonal antibodies.

Four illustrative histograms of flow cytometry data are shown; the horizontal logarithmic scale is divided into intervals of increasing fluorescence intensity, the vertical scale shows the frequency of cells at each level of relative fluorescence.

(a) When cells are reacted with an irrelevant monoclonal antibody, about 95% of the total population sampled (usually 10 000 cells) should fall within the first few channel numbers (to the left of the vertical line) and so define the proportion of lymphocytes that bind the irrelevant monoclonal antibody and fluorescent conjugate (if an indirect staining technique is used) non-specifically.

(b) Lymphocytes in this sample have been stained with a monoclonal antibody that binds to their surface membrane. To analyse the distribution, it is arbitrarily divided into two by placing the cursor (vertical line) in the same position as in histogram (a). Channels to the left of the line contain the ‘non-specifically’ stained population, whereas channels to the right contain ‘specifically’ stained cells, in this case about 50% of the total population sampled (value obtained by integrating the area under the curve). The modal or peak relative fluorescence, indicated by the arrow, is that showing the greatest frequency in any one channel.

(c) and (d) These histograms show different populations of lymphocytes stained with the same monoclonal antibody as in (b). Although approximately the same proportion of lymphocytes are positively stained, the population in (c) is brightly stained (modal fluorescence shifted to the right) whereas the population in (d) is weakly stained (modal fluorescence shifted to the left). If the antibody defined a lymphocyte receptor, then 50% of both populations in (c) and (d) would have this receptor but cells more frequently have receptors in (c) than in (d).

binding sites per cell in the positive population, it is most convenient to use a continuous-flow cytometer. An illustrative example is shown in Fig. 8.10.

8.6.2 Antibody-induced capping

This is a useful technique because: (i) the antibody can induce capping showing that the antigen is mobile in the membrane (or not!), which in turn can be informative; and (ii) it is a gentle and highly selective way to initiate resynthesis of the molecule of interest (e.g. compared to pro- teinase treatment).

8.6MODIFICATION OF CELL-SURFACE ANTIGENS

Table 8.1 Enzymes for modification of cell-surface antigens on viable cells Enzyme

Working concentration

Specificity

Proteinases Pronase

0.5 mg/ml ⎫

Multiple sites of hydrolysis

Papain*

along the polypeptide chain Glycosidases†

0.3 mg/ml ⎭

Neuraminidase

N-terminal sialic acid Endoglycosidase H

0.5 iu/ml

5.0 iu/ml

Linkage between oligosaccharide and protein

* Add also 5 mg/ml cysteine–HCl to activate the enzyme. † Use preparations of mixed glycosidases with caution, lack of modification of staining might be due to an insufficient concentration of a crucial glycosidase. In any case, endoglycosidase H has now largely replaced crude mixtures of glycosidases for this treatment, because the latter is frequently contaminated with proteinases.

Briefly, it involves the incubation of the cell population with antibody at 37°C for varying time periods. If modulation is incomplete or absent with the monoclonal antibody alone, then add an anti-immunoglobulin antibody as well as the monoclonal in a parallel assay. Even so, modulation of surface-antigen expression does not occur with all monoclonal antibodies.

8.6.3 Proteinase- or glycosidase-sensitive epitopes

MATERIALS As in Section 8.2, but in addition: Cells carrying the antigen of interest Proteinases or glycosidases, as Table 8.1 Tissue culture medium Fetal bovine serum (FBS) Phosphate-buffered saline (PBS), containing 10 m M sodium azide

Note: Azide is a dangerous chemicalado not discard down the sink.

METHOD

1 Count cells and determine their viability by dye exclusion. Adjust to 5 × 10 6 /ml in serum- free tissue culture medium. If necessary remove dead cells.

2 For each time point of the assay, mix 1 ml cell suspension with 1 ml enzyme (see Table 8.1), or 1 ml serum-free medium alone.

3 Incubate for 60 min in 37°C water bath with occasional mixing.

4 Add 8 ml ice-cold tissue culture medium containing 10% FBS to stop the reaction.

5 Wash twice by centrifugation (150 g for 10 min at 4°C) and label by indirect immunofluorescence for flow cytometry.

TECHNICAL NOTE Always purchase the enzyme in its purest available form; this limits the number of contaminating enzymes and therefore the number of confusing side reactions.

C H A P T E R 8: Lymphocyte structure

Table 8.2 Inhibitors of protein synthesis, glycosylation and secretion

Working

Inhibitor Stock solution

1.0 mg/ml

1.5 µg/ml

Disrupts RNA.

dihydrochloride in water Forms amino acyl puromycin resulting in premature chain termination

Cyclohexamide

Blocks peptide synthesis by interfering in water

1.0 mg/ml

20 µg/ml

with the ribosomes Tunicamycin

Prevents addition of sugars to hydroxyl in DMSO*

1.0 mg/ml

1.0 µg/ml

and amino groups of polypeptides Monensin

1 mm in water

100 nm

Blocks processing of polypeptides by the Golgi apparatus, thus preventing secretion

* This inhibitor is dissolved in dimethyl sulphoxide (DMSO); add an equal volume of DMSO alone to a parallel cell culture to control for potential non-specific inhibition of resynthesis.

8.6.4 Inhibition of resynthesis

MATERIALS As 8.6.3 above, but in addition: Inhibitors as Table 8.2 Bovine serum albumin (BSA)

METHOD

1 After enzyme or antibody treatment, wash cell suspensions three times by centrifugation (150 g for 10 min at room temperature) in tissue culture medium containing 10% FBS.

2 Determine cell viability by dye exclusion and adjust to 10 6 cells/ml.

3 Prepare separate aliquots of enzyme-treated cells alone, enzyme-treated cells plus appropriate inhibitor (Table 8.2) and, where necessary, enzyme-treated cells plus solvent used to dissolve the enzyme inhibitor.

4 Culture overnight at 37°C in a humidified atmosphere containing 5% CO 2 in air.

5 Harvest the cells and wash twice by centrifugation using PBS containing 1% w/v BSA and

10 m M sodium azide.

6 Label by indirect immunofluorescence for flow cytometry.

8.6.5 Periodate treatment

MATERIALS As 8.6.3 above, but in addition: Sodium metaperiodate Phosphate-buffered saline (PBS)

8.6MODIFICATION OF CELL-SURFACE ANTIGENS

METHOD

1 Dissolve sodium metaperiodate in PBS to 0.1 M , use fresh and keep in the dark.

2 Determine cell viability and adjust to 10 6 cells/ml.

3 For each time point, allow 2 × 1 ml aliquots of cells.

4 Add 10 µl stock periodate solution to 1 aliquot; the other will serve as untreated control.

5 Incubate at 4°C between 30 min and overnight.

6 Recover and wash cells by centrifugation (150 g for 10 min at room temperature) in PBS containing 10 m M sodium azide and 1% w/v BSA.

7 Label by indirect immunofluorescence for flow cytometry. TECHNICAL NOTE

Under the mild conditions described here, cell-surface sialic acid residues are specifically oxidized and cell viability should be virtually unaffected. However, the reaction is relatively inefficient. If you are still in doubt, having obtained a negative result, we recommend that you use low-pH periodate pretreatment of an immunoblot, thus permitting the efficient oxidation of cis-vicindal diol groups.

8.6.6 Double antibody binding

For a proper assessment of additive or competitive binding, both antibodies should be used under saturating (plateau) conditions, and so must have been titrated. Aliquots of cells carrying the antigen of interest should be reacted either with antibody alone or with the two as a 1 : 1 mixture (label by indirect immunofluorescence for flow cytometry). Non-competitive binding of the two antibodies should result in a positive displacement of modal fluorescence intensity (peak staining shifted right). Competitive binding should result in the antibody mixture having a peak intensity equal to the peak of the brighter of the two antibodies alone.

It should be noted that it is desirable to label with each antibody at a consistent dilution, whether it is used alone or used in an antibody mixture, and yet maintain an equivalent protein concentration in each case. This may be achieved by diluting the monoclonal antibodies into an irrelevant protein solution, e.g. 0.1% fetal bovine serum. It is not a valid way of comparing dif- ferent isotypes: IgM and IgG antibodies with precisely the same variable regions would not react with the same binding avidity or permit the same specificity and sensitivity of detection.

8.6.7 Competition with labelled standard

Label a reference monoclonal antibody by direct conjugation to a fluorochrome or by hapten conjugation for hapten sandwich labelling, then carry out a more precise assessment of competit- ive or non-competitive binding using different concentrations of the monoclonal as required for proper immunoassay. Where one is trying to compile a reference set of new monoclonal antibod- ies, it is desirable to avoid reselection of commonly occurring monoclonal antibodies. It is often possible to incorporate recurring antibodies into the primary screen and so favour the detection of less immunodominant clones. For example, a binding assay designed to detect IgG mono- clonals by the use of a labelled anti-IgG Fc reagent could be ‘spiked’ by the addition of the F(ab′) 2 fragment of an already banked monoclonal.

C H A P T E R 8: Lymphocyte structure

TECHNICAL NOTES • The use of a control for non-specific binding (fluorescein-conjugated irrelevant monoclonal

antibody of the same isotype) is crucial throughout all the manipulations described above. Chemical or enzyme treatment of a cell population might increase or decrease non-specific binding in an unpredictable manner.

• Results from these assays must be interpreted with the same caution as any data using these

highly sensitive antibody probes. For example, lack of binding to a test-cell population might mean that the epitope is absent or merely inaccessible. Similarly, enzyme treatment of a cell surface could increase or decrease antibody binding by changing the accessibility of the binding site rather than through a direct effect on the epitope. It is recommended that you optimize the staining protocol used in flow cytometry for the

individual experiments and cells to be investigated.