Hapten sandwich labelling

4.8 Hapten sandwich labelling

Two monoclonal antibodies from the same species, e.g. two mouse antibodies, can be used in the same binding reaction as direct conjugates, e.g. with one labelled with fluorescein and the other with rhodamine. Frequently direct fluoresence is too insensitive and the indirect technique is necessary. This presents difficulties since both mouse antibodies would look alike to the sec- ondary (mouse-revealing) reagent. However, indirect fluorescence can be achieved by modifying each monoclonal antibody to make it antigenically distinct.

If two monoclonal antibodies are derivatized, one with dinitrophenol (DNP) and the other with penicilloyl (Pen), they may be detected independently using, for example, fluorescein- conjugated anti-DNP and rhodamine- or phycoerythrin-conjugated anti-Pen.

To obtain reliable results from this system, considerable time and care is required for stand- ardization; however, the method can yield considerable amplification in systems where the epitopes to be detected by the first antibodies are at low density. These techniques are also useful for coupling haptens to carrier molecules for use in experiments on the immune response.

Entirely synthetic antigens and chemically modified proteins have been used to investigate the nature of the lymphocyte surface receptor for antigen and to uncover the intricacies of T- and B-lymphocyte cooperation.

The techniques described in this section have had wide application in immunology.

4.8.1 Dinitrophenylation of immunoglobulin or protein antigen

A hapten is a small molecule that will bind to B cells or preformed antibody. One of the potential problems is that a hapten alone is too small to cross-link lymphocyte cell-surface receptors and so will not stimulate B-cell differentiation to plasma cells and antibody production. The B-cell response to antigen, in the majority of cases, requires cooperation by T cells. Again because of its

C H A P T E R 4: Antibodies as probes

Fig. 4.5 Chemicals used for dinitrophenyl derivatization of

F SO 3 .Na protein carriers. 2,4-Dinitro-1-

fluorobenzene (DNFB) is much more

NO 2 NO 2

highly reactive than dinitrobenzene sulphonate (DNBS) and is used when rapid and higher molar substitution

NO 2 NO 2 ratios are required. Both chemicals are

2,4-dinitrobenzenesulphonate.Na extremely potent skin sensitizers so

2,4-dinitro-1-fluorobenzene

(DNBS) avoid personal contamination.

(DNFB)

small size, around 400–800 Da, the hapten cannot stimulate two lymphocytes simultaneously. This may be overcome by hapten conjugated to an immunogenic protein. The T cells will recognize this protein, or carrier molecule, and so cooperate with B cells to produce antihapten antibody (anticarrier antibody is also produced). These defined antigens are powerful tools for investigat- ing cell interactions in the immune response. Probably the most commonly used hapten is dini- trophenyl (DNP) which is conjugated to protein via one of its two reactive forms shown in Fig. 4.5.

Dinitrofluorobenzene is highly reactive with the amino groups of proteins under alkaline conditions where the peptide bond is quite stable. It is used when high substitution ratios are required.

High substitution ratios

MATERIALS AND EQUIPMENT Protein antigen, e.g. keyhole limpet haemocyanin (KLH)

1 M sodium bicarbonate 2,4-dinitrofluorobenzene (DNFB) (Caution: an extremely potent skin-sensitizing agent) Sephadex G-25 column (see Appendix B.1) UV spectrophotometer

METHOD

1 Dissolve 100 mg KLH in 1 M sodium bicarbonate (minimum initial concentration 10–20 mg/ml).

2 Add 0.5 ml DNFB (take care as DNFB is an extremely potent skin-sensitizing agent).

3 Mix vigorously on a magnetic stirring platform for 45 min at 37°C.

4 Separate the DNP–KLH conjugate from the free DNFB on a Sephadex G-25 column (see Appendix B.1).

5 Determine the number of DNP groups per KLH molecule using the conversion: DNP: at 360 nm, absorbance of 1.0 (1-cm cuvette) is equivalent to 0.067 mmol DNP; KLH: at 278 nm, absorbance of 1.0 (1-cm cuvette) is equivalent to 0.00018 mmol KLH. The presence of dinitrophenyl groups on the protein accounts for approximately 40% of the absorbance at 278 nm. This is allowed for in the conversion.

TECHNICAL NOTES • KLH tends to self-associate so it is not possible to assign an accurate molecular weight. The

above calculations assume an average molecular weight of 3 × 10 6 .

• Removal of KLH molecular complexes by ultracentrifugation tends to reduce its immunogenicity.

4.8HAPTEN SANDWICH LABELLING

Low substitution ratios

MATERIALS AND EQUIPMENT Immunoglobulin, e.g. fowl g-globulin (FgG)

0.15 M potassium carbonate Dinitrobenzene sulphonate (DNBS), sodium salt recrystallized Sephadex G-25 column (see Appendix B.1) UV spectrophotometer

METHOD

1 Dissolve 100 mg FgG in 5 ml 0.15 M potassium carbonate.

2 Add 20 mg Na DNBS and mix overnight at 4°C.

3 Prepare a column of Sephadex G-25 and equilibrate against phosphate-buffered saline (PBS) (see Appendix B.1.5).

4 Add the DNP–FgG mixture to the column and pump through, adding more PBS when required.

5 Collect the first visible band to elute from the column. This is the DNP–FgG conjugate. The free DNP is retained at the top of the column.

6 Collect 1.5 times the original sample volume.

7 Dilute DNP–FgG solution 1 : 20 with PBS, and read absorbance in the spectrophotometer at 280 and 360 nm.

Calculation of DNP : Fg G ratio DNP: at 360 nm, absorbance of 1.0 (1-cm cuvette) is equivalent to 0.067 mmol DNP.

FγG: at 280 nm, absorbance of 1.0 (1-cm cuvette) is equivalent to 0.0029 mmol FγG. (The DNP interferes with the absorbance reading at 280 nm. This is allowed for in the conversion factor.)

The chemical and antigenic properties of carrier proteins are often altered after hapten sub- stitution. FγG, for example, is irreversibly denatured and becomes insoluble at ratios greater than DNP 40 FγG. With the method described for dinitrophenylation of FγG you should obtain DNP 3–4 FγG. There is good hapten and carrier priming when mice are immunized with conjugates with these molar ratios. With DNP 15–20 FγG, carrier priming is greatly reduced, whereas with DNP 30–35 FγG, direct (IgM) plaques alone are detected; there is no switching to indirect (IgG) plaque formation in the antibody response to the hapten.

The KLH molecule can accept up to 100 hapten groups before carrier priming is affected.

4.8.2 Penicilloylation of protein

MATERIALS Penicillic acid 95% ethanol Protein solution (5–10 mg/ml)

0.1 M phosphate buffer, pH 7.5 Phosphate-buffered saline (PBS)

C H A P T E R 4: Antibodies as probes

METHOD

1 Prepare a 1 m M solution of penicillic acid in 95% ethanol and add 1 ml to 7 ml of protein solution.

2 Leave to mix overnight at room temperature.

3 Dialyse against five changes of PBS to remove unreacted penicillic acid or by desalting using Sephadex G-25 (see Appendix B.1.4–B.1.5).

Estimation of penicilloyl substitution

MATERIALS AND EQUIPMENT p-(hydroxymercuri) benzoate

0.1 M sodium hydroxide

0.1 M carbonate buffer, pH 7.0 Derivatized protein solution UV spectrophotometer

METHOD

1 Dissolve 0.5 g p-(hydroxymercuri) benzoate in minimum volume of 0.1 M sodium hydroxide.

2 Dilute with 0.1 M carbonate buffer, pH 7.0, to obtain a stock solution of approximately

1.5 × 10 –2 M .

3 Determine the precise concentration of this solution spectrophotometrically (molar extinction coefficient, E 1 cmM , through a 1-cm light path at 232 nm is 1.69 × 10 4 ).

This solution will keep for months if stored in the dark at 4°C. Titration of penicilloyl groups

1 Dilute the stock solution of p-(hydroxymercuri) benzoate with carbonate buffer, to obtain a

2 × 10 –3 m solution.

2 Dilute the derivatized protein solution 1 : 10 with carbonate buffer and add 1.0 ml to a spec- trophotometer cuvette.

3 Read absorbance at 280 nm.

4 Add 0.1 ml of diluted p-(hydroxymercuri) benzoate solution to the same cuvette, mix and leave at room temperature for 10 min. Determine the new absorbance value. The difference in the two spectrophotometer readings is due to the p-(hydroxymercuri) benzoate

reacting with the penicilloyl groups to form a penamaldate derivative which absorbs at 280 nm. The molar extinction coefficient of penamaldate at 280 nm, 1 cm light path, is 2.38 × 10 4 .

Calculation of substitution ratio Protein: the interference of the penicilloyl groups with the estimation of protein absorbance at

280 nm is insignificant. The extinction coefficients given for underivatized proteins in the Appendix may be used without correction.

4.8HAPTEN SANDWICH LABELLING

Penicilloyl: at 280 nm, absorbance of 1.0 (1-cm cuvette) is equivalent to 0.0526 m M penamaldate. (An average molar extinction coefficient of 1.9 × 10 3 has been used for this calculation as the relationship between an increase in the molar substitution ratio and absorbance is not linear.)

TECHNICAL NOTES • The p-(hydroxymercuri) benzoate stock solution may form a slight precipitate when carbonate

buffer is added to the original solution in sodium hydroxide. This should be removed by centrifugation.

• Remember to allow for both the original dilution of the derivatized protein solution and the dilution due to benzoate addition when calculating the final penamaldate absorbance value. • The rate of substitution varies with pH and protein concentration. At pH 11.0 and a 30–50 molar excess of penicillic acid with respect to the free amino groups on the protein, there is an almost quantitative substitution of the protein lysyl groups.

4.8.3 Chemical derivatization of first antibodies

MATERIALS Monoclonal antibodies Materials for DNP labelling (see Section 4.8.1) Materials for Pen labelling (see Section 4.8.2) Sephadex G-25 mini-columns (packed in Pasteur pipettes) Mini-concentrator for multiple samples

METHOD

1 Isolate the antibody protein either by ammonium sulphate precipitation (see Section 1.1.2), or by protein A affinity chromatography (see Section 1.4.2).

2 Prepare the derivatization mixture for DNP or Pen (as above) labelling using 20 mg antibody protein in a total volume of 2 ml, and the other chemicals pro rata.

3 Remove 0.2 ml of sample at each of the following time points: 0, 0.25, 0.5, 1.0, 1.5 and

2.0 h and re-isolate the antibody by chromatography through a Sephadex G-25 column. (Use a fresh equilibrated column for each sample.)

4 At the end of the time course determine the molar substitution ratios for DNP or Pen as above.

5 Plot a graph of molar substitution ratio against time, as in Fig. 4.6.

The hapten-conjugated antibodies tend to work best at a substitution ratio of between 15 and

25 mol/mol. Having determined the time and conditions for optimum substitution, repeat the derivatizations for a single time point using sufficient antibody protein for the intended use.

TECHNICAL NOTES • These chemically modified antibodies are very stable if stored at –20°C in the dark. • The rate of reaction is temperature dependent. If greater control over derivatization is required,

perform the reaction at 4°C. It will take about 16 h to obtain the maximum values shown in Fig. 4.6.

• If the antibodies are against pure antigens, it is possible to carry out the derivatization reactions while the antibody is bound to an affinity column, thus protecting the binding site. Subsequent

C H A P T E R 4: Antibodies as probes

DNP substitution Penicilloyl substitution

Room temp.

10 Room temp.

Hapten: protein ratio (mol/mol)

Hapten: protein ratio (mol/mol)

0 0 0 1 2 0 1 2 Time (h)

Time (h)

Fig. 4.6 Time course of dinitrophenyl and penicilloyl substitution of mouse IgG 1 monoclonal

antibodies. Each time point shows the hapten : protein molar ratio determined for derivatization reactions carried out at room temperature or 37°C.

washing of the column with buffer prior to elution of the bound antibody eliminates the need for dialysis to remove unreacted hapten.

4.8.4 Preparation of anti-DNP and anti-Pen fluorescent conjugates

MATERIALS Two cheap proteins, available in pure form; for example, ovalbumin and bovine serum albumin

(BSA) Materials for DNP and Pen labelling (see Sections 4.8.1 and 4.8.2) Materials for fluorochrome conjugation (see Section 4.2)

METHOD

1 Prepare two hapten-carrier conjugates; for example, DNP–ovalbumin and Pen–BSA.

2 Immunize rabbits, or larger animals, with the conjugates independently, to obtain good antisera.

3 Isolate the IgG fraction of each antiserum (see Section 1.3) and conjugate one with fluorescein isothiocyanate (FITC), and the other with rhodamine isothiocyanate (RITC) or phycoerythrin (PE).

4.8.5 Determination of optimum reaction conditions

MATERIALS DNP- and Pen-labelled monoclonal antibodies (see Section 4.8.3) Fluorochrome-labelled anti-DNP and anti-Pen (see Section 4.8.4) Target cells or tissues carrying antigens of interest

4.8HAPTEN SANDWICH LABELLING

It is necessary to determine the optimum reaction conditions for the most intense specific stain- ing and the lowest non-specific background. The major variables will be: (a) molar substitution ratio of hapten : antibody; (b) concentration of antibody protein; and (c) dilution of fluorescent conjugates. In practice, a molar substitution ratio of 20 : 1 (hapten : antibody protein) and a dilu- tion of 1 : 20–1 : 40 of fluorescent conjugate has been found to give generally acceptable results. More precise refinements can be introduced as experience with the system increases. The concen- tration of first antibody will vary widely, both with the quality of the monoclonal and with the epitopes to be detected.

Carry out standardization as described in Section 4.2.6 according to the Protocol below. Remember to use a concentration range of haptenated antibodies to obtain optimum results.

Protocol. Tube number

Cells carrying antigens A and B

+ + + DNP20 anti-A monoclonal

– – + Pen20 anti-B monoclonal

– – + FITC anti-DNP (1 : 30 dil.)

+ – + RITC (or PE) anti-Pen (1 : 30 dil.)

– + + FITC, fluorescein isothiocyanate; RITC, rhodamine isothiocyanate; PE, phycoerythrin.

TECHNICAL NOTES

• As a guide, use 5–50 µg DNP or Pen antibody protein as an initial concentration range. • Tubes 3–6 are specificity controls; 3 and 4 to ensure that the haptens and conjugates do not

cross-react, 5 and 6 to ensure that the fluorescent conjugates do not react directly with the tar- get cells.

• Tube 7 contains the full set of reactants ad the first antibodies as a mixture, but then wash thoroughly before adding the fluorescent conjugates. Remember to increase the volume of reactants to maintain the same protein concentration throughout. An increase in the protein concentration (as opposed to total protein) in tube 7 could give a higher non-specific back- ground than with tubes 1–6. This method can work well when sensitive double labelling is required either for microscopy or

flow cytometry. The same general approach can be used with other detection systems, e.g. enzyme immunoconjugates.