Preparation of fluorochrome-conjugated antisera

4.2 Preparation of fluorochrome-conjugated antisera

Although a wide diversity of good quality conjugates is commercially available, the techniques in the following sections will be invaluable if you have to prepare and standardize your own con- jugates for specialist applications.

4.2.1 Fluorescein conjugation technique

MATERIALS AND EQUIPMENT Antiserum (monoclonal or polyclonal)

Saturated ammonium sulphate, pH 7.2

0.25 M carbonate buffer, pH 9.0 Sephadex G-25 column

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

UV spectrophotometer Fluorescein isothiocyanate Phosphate-buffered saline (PBS)

METHOD

1 Precipitate the antiserum with 40% saturated ammonium sulphate.

2 Dialyse the g-globulin fraction of the antiserum against 0.25 M carbonate buffer, pH 9.0, using a Sephadex G-25 column (see Appendix B.1.4–B.1.5).

3 Determine the protein concentration of the solution and adjust to 20 mg/ml (see Appendix B.5.1–B.5.3).

4 Add 0.05 mg fluorescein isothiocyanate per mg of total protein.

5 Mix overnight at 4°C.

6 Separate the conjugated protein from the free fluorochrome by passing the mixture down a Sephadex G-25 column equilibrated with PBS.

Conjugation with tetramethylrhodamine isothiocyanate is done under the same conditions but it is necessary to separate rhodamine-conjugated antisera from free rhodamine on a DEAE (diethylaminoethyl) ion-exchange column (see Section 1.3 and Appendix B.1).

4.2.2 Calculation of fluorochrome : protein ratio

This should be done routinely every time a new conjugate is made. The presence of the fluorochrome interferes with the absorbance of the protein at 280 nm; this is allowed for in the formula.

2.87 × abs 495 nm

Fluorescein : protein ratio =

abs

− × 2 80 nm . 0 35 abs 495 nm

Unless you use crystalline rhodamine for conjugation, which we do not recommend, it is not possible to make the same correction when calculating the rhodamine : protein ratio.

abs

Rhodamine : protein ratio =

If you intend to use the conjugate to stain fixed material the fluorochrome : protein ratio should

be low (2 : 1); however, antisera used to stain viable cells, where the specific and non-specific fluorescence is much weaker, should have a higher conjugation ratio (2–4 : 1).

4.2.3 Conjugation with phycoerythrin

Phycoerythrin is one of a family of phycobiliproteins which are crucial to the light-harvesting apparatus of blue-green algae, red algae and the cryptomonads. It contains multiple bilin chro- mophores, so can absorb efficiently over a relatively wide spectral range and emits with a high quantum yield of fluorescence. It can be conjugated to immunoglobulin and protein A using the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) (see Section 4.6.3).

4.2PREPARATION OF FLUOROCHROME-CONJUGATED ANTISERA

Comparison of the useful absorption and emission wavelengths of fluorescein and phycoery- thrin illustrates the advantages of this dye combination, particularly for flow cytometry:

Wavelengths shown in nm.

The two dyes may be excited at the same wavelength and their emissions resolved by the appro- priate combination of long- and short-pass filters.

Phycoerythrin may be conjugated to immunoglobulin and protein A using the heterobifunc- tional cross-linking agent N-succinimidyl-3-(2-pyridylthio) propionate (SPDP). Although it is commercially available in crystalline form, the chemistry of derivatization and purification is sufficiently arduous to strongly recommend the purchase of commercial conjugates.

4.2.4 Fractionation of fluorochrome-conjugated antisera

The fluorochrome : protein ratio calculated above is only an average determination; some pro- tein molecules will have more fluorochrome and others less.

As each fluorochrome molecule is added to the protein molecule there is a net decrease in charge. Consequently, conjugated antisera may be fractionated according to their substitution ratio by ion-exchange chromatography using an elution gradient of increasing ionic strength.

For fluorescein-conjugated proteins, adsorb the conjugate to a DEAE ion exchanger in 0.005 m phosphate buffer, pH 8.0, and elute with a linear gradient (limit 0.2 m) phosphate buffer, pH 8.0 (see Section 1.3).

4.2.5 Indirect versus direct immunofluorescence

Indirect immunofluorescence using an unlabelled antiserum detected by a second, fluorochrome- conjugated antiserum is much more sensitive than direct immunofluorescence where one antiserum alone is used. The direct technique gives excellent results with an incident light UV microscope or flow cytometric analysis and can save a lot of time. With the indirect technique, binding of a single first antibody can act as a target for up to eight secondary antibodies. This amplification is not always all gain, as the background due to non-specific binding can also increase.

The techniques of fluorochrome conjugation can be used with human, goat, sheep and rabbit antisera. They are, however, unsatisfactory for the conjugation of mouse antisera because of excessive denaturation of the antibody molecules.

Biotinylation offers better prospects for ‘in-house’ labelling of mouse antibodies, especially as the same biotin-derived antibody can then be detected with avidin molecules conjugated with fluorochrome, radioisotope or enzyme labels.

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

Mouse alloantisera and monoclonal antibodies can only be used as direct conjugates when staining mouse lymphocytes, as the anti-immunoglobulin second antibody would react directly with the B-cell receptors for antigen. To overcome this problem, it is advised that you use hapten sandwich labelling, whereby monoclonal antibodies required for single- or double-fluorochrome immunofluorescence may be chemically derived with different haptens and antibody binding visualized using antihapten sera conjugated with different fluorochromes.

4.2.6 Standardization of conjugated antisera

This should be done before attempting to use immunoconjugates in detection systems, even if you are using a commercially prepared conjugate. Determine the titration range over which the antiserum gives a plateau of staining values and then work within this titration range.

Protocol for rabbit anti-mouse Ig indirect immunofluorescent staining Details of the staining technique are found in Section 8.2.1. A similar protocol may be used for

standardization of any immunoconjugate provided the appropriate substrates are used. MATERIALS AND EQUIPMENT

Mouse thymocytes and lymph node cells Unconjugated antiserum Fluorescein-coated second antibody

METHOD

1 Incubate mouse thymocytes and lymph node cells with a series of dilutions of the unconjugated antiserum starting at 1 : 5 (Fig. 4.1).

2 Detect the antibody with fluorescein-labelled goat or sheep anti-rabbit g-globulin (1 mg/ml).

Key:

Lymph-node cells with rabbit anti-mouse Ig

Thymus cells with rabbit anti-mouse Ig

20 Thymus cells with normal rabbit serum

10 Prozone

Plateau

Fig. 4.1 Indirect immunofluorescent staining of mouse thymus and

lymph-node lymphocytes. Lymph- node cells showed no prozone with normal rabbit serum. The binding of

Immunofluorescent lymphocytes (%)

the rabbit antiserum was detected by a

1:40 fluorescein-conjugated goat anti-rabbit

Serum dilution

IgG serum. 4.2PREPARATION OF FLUOROCHROME-CONJUGATED ANTISERA

TECHNICAL NOTE Remember to include a preimmunization bleed or pooled normal serum as control (normal rabbit serum aNRS). If you are using a monoclonal as the first antibody then it is essential to use a monoclonal with an unrelated antigen-binding specificity, but the same isotype, as a negative control.

Evaluation of results Plot a graph of percentage staining for each antiserum dilution as shown in Fig. 4.1. As can be

seen from Fig. 4.1, at low dilutions there is an elevated percentage staining, or prozone, before the plateau. This is probably caused by non-specific sticking of serum proteins at high concentration (cf. staining with NRS). Obviously the best dilution at which to use this antiserum would be between 1 : 10 and 1 : 15. One can be sure that the immunoconjugate is not limiting, but still economize in the use of antiserum.

4.2.7 Specificity of immunofluorescent staining

Antisera, their subfractions and even purified monoclonal antibodies are biological materials and not chemical reagents. Many antibodies do not behave as expected so be aware of the limitation on the use of these conjugates, especially in a sensitive system such as immunofluorescence. We list below some pitfalls of this technique and their correction. For maximum sensitivity you should use an epifluorescent microscope in the dark and ensure that the microscope has the correct filter combinations for the fluorochrome in use.

Everything staining everywhere

It is probable that one of the antisera is recognizing species or cell-surface determinants other than immunoglobulin. If both control and anti-Ig slides show total staining then the lack of specificity is probably due to the conjugate. If only one of the slides shows high staining it is prob- ably one of the unconjugated sera. We suggest that you absorb the offending serum with liver membranes as follows.

Absorbing serum with liver

MATERIALS AND EQUIPMENT Mouse liver Tea-strainer or 63-µm steel sampling sieve Ice-cold tissue culture medium

METHOD

1 Force chopped mouse liver through a tea-strainer or a 63-µm steel sampling sieve into tissue culture medium on ice.

2 Wash the membrane suspension 10–15 times by centrifugation (500 g for 20 min at 4°C)

until the absorbance of the supernatant is below 0.1 (E 1 c 28 m 0 nm ).

3 Mix a volume of the packed cell membranes with an equal volume of the serum to be absorbed.

Continued

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

4 Leave the suspension to mix at 4°C overnight.

5 Spin off the cell membranes (500 g for 20 min at 4°C) and retest the antiserum. TECHNICAL NOTE

If problems of non-specific staining are encountered these can be due either to non-specific adsorp- tion (due to the forces which can cause any two protein molecules to interact) or to inappropriate antibody binding. Inappropriate binding can apply to the first or second (conjugated) antibody but only in the case of a polyclonal serum can this be removed by absorption. Similar absorption of a purified monoclonal antibody might leave only phosphate-buffered saline! Non-specific absorption is more frequently a problem with the fluorochrome-conjugated antibody, not only because it is usually used at higher concentration than the first (these forces of non-specific interaction are con- centration dependent, as determined by the law of mass action) but also because it is more highly charged than a native molecule.

No staining anywhere

Almost certainly you have forgotten to add the conjugate. If you are sure you added the con- jugate, then you have forgotten the positive unconjugated antibody or it does not bind in any case. It is rare for conjugates to become completely inactive during storage. This may also be due to the UV microscope

aa transmitted light microscope is acceptable for stained sections but epi-illumination is essential for cell-surface immunofluorescence. Finally, ensure that the eye-

pieces are of the correct magnification for the lens system; the intensity loss with × 12.5 com- pared with × 6.3 eye-pieces often makes the difference between nothing and superb fluorescence.

Everything staining with bright stars

Your conjugated antiserum is either contaminated with bacteria, or has been frozen or thawed too many times thus producing immune complexes. Ultracentrifuge the conjugate to remove the contamination. Whenever possible, store antisera at 4°C with a preservative (either 0.02% w/v sodium azide or 0.01% merthiolate, final concentration). If it is not desirable to use a preservative, store at –20°C in small aliquots.

Note: Azide is a dangerous chemical—do not discard down the sink.

Negative control serum giving positive staining

Strictly, the negative control serum must be taken from the animal before immunization. Staining indicates either non-specific binding (perhaps due to a high protein concentration, see Technical note above) or the presence of antibodies not elicited by immunization. It is not valid to attempt to absorb out this reactivity. You must purchase or prepare another serum and review the specificity of the whole system with care.

Uptake of exogenous proteins onto cell surfaces

In later sections, cells will be cultured in medium containing a serum supplement. Serum supple- ments can absorb to the surface of cultured cells and as a consequence might give rise to spurious staining reactions. If required, we suggest that you ensure that your conjugates stain specifically by absorbing them in the manner described below.

4.2PREPARATION OF FLUOROCHROME-CONJUGATED ANTISERA

4.2.8 Absorption of antisera with insolubilized antigens

MATERIALS Saturated ammonium sulphate, pH 7.2 Phosphate-buffered saline (PBS) Fetal bovine, human or rat serum (whichever serum supplement is giving problems)

0.14 M sodium chloride (saline) 2.5% v/v glutaraldehyde in aqueous solution (Caution: use protective glasses)

METHOD

1 Precipitate the serum with 50% saturated ammonium sulphate and redissolve the precipitate in a minimum volume of PBS.

2 Dialyse against PBS to remove ammonium sulphate.

3 Measure the protein concentration of the sample and adjust to 20 mg/ml (see Appendix B.5.1–B.5.4).

4 Add glutaraldehyde dropwise to the protein solution while stirring (use 0.5 ml of a 2.5% aqueous solution of glutaraldehyde for each 100 mg of protein to be insolubilized). A gel should form almost immediately.

5 Allow the gel to stand for 3 h at room temperature and then disperse in PBS using a Potter homogenizer.

6 Wash the gel with PBS by centrifugation (500 g for 20 min) until protein cannot be detected in the undiluted supernatant by UV spectroscopy (absorbance less than 0.01, E 1 c 28 m 0 nm ).

7 Mix an equal volume of this immunoadsorbent gel with the anti-Ig serum. Mix at 4°C overnight.

8 Spin off this immunoadsorbent (500 g for 20 min) and store the antiserum at –20°C until used.

It is obviously necessary to ensure specificity of the antiserum under these conditions as one may simply be examining the uptake of proteins from the serum supplement of the tissue culture medium. It is important to use an insoluble immunoabsorbent to avoid the formation of soluble complexes in the absorbed antiserum.