BASIC PROTOCOL 1 ANTIBODY-CAPTURE BIOASSAY FOR IL-12 ACTIVITY
ALTERNATE PROTOCOL 1 LYMPHOBLAST PROLIFERATION ASSAY FOR IL-12 ACTIVITY
SUPPORT PROTOCOL 2 CONJUGATION OF ANTIBODY WITH HORSERADISH PEROXIDASE
Measurement of Human and Mouse Interleukin-12
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Interleukin 12 (IL-12) is a heterodimeric cytokine that can stimulate both the growth and functional activities of T and natural killer (NK) cells. The IL-12 p75 heterodimer is composed of two disulfide-linked subunits with molecular masses of 40 kDa (p40) and 35 kDa (p35).
This unit describes functional assays for measurement of bioactive IL-12 (Basic Protocol 1 and Alternate Protocol 1) and ELISAs for measurement of IL-12 protein (Basic Protocol 2 and Alternate Protocol 2). The functional assays are based on the ability of IL-12 to stimulate proliferation of PHA-activated T lymphoblasts ("PHA blasts"). Basic Protocol 1 is an antibody-capture bioassay that can be used to quantitate human or mouse IL-12 with specificity in serum or culture fluid containing other cytokines or agents that affect lymphocyte function. In this assay, IL-12 that has been bound to immobilized anti-IL-12 antibody stimulates proliferation of PHA-activated human lymphoblasts. Alternate Protocol 1 is another proliferation assay for mouse and human IL-12 employing PHA blasts. It lacks the specificity of Basic Protocol 1 but is simpler, and can be used where interference from other cytokines is not an issue. Support Protocol 1 describes the preparation of PHA blasts for use in either Basic Protocol 1 or Alternate Protocol 1.
The ELISAs are technically simpler to perform than the functional assays, but cannot distinguish bioactive from inactive cytokine. Basic Protocol 2 is an ELISA that specifically measures human and mouse IL-12 heterodimer. However, the production of IL-12 heterodimer is consistently accompanied by the production of a variable (generally [Image]5-fold) excess of the IL-12 p40 subunit. Alternate Protocol 2 is an ELISA that quantitates total (i.e., both free and heterodimer-associated) IL-12 p40 protein. Support Protocol 2 describes conjugation of antibody to horseradish peroxidase (HRPO) for use in either ELISA protocol.
NOTE: All solutions and equipment coming in contact with cells must be sterile, and proper sterile technique should be used accordingly.
ANTIBODY-CAPTURE BIOASSAY FOR IL-12 ACTIVITY
Human or mouse IL-12 is captured from IL-12-containing culture fluid or serum by rat monoclonal anti-human IL-12 or anti-mouse IL-12 antibody adsorbed to the wells of an EIA (enzyme immunoassay) plate. The test fluid is then washed from the wells and replaced with a PHA-activated human lymphoblast suspension. The lymphoblasts proliferate in response to the captured IL-12, and [3H]thymidine incorporation by the lymphoblasts is measured.
Materials
Coating antibody: rat monoclonal anti-human IL-12 antibody 2-4A1 or rat monoclonal anti-mouse IL-12 antibody 5C3 (both available from R. Chizzonite, Hoffmann-La Roche; see APPENDIX 5) Coating buffer: use recipe in UNIT 7.12 but adjust to pH 9.5 Blocking solution: 1% (w/v) BSA in Dulbeccos phosphate-buffered saline with Ca+2 and Mg+2 (D-PBS; Life Technologies) IL-12 reference standard (see recipe) Supplemented medium (see recipe) Unknown sample containing IL-12 2 x105 cell/ml PHA-activated human lymphoblast suspension (see Support Protocol 1)
Multichannel and repeating pipettors 96-well EIA plates with lids (e.g., Nunc Immuno-plate MaxiSorp) 17 x100-mm polystyrene tubes Additional reagents and equipment for labeling cells and determining [3H]thymidine incorporation (APPENDIX 3D) and for quantitation of interleukin activity (UNITS 6.3 & 6.13)
Coat plate with anti-IL-12 and block
1. Thaw aliquot of coating antibody stock and dilute in coating buffer to 5 µg/ml. Using a multichannel pipettor, immediately add 100 µl diluted coating antibody to each well of a 96-well EIA plate. Cover plate with lid and incubate overnight at room temperature.
Not all anti-IL-12 antibodies capable of capturing IL-12 are suitable for use in this assay. For example, the strongly neutralizing 20C2 antibody is an excellent capture antibody for use in an ELISA (see Basic Protocol 2); however, PHA blasts will not proliferate in response to IL-12 captured by the 20C2 antibody.
2. Flick out well contents. Flood plate with water, then shake out water. Repeat this washing procedure two more times.
3. Add 200 µl blocking solution to each well and incubate 1 hr at 37 °C.
Capture IL-12 on antibody-coated plate
4. While plate is incubating with blocking solution, dilute IL-12 reference standard to 5 ng/ml in a 17 x100-mm tube using supplemented medium. Perform four additional serial 5-fold dilutions in supplemented medium (so that the lowest IL-12 concentration is 8 pg/ml). Retain all dilutions.
Assay of IL-12-containing serum may result in lower-than-expected levels of proliferation if the serum is tested at >2% concentration. When this is the case, a standard dose-response curve should be generated by diluting the IL-12 reference standard in supplemented medium containing normal control serum (from an untreated, control-treated, or disease-free animal or human) at a concentration comparable to the serum concentration in the test sample (i.e., in addition to the 5% human AB serum contained in the supplemented medium).
5. Prepare three to five serial 5-fold dilutions of unknown sample in 17 x 100-mm tubes using supplemented medium, to achieve a range of IL-12 concentrations from 5 ng/ml to 10 pg/ml. Retain all dilutions. On the basis of published information or past experience, it is often possible to estimate a range of concentrations within which the concentration of IL-12 in an unknown sample is likely to fall. If there is no basis for such an estimate, the authors routinely screen unknown samples by carrying out three or four 5-fold serial dilutions, starting with a 1:1 dilution for culture supernatants and a 1:9 dilution for serum samples. If necessary, a second assay can then be performed in which the dilutions are adjusted on the basis of the results of the original screening assay.
6. Following the 1-hr incubation (step 3), flick out well contents and wash antibody-coated EIA plate three times as in step 2.
7. Add 100 µl of each dilution of IL-12 reference standard and unknown sample to triplicate wells of coated EIA plate. Add 100 µl supplemented medium (lacking IL-12) to last three wells of plate. Incubate 2.5 to 3 hr at room temperature.
If more than one EIA plate is included in the assay, each plate should receive a set of IL-12 reference standard dilutions and three wells with medium lacking IL-12. Also, if accurate quantitation of IL-12 in the unknown samples is critical, each unknown sample should be assayed on two separate plates and the titers from the two plates averaged.
Detect captured IL-12 by proliferation of added lymphoblasts
8. Flick out well contents and wash plate five times as in step 2.
9. Using repeating pipettor, add 100 µl PHA-activated human lymphoblast suspension (2 x104 cells) to wells of EIA plate. Incubate 2 days in humidified 37 °C, 5% CO2 incubator.
Human PHA blasts respond well to both human and mouse IL-12. Mouse lymphoblasts have not given satisfactory results in this assay.
10. Label cells with [3H]thymidine (APPENDIX 3D), continuing the incubation in step 3 of that procedure for 6 to 7 hr. Harvest using a semiautomated cell harvesting apparatus and determine amount of [3H]thymidine incorporation using a liquid scintillation counter.
11. Construct a standard curve for [3H]thymidine incorporation versus ng/ml IL-12 (see UNITS 6.3 & 6.13) and calculate the concentration of IL-12 in the unknown samples using this standard curve.
Estimate IL-12 titer by using the unknown sample dilution that results in a [3H]thymidine incorporation value that lies on the steep portion of the standard curve. If two unknown sample dilutions yield values for [3H]thymidine incorporation that lie on the steep portion of the standard curve, average the titers obtained from these two dilutions.
LYMPHOBLAST PROLIFERATION ASSAY FOR IL-12 ACTIVITY
Like Basic Protocol 1, this bioassay measures human or mouse IL-12 as a function of the proliferation of PHA-activated human lymphoblasts. It is simpler to use than Basic Protocol 1 because it does not involve capturing the IL-12 with a coating antibody. This method is therefore not specific for IL-12, and is useful primarily with samples that are known to contain IL-12 as the only cytokine. Because other cytokines--e.g., human IL-2, IL-4, and IL-7--also cause proliferation of human PHA blasts, this assay is not suitable for quantitating IL-12 in fluids containing a mixture of cytokines (unless antibodies capable of neutralizing the activity of other cytokines are added). Activity detected in this assay can be shown to be due to IL-12 by addition of a neutralizing anti-IL-12 antibody such as 20C2 (available upon request from R. Chizzonite, Hoffmann-La Roche).
Additional Materials (also see Basic Protocol 1)
4 x 105 cell/ml PHA-activated human lymphoblast suspension (see Support Protocol 1) 96-well flat-bottom microtiter plates with lids (e.g., Costar)
1. Dilute human IL-12 reference standard to 2 ng/ml or mouse IL-2 reference standard to 4 ng/ml in a 17 x 100-mm tube using supplemented medium. Perform three additional serial 5-fold dilutions in supplemented medium and retain all dilutions.
2. Prepare serial dilutions of unknown sample in 17 x 100-mm tubes using supplemented medium to achieve a range of IL-12 concentrations from 2 ng/ml to 20 pg/ml. Retain all dilutions.
3. Using repeating pipettor, add 50 µl PHA-activated human lymphoblast suspension (2 x 104 cells) to each well of a 96-well flat-bottom microtiter plate.
4. Add 50 µl of each dilution of IL-12 reference standard and unknown sample to triplicate wells of the microtiter plate. Add 50 µl supplemented medium (lacking IL-12) to last three wells of plate. Incubate 48 hr in humidified 37 °C, 5% CO2 incubator.
If more than one plate is included in the assay, each plate should receive a set of IL-12 reference standard dilutions and three wells with medium lacking IL-12.
5. Label and harvest cells, measure [3H]thymidine incorporation, and calculate IL-12 content of unknown samples (see Basic Protocol 1, steps 10 and 11).
SUPPORT PROTOCOL 1
PREPARATION OF PHA-ACTIVATED HUMAN LYMPHOBLASTS
Human peripheral blood mononuclear cells (PBMC) are activated by culture with the mitogen phytohemagglutinin (PHA) for a total of 4 days, during which time the T cells become lymphoblasts. On day 3, the cultures are split and IL-2 is added to promote lymphoblast proliferation. On day 4, the lymphoblasts are harvested, washed, and adjusted to the desired cell concentration.
Materials
Supplemented medium (see recipe)
10 mg/ml phytohemagglutinin-P (PHA-P; Difco Laboratories; reconstitute lyophilized material according to manufacturer's instructions)
Human recombinant IL-2 (Table 6.0.1)
75-cm2 tissue culture flasks
15-ml conical centrifuge tube
Additional reagents and equipment for collecting human peripheral blood cells (APPENDIX 3F), isolating human PBMC (UNIT 7.1), and counting viable cells (APPENDIX 3B)
NOTE: All incubations are performed in a humidified 37 °C, 5% CO2 incubator unless otherwise noted.
1. Collect peripheral blood cells from normal donor (APPENDIX 3F). Isolate PBMC by Ficoll-Hypaque gradient centrifugation (UNIT 7.1), performing the optional procedure to remove all platelets.
2. Suspend PBMC in 5 ml supplemented medium and count viable cells using trypan blue (APPENDIX 3B).
3. Add 1 x 107 PBMC in a total volume of 20 ml supplemented medium to a 75-cm2 culture flask. Add 20 µl of 10 mg/ml PHA and shake flask to mix. Incubate horizontally for 3 days.
If PHA-activated lymphoblasts will be required for IL-12 assays on two successive days, a second culture can be set up as in step 3 but without PHA-P. This is incubated overnight and PHA-P is added on the following day. After the cells have incubated for 3 days following the addition of PHA-P, step 4 is carried out.
4. Add 20 ml supplemented medium, shake flask to mix, and transfer 20 ml of the contents to a clean 75-cm2 flask. Add human recombinant IL-2 to flask to a final concentration of 50 U/ml, then incubate 24 hr.
Lymphoblasts from most donors will be in an optimal state for use in the IL-12 bioassay protocols after this incubation, but in general the optimal length of the incubation should be determined empirically (see Critical Parameters).
5. Transfer lymphoblasts from culture flask to a 15-ml conical centrifuge tube. Pellet cells by spinning 5 min in tabletop centrifuge at 400 xg room temperature, then discard supernatant.
6. Wash cells three times by adding 5 ml supplemented medium and centrifuging as described in step 6 for each wash.
These washes remove residual mitogen and IL-2.
7. Resuspend lymphoblasts in 5 ml supplemented medium and count viable cells using trypan blue (APPENDIX 3B).
8. For each plate to be used in Basic Protocol 1, prepare 12 ml of lymphoblast suspension containing 2 x 105 cells/ml in supplemented medium. For each plate to be used in Alternate Protocol 1, prepare 7 ml of lymphoblast suspension containing 4×105 cells/ml in supplemented medium.
BASIC PROTOCOL 2
ELISA FOR HUMAN AND MOUSE IL-12 p75 HETERODIMER
The assay described in this protocol specifically measures IL-12 heterodimer. This ELISA is simpler to perform than the antibody-capture bioassay described in Basic Protocol 1, and the results correlate well with those of IL-12 bioassays.
Human or mouse IL-12 is captured from IL-12-containing culture fluid or serum by rat monoclonal antibody specific for human or mouse IL-12 heterodimer, after the MAb has been adsorbed to the wells of an EIA plate. The test fluid is then washed from the wells, and captured IL-12 is detected using horseradish peroxidase-conjugated rat MAb specific for the human or mouse IL-12 p40 subunit. Unlike Alternate Protocol 2, this assay does not detect free IL-12 p40 monomer, thereby allowing for the measurement of IL-12 p75 heterodimer in the presence of excess IL-12 p40 subunit.
Materials
ELISA coating antibody (see recipe) appropriate for human or mouse IL-12 heterodimer
Coating buffer: use recipe in UNIT 7.12 but adjust to pH 9.5
Wash buffer (see recipe)
Blocking solution (see recipe)
IL-12 reference standard (see recipe)
Unknown sample containing IL-12
Assay buffer (see recipe)
Rat monoclonal anti-human IL-12 p40 subunit antibody 4D6 (available from R. Chizzonite, Hoffmann-La Roche) or rat monoclonal anti-mouse Il-12 p40 subunit antibody 5C3 (available from D. Presky, Hoffman-La Roche), conjugated with HRPO (see Support Protocol 2)
Chromogenic substrate for detecting HRPO (e.g., K-Blue Substrate, ELISA Technologies), prepared according to manufacturer's instructions Stop solution appropriate for substrate employed (e.g., 1 M H3PO4 for K-Blue Substrate)
Multichannel and repeating pipettors
96-well EIA plates with plate sealers (e.g., Immulon II, Dynatech)
ELISA plate reader (e.g., Vmax, Molecular Dynamics) with appropriate filter for HRPO substrate employed
Coat plate with heterodimer-specific anti-IL-12 antibody and block
1. Thaw aliquot of appropriate ELISA coating antibody stock. If an anti-human IL-12 antibody is being used, dilute to 2.5 µg/ml in coating buffer; if an anti-mouse IL-12 antibody is being used, dilute to 5 µg/ml in coating buffer.
2. Using a multichannel pipettor, immediately add 100 µl diluted coating antibody to each well of a 96-well EIA plate. Seal plate with plate sealer and incubate overnight at 4 °C.
3. Wash plate three times by repeatedly aspirating or flicking out well contents and filling wells with wash buffer.
4. Add 200 µl blocking solution to each well and incubate either 1 hr at 37°C or overnight at 4°C.
Antibody-coated plates can be stored at least 2 weeks at 4°C in blocking solution before use.
Capture IL-12 on antibody-coated plate
5. Dilute IL-12 reference standard to 1 ng/ml in assay buffer. Prepare six additional serial 2-fold dilutions in 1.5-ml microcentrifuge tubes (so that the lowest IL-12 concentration is 15.6 pg/ml). Retain all dilutions.
6. Using 1.5-ml microcentrifuge tubes prepare 2-fold serial dilutions of unknown sample in assay buffer, using a starting dilution that will allow a range of IL-12 concentrations from 1 ng/ml to 0.1 ng/ml. Retain all dilutions.
As noted previously (see Basic Protocol 1, step 5) it is often possible to estimate the likely range of concentrations within which an unknown sample will fall. If this is not possible, a screening assay is performed in which four serial 5-fold dilutions of the unknown sample are tested, starting with a 1:1 dilution for either culture supernatants or serum samples. If necessary, a second assay can subsequently be performed in which the dilutions are adjusted on the basis of the results of the original screening assay.
7. Following incubation with blocking solution, aspirate or flick out well contents from antibody-coated plate, then wash plate three times as in step 3.
8. Add 100 µl of each dilution of IL-12 reference standard and unknown sample to duplicate wells of the coated EIA plate. Add 100 µl assay buffer to at least two wells of the EIA plate for determination of background. Incubate either 2 hr at room temperature with shaking or overnight at 4 °C.
Detect captured IL-12 using HRPO-conjugated antibody
9. Aspirate or flick out well contents from plate, then wash plate five times as in step 3.
10. Dilute HRPO-conjugated 4D6 (anti-human IL-12 p40 subunit) to 250 ng/ml or HRPO-conjugated 5C3 (anti-mouse IL-12 p40 subunit) to 500 ng/ml in assay buffer. Add 100 µl of the diluted antibody to each well and incubate 2 hr at room temperature.
11. Wash plate five times as in step 3, then add 100 µl chromogenic substrate for detecting HRPO to each well. Incubate 10 to 15 min at room temperature.
12. Add 100 µl stop solution to each well, then read plates in ELISA plate reader at appropriate wavelength for HRPO substrate employed.
13. Construct a standard curve (absorbance versus pg/ml IL-12) and calculate the concentration of IL-12 in the unknown samples using this standard curve.
See UNIT 6.14 for calculation of results in cytokine ELISA.
ALTERNATE PROTOCOL 2
ELISA FOR HUMAN AND MOUSE IL-12 p40 SUBUNIT
This assay specifically measures the p40 subunit of IL-12, in both its heterodimer-associated and free forms. It is identical in format to the IL-12 heterodimer ELISA described in Basic Protocol 2; however, a capture (coating) antibody is used that recognizes the IL-12 p40 subunit, whether heterodimer-associated or free, instead of the IL-12 heterodimer-specific coating antibody used in Basic Protocol 2. In combination with the assay described in Basic Protocol 2, this technique can be used to evaluate the presence of free p40 subunit as well as IL-12 p40 homodimer, a known antagonist of IL-12 action. This Alternate Protocol is not well-suited for measurement of IL-12 heterodimer, as it is known that the p40 subunit of IL-12 is generally produced in excess of the p35 subunit, both in vitro and in vivo. As a result, small amounts of IL-12 p40 may be measurable in some samples even though the amount of IL-12 heterodimer is below the limit of detection.
The methodology is identical to that of Basic Protocol 2, except that in step 1 an ELISA coating antibody (see recipe) appropriate for the IL-12 p40 subunit is used. As in Basic Protocol 2, this is diluted in coating buffer to 2.5 µg/ml for anti-human IL-12 or 5 µg/ml for mouse IL-12.
CONJUGATION OF ANTIBODY WITH HORSERADISH PEROXIDASE
In this protocol, horseradish peroxidase (HRPO) is activated by addition of sodium periodate and reacted with the antibody in the presence of sodium borohydride. The keys to this conjugation procedure are: (1) addition of 0.4 M NaHCO3 (pH 10.5) to the dialyzed antibody solution and (2) stabilization of the HRPO-conjugated antibody with the stock buffer.
Materials
Antibody to be conjugated (0.5 to 4 mg/ml stock solution)
50 mM NaHCO3 (pH 8.0; adjust with 10 N NaOH), 4 °C
Horseradish peroxidase (HRPO; purity grade 1, Boehringer-Mannheim)
0.1 M sodium periodate (107 mg NaIO4 dissolved in 5 ml distilled H2O)
0.5 M ethylene glycol (0.15 ml ethylene glycol dissolved in 5 ml of distilled H2O)
5 mM sodium acetate (pH 4.5), 4 °C
0.4 M NaHCO3, pH 10.5 (adjust with 10 N NaOH)
0.1 M sodium borohydride (37 mg NaBH4 dissolved in 10 ml distilled H2O)
0.154 M sodium chloride
Stock buffer (see recipe)
Dialysis membranes (15,000 MWCO, 16-mm diameter and 50,000 MWCO, 22-mmdiameter)
Additional reagents and equipment for dialysis (APPENDIX 3H)
Dialyze antibody and activate HRPO
1. Using a 15,000 MWCO dialysis membrane, dialyze 5.0 mg of antibody to be conjugated overnight at 4 °C against 1 liter of 50 mM NaHCO3, pH 8.0, changing the solution once.
2. Add 5.0 mg HRPO to 1.5 ml distilled water in a 10-ml glass flask. Incubate 30 min at room temperature.
3. Add 0.25 ml of 0.1 M sodium periodate. Incubate 20 min at room temperature.
4. Add 0.25 ml of 0.5 M ethylene glycol to quench sodium periodate. Incubate 10 to 20 min at room temperature.
5. Using a 15,000 MWCO dialysis membrane, dialyze reaction mixture overnight at 4 °C against 1 liter of 5 mM sodium acetate, pH 4.5, changing the solution once.
6. Measure volume of dialyzed activated peroxidase solution. Calculate mg/ml HRPO, assuming no loss from the 5 mg of starting material.
Conjugate HRPO to antibody and stabilize conjugate
7. Transfer 5.0 mg of dialyzed antibody to a 50-ml glass flask. Add 4.0 ml of 0.4 M NaHCO3, pH 10.5 and mix.
Antibody quantities of 0.5 to 5.0 mg have been conjugated using this procedure. The key is to maintain the following ratio of reagents: 1.25 mg antibody: 1 ml of 0.4 M NaHCO3, pH 10.5; 1.0 mg antibody:1.0 mg HRPO, and 10 mg antibody:1 ml of 0.1 M NaBH4.
8. Add the 5.0 mg of activated, dialyzed peroxidase (step 5) to the antibody solution (step 7). Incubate 2 hr at room temperature.
9. Add 0.50 ml of 0.1 M sodium borohydride to the reaction mixture and incubate 4 to 8 hr at 4 °C.
10. Dialyze reaction mixture overnight at 4 °C against 0.154 M sodium chloride using an MWCO 50,000 dialysis membrane.
11. Dilute conjugated protein solution 1:1 with stock buffer. Store at 4 °C in a light-tight container.
The phenol-containing stock buffer serves to stabilize the conjugated antibody. HRPO-conjugated antibodies stored in stock buffer at 4 °C in the dark may be used for [Image]12 months.
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 5.
Assay buffer
1% (w/v) BSA
0.5 M NaCl
0.02 M Na2HPO4
0.05% (v/v) Tween 20
0.01% (w/v) thimerosal
Adjust to pH 6.5 with concentrated HCl
Store up to 4 weeks at 4 °C
Blocking solution
Dulbeccos phosphate-buffered saline (D-PBS) containing:
3% (w/v) BSA
0.01% (w/v) thimerosal
Store up to 4 weeks at 4 °C
ELISA coating antibody
For IL-12 p75 heterodimer assay (Basic Protocol 2): Use rat monoclonal anti-human IL-12 heterodimer antibody 20C2 (available from R. Chizzonite, Hoffmann-La Roche) for human IL-12 or rat monoclonal anti-mouse IL-12 heterodimer antibody 9A5 for mouse IL-12 (available from R. Presky, Hoffmann-La Roche).
For IL-12 p40 subunit assay (Alternate Protocol 2): Use rat monoclonal anti-human IL-12 p40 subunit antibody 2-4A1 (available from R. Chizzonite, Hoffmann-La Roche) for human IL-12 or rat monoclonal anti-mouse IL-12 p40 subunit antibody 5D9 for mouse IL-12 (available from D. Presky, Hoffmann-La Roche).
Store stock solutions frozen in aliquots at -20 °C. See APPENDIX 5 for address and phone number of Hoffmann-La Roche.
Human AB serum
Divide human AB serum (Irvine Scientific) into aliquots in 50-ml Oak Ridge centrifuge tubes and centrifuge 30 min at 27,000 xg (e.g., 15,000 rpm in Sorvall SS-34 rotor), 4 °C. Remove clear serum with pipet, leaving behind the layer of fat that floats to the top during centrifugation. Sterilize through 0.2-µm filter, divide into aliquots, and store indefinitely at -20 °C until use.
Human AB serum used in these assays should not be heat-inactivated.
IL-12 reference standard
Human and mouse IL-12 are available upon request from M. Gately at Hoffmann-LaRoche (see APPENDIX 5). The standard solutions should be divided into aliquots and stored at -20 °C.
IL-12 appears to be stable when frozen and thawed. Activity may decline gradually upon prolonged storage at 4 °C in serum-containing media.
Stock buffer
0.2 M Tris-Cl, pH 7.5 (APPENDIX 2)
1% (w/v) BSA
0.2% (v/v) phenol
0.05% (w/v) thimerosal
Store up to 4 weeks at 4 °C
Supplemented medium
Mix equal volumes of complete RPMI medium and complete DMEM medium (both without serum; APPENDIX 2), but include the following ingredients: 10 mM HEPES, 0.006% (w/v) L-arginine monohydrochloride, 0.1% (w/v) dextrose, and 5% (v/v) human AB serum (see recipe, above).
Wash buffer
Dulbeccos phosphate-buffered saline (D-PBS) containing:
0.05% (v/v) Tween 20
0.01% (w/v) thimerosal
Store up to 2 months at room temperature
Background Information
Interleukin 12, originally called natural killer cell stimulatory factor (NKSF; Kobayashi et al., 1989) and cytotoxic lymphocyte maturation factor (CLMF; Stern et al., 1990), is a heterodimeric cytokine composed of two disulfide-bonded subunits with molecular masses of 40 kDa and 35 kDa. The p35 subunit of IL-12 shares amino acid sequence homology with IL-6 and granulocyte colony-stimulating factor (G-CSF; Merberg et al., 1992), whereas the p40 subunit bears homology to the extracellular domain of the IL-6 receptor and to the ciliary neurotrophic growth factor receptor (Gearing et al., 1991; Schoenhaut et al., 1992). These relationships have led to speculation that IL-12 might be analogous to a secreted cytokine/cytokine receptor complex (Gearing et al., 1991). However, membrane-bound forms of IL-12 or of either of its two subunits have not yet been identified.
The cDNAs encoding both human IL-12 (Wolf et al., 1991; Gubler et al., 1991) and mouse IL-12 (Schoenhaut et al., 1992) have been cloned. Based on the predicted amino acid sequences, the human and mouse p40 IL-12 subunits are 70% identical, whereas the human and mouse p35 subunits display 60% amino acid sequence identity. Mouse IL-12 is biologically active on activated human T and natural killer (NK) cells, but activated mouse T and NK cells do not respond to human IL-12 (Schoenhaut et al., 1992).
Studies performed in vitro have shown that IL-12 can exert a number of effects on T and NK cells. IL-12 promotes differentiation of naive T helper (TH) cells into type 1 TH (TH1) cells that secrete interferon and IL-2 and promote cell-mediated immunity (Manetti et al., 1993; Hsieh et al., 1993). In addition, IL-12 can serve as a costimulus for activating already differentiated TH1 cells to proliferate and secrete IFN (Murphy et al., 1994) and can alter cytokine production by antigen-stimulated memory T cells, resulting in increased secretion of type 1 cytokines and decreased secretion of type 2 cytokines (Marshall et al., 1995). IL-12 can enhance the lytic activity of nonspecific NK/lymphokine-activated killer (LAK) cells (Kobayashi et al., 1989; Stern et al., 1990) and facilitate specific cytotoxic T lymphocyte (CTL) responses (Gately et al., 1992). Both activated T and NK cells proliferate in response to IL-12 (Gately et al., 1991). However, unlike IL-2, IL-12 causes only minimal proliferation of resting peripheral blood mononuclear cells (PBMC) or isolated NK cells (Gately et al., 1991; Naume et al., 1992; Robertson et al., 1992).
PBMC activated by culturing with a variety of mitogens, anti-CD3 antibody, or IL-2 acquire the ability to proliferate in response to IL-12, correlating with the up-regulation of IL-12-receptor expression (Desai et al., 1992). Cloned, IL-2-dependent lines of both CD8+ CTL and CD4+ TH cells have been found to proliferate in response to IL-12 (Gately et al., 1991). IL-12 has been shown to induce the secretion of IFN from both resting and activated T and NK cells (Kobayashi et al., 1989; Chan et al., 1991) and the secretion of tumor necrosis factor (TNF-[Image]) from isolated NK cells (Naume et al., 1992). IL-12 has also been shown to enhance the expression of a number of surface antigens and receptors on isolated NK cells (Robertson et al., 1992; Naume et al., 1992).
IL-12 was shown to be capable of selectively inhibiting IgE synthesis by both IFN-[Image]-dependent and IFN-[Image]-independent mechanisms (Kiniwa et al., 1992). It is not known whether the latter mechanism is mediated via a direct interaction of IL-12 with IgE-secreting B cells or indirectly via some other cell type. Recently, IL-12 was reported to enhance proliferation and secretion of IgM, IgG, and IgA antibodies by activated human peripheral blood B cells (Jelinek and Braaten, 1995).
IL-12 was originally isolated from the supernatant fluids of cultures of activated human B lymphoblastoid cell lines (Kobayashi et al., 1989; Stern et al., 1990). Monocytes and macrophages appear to be the primary normal cell source of IL-12 (D'Andrea et al., 1992); however, IL-12 can also be produced by other cell types, including neutrophils, keratinocytes, and dendritic cells (Trinchieri, 1995). Production of IL-12 both in vitro (Stern et al., 1990; D'Andrea et al., 1992) and in vivo (Heinzel et al., 1994) has invariably been accompanied by production of excess p40 subunit. Preliminary studies have indicated that this excess IL-12 p40 is mostly p40 monomer but also contains a small amount of p40 homodimer (M. Gately and R. Warrier, unpub. observ.). Both human IL-12 p40 homodimer (Ling et al., 1995) and mouse IL-12 p40 homodimer (Gillessen et al., 1995) have been shown to act as IL-12 antagonists.
The abilities of IL-12 to induce IFN-[Image] secretion, to activate LAK cells, and to induce proliferation of activated T lymphoblasts have all been used as the basis of assays for quantitating IL-12 bioactivity. In the authors' experience, bioassays based on the ability of IL-12 to induce proliferation of activated T lymphoblasts are technically the simplest, yet yield reproducible, quantitative results. Although the simple lymphoblast proliferation assay described in Alternate Protocol 1 suffers from a lack of specificity, the antibody-capture bioassay described in Basic Protocol 1 overcomes this problem by measuring lymphoblast proliferation in response to IL-12 specifically bound to an immobilized anti-IL-12 monoclonal antibody. The use of a human T leukemia cell line, Kit225/K6, has been reported as an alternative to the use of PHA-activated lymphoblasts in the antibody-capture bioassay for measurement of human IL-12 (Zhang et al., 1994). However, this cell line has proven difficult to work with because of instability in its responsiveness to IL-12; furthermore, it cannot be used to measure mouse IL-12 in the assay. Variants of the antibody-capture bioassay that use IFN production rather than lymphoblast proliferation as the readout have been described for both human IL-12 (D'Andrea et al., 1993) and mouse IL-12 (Skeen and Ziegler, 1995).
Heterodimer-specific ELISAs represent a technically simpler alternative to the antibody-capture bioassay for quantitating human and mouse IL-12. The 9A5 anti-p75 heterodimer MAb used as the capture antibody in the heterodimer-specific mouse IL-12 ELISA does not react with either mouse IL-12 p40 monomer or mouse p40 homodimer (D. Presky and V. Wilkinson, unpub. observ.). On the other hand, the 20C2 anti-p75 heterodimer MAb used as the capture antibody in the heterodimer-specific human IL-12 ELISA is nonreactive with human p40 monomer but displays weak cross-reactivity with the human IL-12 p40 homodimer (M. Gately and P. Ling, unpub. observ.). Nonetheless, as noted above, it appears that normal cells produce only very small amounts of IL-12 p40 homodimer. In analyzing many IL-12-containing samples from a variety of sources, the authors have observed good concordance between the results of the heterodimer-specific ELISA and the antibody-capture bioassay for both human and mouse IL-12.
The antibody-capture bioassay is ~5- to 10-fold more sensitive than the heterodimer-specific ELISA, and possesses the additional advantage of measuring only biologically active IL-12. Because IL-12 p40 is usually produced in substantial excess (up to 100-fold) of the heterodimer, use of the IL-12 p40-specific ELISA may reveal the presence of measurable IL-12 p40 in samples containing heterodimer in amounts below those detectable in either the heterodimer-specific ELISA or the antibody-capture bioassay. Whether IL-12 p40 can be produced in the absence of IL-12 heterodimer is unknown; however, it has been reported that expression of IL-12 p40 and p35 mRNAs were largely localized to different areas of the spleen in mice in vivo (Bette et al., 1994). Production of bioactive IL-12 heterodimer requires expression of both IL-12 p40 mRNA and p35 mRNA within the same cell (Wolf et al., 1991; Gubler et al., 1991). In screening unknown samples for the presence of IL-12, the authors routinely run the p40-specific ELISA in combination with the antibody-capture bioassay or the heterodimer-specific ELISA. The ratio of total p40 to heterodimer can vary widely from sample to sample; hence, use of an IL-12 p40-specific ELISA alone is not adequate for estimating the IL-12 content of unknown samples.
Assays of IL-12 activity should always contain an IL-12 reference standard to which unknown samples can be compared. An international unit of IL-12 activity has not yet been defined. In the existing literature on IL-12, various investigators have defined arbitrary units of IL-12 activity that are not equivalent, and this should be taken into account when comparing results from different laboratories. Definition of an international unit of IL-12 activity would be useful.
Critical Parameters
Functional assays
In contrast to the bioassays for IL-1 (UNIT 6.2), IL-2 and IL-4 (UNIT 6.3), IL-3 (UNIT 6.4), and IL-6 (UNIT 6.6)--all of which use cloned cell lines as the responder population--an IL-12-responsive cell line suitable for routine use in a bioassay to quantitate human IL-12 activity has not yet been identified. This has necessitated the use of human PBMC (activated with PHA to become PHA blasts), for which considerable individual-to-individual variation in results may be observed. It is critical that human PHA blasts be used at a time when they will proliferate well in response to IL-12 but display minimal proliferation in the absence of added cytokine. For most individuals, PHA blasts prepared as described in Support Protocol 1 are best used on day 4 after PHA activation. Spontaneous proliferation in the absence of added cytokine is usually too high if PHA blasts are used in the assay on day 3. PHA blasts on day 5 after PHA activation are often usable and sometimes optimal. However, for some donors, IL-12 responsiveness of lymphoblasts may decrease dramatically on day 5.
If IL-12 assays are to be performed routinely, it is recommended that PHA blasts be screened from a panel of normal donors for responsiveness to IL-12 in the proliferation assay both on day 4 and on day 5 after PHA activation. Donors whose PHA blasts give a good signal-to-background ratio (i.e., the ratio of maximum proliferation in the presence of IL-12 to proliferation observed in the absence of added cytokine) in the assay may then be used repetitively to obtain reliable results. The identification or engineering of a stable, IL-12-responsive cell line with good growth characteristics could eliminate this problematic aspect of the IL-12 bioassay. A mouse T cell line, CT.4S, responsive to mouse IL-12 has recently been identified (UNIT 6.3; Xu et al., 1995). Whether this cell line can be used in an antibody-capture bioassay for mouse IL-12 is unknown at present. Because of the species specificity of human IL-12, this mouse cell line would not be expected to respond to human IL-12.
Substantial differences have been observed in the ability of human AB sera obtained from various suppliers to support the generation of large numbers of viable human PHA-activated lymphoblasts. However, human AB serum from Irvine Scientific consistently gives good results. It may be possible to substitute fetal bovine serum for human AB serum in this assay. If any such changes in the composition of the culture medium are made, the effects of such changes on the kinetics of development of IL-12 responsiveness and the decline of cytokine-independent proliferation should be examined so that human PHA blasts are used in the assay at a time when an optimal signal-to-background ratio results.
As in any cytokine bioassay, the specificity of the IL-12 assay is critical. It should be confirmed that activity detected in an unknown sample is actually mediated by IL-12 by demonstrating that addition of a neutralizing anti-IL-12 antibody to the bioassay inhibits cytokine-induced proliferation (Chizzonite et al., 1991).
ELISAs
The sensitivity of the ELISAs is dependent on achieving a low assay background. Therefore, adequate blocking of nonspecific protein binding to the EIA plates must be performed using 3% BSA. In addition, the authors have found that different lots of HRPO-conjugated antibody can result in different assay sensitivities. Therefore, it is important to evaluate each lot of HRPO-conjugated antibody that is produced in Support Protocol 2 for use as the detection reagent in Basic or Alternate Protocol 2. When conjugating antibodies with HRPO, care should be taken to maintain the correct ratios of reagents during the reactions (see Support Protocol 2, annotation to step 7). It is also important that HRPO-conjugated antibodies be stabilized with stock buffer containing 0.2% phenol and stored at 4 °C in the dark.
Troubleshooting
The problems most often encountered with the ELISAs involve plate spotting (i.e., random wells with high absorbance), high assay backgrounds, and insufficient assay sensitivity. Both plate spotting and high assay backgrounds can be indications that the antibody-coated plates have been insufficiently blocked with the 3% BSA blocking solution or that they have been stored too long before use. Freshly coated and blocked EIA plates should be prepared in the event of these problems, and this will usually result in an improvement. High assay backgrounds and insufficient assay sensitivity can be caused by problems with the HRPO-conjugated antibody detection reagent. Sometimes, such problems can be corrected either by centrifuging the stock HRPO-conjugated antibody at high speed (100,000 xg for 30 min) prior to diluting it in assay buffer, or by filtering the diluted HRPO-conjugated antibody through a 0.2-µm filter. Individual lots of HRPO-conjugated antibody should be compared to identify the best lot for use as detection reagent.
Anticipated Results
Results of a representative antibody-capture bioassay are illustrated in Figure 6.16.1. Half-maximum activity is generally observed at IL-12 concentrations of 100 to 400 pg/ml, and maximum proliferation is induced by [Image]1 to 5 ng/ml IL-12. [3H]thymidine incorporation in the absence of added cytokines generally varies between 1000 and 20,000 cpm, and the signal-to-background ratio (the ratio of maximum proliferation in the presence of IL-12 to proliferation observed in the absence of added cytokine) is usually 2.5 to 10.
Representative standard curves for the ELISAs are shown in Figure 6.16.2. The sensitivity of the ELISA technique is ~40 to 100 pg/ml IL-12. In the p40-specific ELISA (Alternate Protocol 2), values derived from standard curves such as those shown in Figure 6.16.2, in which IL-12 heterodimer was used as the standard, should be multiplied by the fraction 40/75 to estimate the total p40 protein (both free and heterodimer-associated). This represents the approximate fraction of the heterodimer that is p40 protein. Such estimates should be regarded as only general approximations of the amount of p40 protein in the unknown sample, because the IL-12 p40-specific ELISA may detect p40 monomer, p40 homodimer, and heterodimer-associated p40 with different sensitivities.
Time Considerations
Functional assays. To prepare PHA blasts for the functional assays as in Support Protocol 1, cultures of PBMC in PHA must be set up 4 or 5 days prior to setup of the IL-12 assay. Isolation of human PBMC and culture setup for Support Protocol 1 require 1 to 1.5 hr. Depending upon the number of samples to be assayed for IL-12, setup of the simple lymphoblast proliferation assay (Alternate Protocol 1) requires 1 to 2 hr. Setup of the antibody-capture bioassay requires ~[Image] day. Thus, the entire assay from setup of the PMBC cultures in PHA to harvesting of the assay requires 6 to 7 days to complete. The amount of time during this interval that is actually spent performing the assay is ~7 to 8 hr for Basic Protocol 1 or ~3 to 4 hr for Alternate Protocol 1.
ELISA. Conjugation of antibodies with HRPO (Support Protocol 2) requires 3 to 4 days, much of which is involved in overnight dialysis of the reaction mixtures. EIA plates must be coated with antibody at least 1 day before use, but antibody-coated plates can be stored at least 2 weeks at 4 °C in blocking solution before use. The ELISAs can take 2 to 4 days to perform, depending on the length of time the samples are incubated in the EIA plate (2 hr at room temperature or overnight at 4 °C). Of this 2 to 4 day period, ~6 hr of hands-on time is required.