Sandwich enzyme immunoassay for murine IL-3

SANDWICH

ENZYME IMMUNOASSAY MURINE IL-3

Hermann J. Ziltener,*

Ian Clark-Lewis,

FOR

Stacey L. McDonald

A reproducible, sensitive immunoassay for murine interleukin-3 (IL-3) has been developed using two preparations of polyclonal antipeptide antibodies. Rabbits were immunized with the N-terminal peptide l-29 (IL-3) coupled to KLH and the antibodies were affinity purified on immobilized peptide l-29 (IL-3). This antibody preparation showed good reactivity with native IL-3, and was used to coat polyvinyl microtiter trays. IL-3 captured by this first antibody was detected by the addition of anti-IL-3 serum (second antibody) raised in sheep against synthetic full length IL-3 (l-140). This test reliably detects IL-3 from every source tested (T cells, WEHI-3B cells, recombinant material from transfected COS 7 cells or murine myeloid FDC-Pl cells transfected with an IL-3 containing retrovirus) with a sensitivity to 2 to 4 U/ml of bioactive IL-3 or about 60 pg synthetic IL-3/ml. The test is performed within 5 to 6 b compared to 2 to 3 d of a standard bioassay. o 1989 by W.B. Saunders Company.

Interleukin-3, a lymphokine produced by the activated T cell, acts on a wide variety of hemopoietic cells including pluripotential stem cells, multiple types of progenitor cells, and certain mature cells, e.g. mast cells and macrophages. Despite its effect on cells of every hemopoietic lineage, IL-3 is not thought to be involved in steady state hemopoiesis, but it is seen as a link between the immune system and the hemopoietic system in that immunogenic activation of T cells leads to enhanced defense and repair mechanisms (Schrader et al., 1988). Murine IL-3 has been described, purified, and characterized by many different groups and several detection systems based on IL-3 bioactivity were developed. These include the induction of the enzyme 2Oa hydroxysteroid dehydrogenase (Ihle et al., 1982), histamine release (Dy et al., 1981), colony forming assays (Cutler et al., 1985), or the proliferation of IL-3 dependent cells (Schrader and Nossal, 1980). The specificity, sensitivity, and effort to perform these assays varied considerably and the use of IL-3 dependent cell lines (e.g. R6-X.E4 [Crapper et al., 19841, FDC-Pl [Dexter et al., 19801, 32D [Greenberger et al., 19831)

The Biomedical Research Centre, 2222 Health Sciences Mall, UBC, Vancouver, B.C. Canada V6T lW5. *To whom reprint requests should be addressed. Q 1989 by W.B. Saunders Company. 1043-4666/89/0101-0004$5.00/0 KEY WORDS: Sandwich enzyme immunoassay/antipeptide ies/interleukin-3

antibod-

emerged as the most widely used method for IL-3 detection. Although this method is very sensitive, detecting as little as 0.4 pg IL-3/ml or 0.1 U IL-3 (ClarkLewis et al., 1984), it is labor intensive and time consuming. Furthermore, the cell lines used can be affected by inhibitors present in the sample particularly in serum and other biological fluids. Moreover, there is always the possibility that the response is affected by factors other than IL-3. Many IL-3 dependent lines have given rise to variants that respond to other factors. FDC-Pl will respond to IL-3 and granulocyte-macrophage colony stimulating activity (GM-CSF) and granulocyte colony stimulating factor (G-CSF), FDC-P2 reacts to IL-3 and IL-2, DA-1 is responsive to IL-3, leukemia inbitory factor (LIF), and erythropoietin (EPO), and 32D responds to IL-3 and G-CSF. Many lines also show synergistic effects with other factors such as IL-4. Sandwich immunoassays using monoclonal or polyclonal antibodies have recently offered an alternative approach of quantitating cytokines or similar proteins in much shorter time and with great specificity and good reproducibility (Ferrua et al., 1988; Budd and Smith, 1986; Cebon et al., 1988; Watari et al., 1989; Chretien et al., 1989), and thus potentially overcoming problems associated with bioassays as discussed above. Here we describe an immunoassay for murine IL-3 using polyclonal antipeptide antibodies raised in rabbits and sheep. RESULTS Serum from a sheep immunized with synthetic murine IL-3 (l-140) had a high titer (6 x 106) of CYTOKINE,

Vol. 1, No. 1 (November), 1989: pp 56-61

Immunoassay for murine IL-3 / 57

antibodies that specifically bound synthetic murine IL-3 in an ELISA assay and did not bind to synthetic human IL-3 or human GM-CSF. The titer of antibodies that bound to peptide 1-29 (IL-3) was 5 x lo3 (data not shown). The fact that the l-29 titer was 1,OOO-fold lower was consistent with our experiments indicating that peptide l-29 does not encompass an immunodominant epitope when IL-3 (l-140) is used as an immunogen (unpublished data). The serum was further tested for reactivity with native IL-3 by its ability to neutralize the growth stimulating effects of IL-3 on the IL-3 dependent cell line FDC-PI (Fig. 1). Serial dilutions of sheep serum were added to FDC-Pl cells cultured either with 1 U of IL-3 or 1 U of GM-CSF. A dilution of up to 1:200 of this serum was able to completely neutralize the response of the cells to 1 U of IL-3. This inhibition was specific for IL-3 mediated growth as the action of GM-CSF on the same cells was not affected (Fig. 1). Immunization of rabbits with l-29C (IL-3)-KLH conjugate induced high titers of anti-l-29 (IL-3) antibodies that could reproducibly be affinity-purified with yields of 0.2 to 0.4 mg antibody/ml serum from New Zealand white rabbits or yields of 0.5 to 1.25 mg antibody/ml serum from Dutch Belted rabbits. Affinity columns were prepared with these purified antibodies and were shown to deplete over 99% native WEHI-3B derived IL-3 as described previously (Ziltener et al., 1987) (data not shown). A sandwich assay was developed using the rabbit polyclonal anti-l-29 (IL-3) antibodies as the solid phase and sheep anti-IL-3 serum as the second layer. Fig. 2 shows the quantitation by the immunoassay of an HPLC purified preparation of synthetic IL-3 that was dried overnight and carefully weighed. The detection limit for synthetic IL-3 calculated from the value of the absorbance that is three standard deviations greater than the

Figure 1. The cell line FDC-PI responsive to either IL-3 or GM-CSF was cultured for 3 d in the presence of either 1 U/ml IL-3 (closed square, W) or 1 U/ml GM-CSF (open square, 0) and titrated amounts of sheep anti-( l-140) IL-3 antiserum.

pg IL3/ml

Figure 2. Standard curve of synthetic (l- 140) IL-3. x-axis and y-axis are displayed in the logarithmic scale showing the endpoint of the titration curve.

background reading was 60 pg/ml(3.7 PM), corresponding to an absolute amount of 3 pg. IL-3 obtained from four different sources was tested in the immunoassay and in the standard bioassay and plotted (Fig. 3) to emphasize the relative behavior of samples in the two assays. The samples were conditioned medium derived from (A) Con A stimulated T cells; (B) myeloid leukemia WEHI-3B; (C) recombinant material derived from COS 7 cells and (D) from the myeloid cell line FDC-Pl transfected with a retrovirus expressing the IL-3 gene. The bioassay was, in all four cases shown, lo- to 1OO-fold more sensitive than the immunoassay. The immunoassay data shown in Fig. 3 (A-D) were replotted in Fig. 4 in order to compare the immunoreactivity of the different IL-3 samples with the units of IL-3 bioactivity. The signal levels in the immunoassay correspond to similar levels of bioactivity in each case and the detection limit of the immunoassay was found to be between 2 to 4 U of bioactivity for all four samples tested. Specificity of the immunoassay was tested by fractionating a sample of WEHI-3B conditioned medium using an affinity column made with the anti-IL-3 monoclonal antibody (MAb) 2Ell. MAb 2Ell recognizes IL-3 at an epitope containing amino acid residues 130-135 of native IL-3 close to the C-terminus and depletes IL-3 bioactivity to about 95% from conditioned media applied as shown in an earlier study (Ziltener, Clark-Lewis et al., 1988). Fig. 5 shows that this IL-3 depleted WEHI-3B conditioned medium had a 95% reduced signal in the immunoassay compared to the signal obtained with untreated WEHI-3B conditioned medium. When bound material was eluted from the MAb 2Ell affinity column, IL-3 was recovered with a yield of about 90%, as measured in the immunoassay (Fig. 5).

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Figure 3. Comparison of IL-3 quantitation by [‘Hlthymidine incorporation (open square, 0) and by sandwich immunoassay (closed square, n ). Samples were: (A) Con A activated T cells, (B) WEHI-3B conditioned medium lOxconcentrated, recombinant materials derived from (C) FDC-Pl transfected with an IL-3 containing retrovirus, or (D) from COS 7 cells transfected with IL-3 cDNA.

DISCUSSION The present study describes the use of antipeptide antibodies raised against selected amino acid sequences of the murine IL-3 molecule. Antibodies raised against the l-29 (IL-3) N-terminal peptide showed a high affinity for native IL-3 as shown in earlier studies by its ability to neutralize IL-3 bioactivity and its use as a potent reagent for the affinity purification of IL-3 from several sources (Ziltener et al., 1987; Ziltener, Fazekas de St. Groth et al., 1988). Immunization of rabbits with l-29C (IL-3)-KLH complex induced reproducible high anti-l-29 antibody titers (over 106). These antibodies were therefore chosen as the solid-phase (first) antibody in an immunoassay for IL-3. It was interesting to note that Dutch Belted rabbits developed higher anti-l-29 (IL-3) antibody titers than New Zealand white rabbits, corresponding to a higher yield of specific antibody per ml serum. Although the Dutch Belted rabbits have only about half the weight of New Zealand white rabbits and

therefore yield only about half the amount of serum, the overall yield of antibody from Dutch Belted rabbits was still about 50% better. Affinity purified anti-l-29 (IL-3) antibodies from both rabbit strains were indistinguishable in their reactivity with native IL-3. Immunization with synthetic IL-3 induced reproducibly high anti-IL-3 antibody titers in several species tested (sheep, rabbits and chicken, Ziltener unpublished observations) and even proved to be an immunogen in mice, inducing autoantibodies against native IL-3 (Ziltener, Clark-Lewis et al., 1988). Immunization of a sheep with synthetic l-140 (IL-3) gave rise to serum with high anti-IL-3 antibody titers and a strong neutralizing ability for native IL-3 (Fig. 1). This antiserum was chosen as the source for the indicator (second) antibody for the detection of captured IL-3. The use of antibodies raised in two different species (rabbit and sheep) greatly facilitates the preparation of the second antibody, as it can be used as an appropriate dilution of the serum and does not need to be purified

Immunoassay for murine IL-3 / 59

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Figure 4. Comparison of units of IL-3 bioactivity with signal in the immunoassay. Immunoassay results obtained with the four IL-3 samples shown in figure 3 were replotted with the x-axis displaying the amount of units of IL-3 bioactivity present in the sample and the y-axis displaying the corresponding signal of the immunoassay.

and labeled. This allows a third developing reagent (e.g., a peroxidase coupled rabbit anti-sheep antibody) to detect specifically the second but not the first antibody. Additionally, use of the third antibody results in an amplifying effect similar to that obtained with biotinavidin complexes. The detection limit for HPLC purified synthetic

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Figure 5. IL-3 from WEHI-3B conditioned medium was purified using anti-IL-3 MAb 2Ell. The IL-3 content of the fractions was tested using the sandwich immunoassay. Untreated WEHI-3B conditioned medium (open square, q ), WEHI-3B conditioned medium IL-3 depleted using MAb 2Ell (open circle,o) and affinity purified

IL-3 elutedfrom MAb 2Ell (closedsquare,n ).

IL-3 was found to be approximately 60 pg IL-3/ml under the assumption that all the IL-3 was detectable in the assay. The detection limits for the four samples of biosynthetic IL-3 corresponded to concentrations containing 2 to 4 U of biological activity per ml. This suggests that 1 U of IL-3 bioactivity corresponds to 15 to 30 pg/ml of native IL-3. This result compares favorably to a value of 4 pg/ml previously obtained for biochemically purified IL-3, given that as discussed, there were uncertainties in the precise quantitation because of the small amounts of material obtained (Clark-Lewis et al., 1984). A requirement for the sandwich immunoassay is the availability of two specific antibodies which will react simultaneously with the molecule of interest without competition with each other. The most obvious way to achieve this is the use of monoclonal antibodies or a combination of monoclonal antibody and polyclonal antibody. However, the preparation of suitable high affinity monoclonal antibodies in large quantities is not always straightforward. Antipeptide antibodies with predetermined specificity may offer an alternative to monoclonal antibodies and have proven useful in the immunoassay discussed here. The assay described has the advantage of rapid and specific quantitation of IL-3. It should be applicable for the measurement of IL-3 from biological sources which are difficult to measure in a bioassay for reasons of inhibitory effects or specificity. The sensitivity of the assay described is comparable to systems described for IL-2 using monoclonal antibodies (Budd and Smith, 1986) or for IL- 1 using polyclonal antisera (Ferrua et al., 1988). The reduced sensitivity of the immunoassay compared to the bioassay remains the major drawback of the sandwich immunoassay. Clearly, improvements in the quality of antibodies and amplification without affecting background could improve the sensitivity. The use of antipeptide antibodies of predetermined specificity in a sandwich immunoassay is a valid alternative to other existing methods. In principle this method should be generally applicable to cytokines and other proteins for which sequence information is available. Similar tests for other lymphokines are also at present being developed.

MATERIALS

AND

METHODS

Micro test III flexible assay plates 96 U-bottom wells Falcon 3911 were obtained from Becton Dickinson labware (Oxnard, CA). 2,2’-azinodi-(3-ethylbenzthiazoline sulfonic acid) (ABTS) was from Sigma (St. Louis, MO); peroxidase coupled rabbit anti-sheep antibody was from Kirkegaard and Perry Laboratories Inc. Peptidescorresponding to murine IL-3 residues l-29 and full length IL-3 (l-140) were synthesized by

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solid phase methods (Clark-Lewis et al., 1986). Medium conditioned by high density cultures (5 x 10’ to lo6 cells/ml) of the myelomonocytic leukemia WEHI-3B and medium conditioned by high density cultures (5 x lo5 to lo6 cells/ml) of the myeloid cell line FDC-PI (Dexter et al., 1980) transfected with an IL-3 containing retrovirus (the kind gift of Dr. Tony Pawson) were collected by centrifugation and concentrated lo-fold using an Amicon hollow fiber system (Mr cutoff > 10,000). Supernatants of a T cell clone P41.1 (Ziltener et al., 1987) containing maximal amounts of IL-3, were cultured at 3 x 106/ml in RPM1 supplemented with 1% fetal calf serum in loo-ml dishes; lymphokine production was induced by adding concanavalin A (ConA) (Calbiochem) at 5 bug/ml and supernatants were removed after 18 to 24 h.

Rabbit Anti-Z-29 (IL-3) Antibodies Peptide l-29 (IL-3) was coupled to keyhole limpet hemocyamine (KLH) via a cysteine added at position 30 as fully described elsewhere (Ziltener et al., 1987). New Zealand white or Dutch Belted rabbits were injected in multiple subcutaneous sites with a total of 0.5 mg of peptide coupled to KLH and emulsified in complete Freunds adjuvant. The rabbits were bled after four weeks and boosted intramuscularly with 200 rg of peptide-KLH emulsified in incomplete Freunds adjuvant. Rabbits were then bled and boosted at 3-week intervals. Anti-l-29 (IL-3) antibody titers were measured in an ELISA using polyvinyl microtiter plates coated with synthetic IL-3 (5 pg IL-3/ml PBS). The antibody titer was defined as the reciprocal value of the last dilution which gave an absorption reading three. standard deviations greater than the background reading. Peptide (l-29C) IL-3 was coupled to activated thiolsepharose(Pharmacia) via cysteine. Rabbit sera were applied to the column which was then washed with PBS (pH 7.3,0.13 M) and the peptide-specific antibodies were eluted with glycine/HCI buffer (pH 2.5, 0.1 M). Eluates were dialyzed against PBS and the antibody concentration estimated by measuring OD 280 mm, assuming 1 mg of protein/ml to have OD,*, of 1.4. Antibody preparations were stored as aliquots at - 7ooc.

SheepAnti-IL-3 Antiserum A sheep was injected in multiple subcutaneous siteswith a total of 600 rg of synthetic (l-140) IL-3 emulsified in complete Freunds adjuvant. After four weeks the animal received booster injections (2 sites intramuscularly) with 300 pg of synthetic (l-140) IL-3 emulsified in incomplete Freunds adjuvant. The sheep was then regularly bled and boosted at approximately 3-week intervals. Anti-IL-3 titers were measured in an ELISA as described above.

Sandwich Enzyme Immunoassay Microtiter plates were coated overnight with affinity purified anti- l-29 (IL-3) antibody (5 bg antibody/ml PBS, 50 rl/well). The plates were washed in PBS pH 7.3 containing 0.5% skim milk powder, and IL-3 sampleswere serially diluted in PBS skim milk and 50 ~1transferred to the microtiter trays. After 3 h at room temperature the trays were washed three times in PBS skim milk, and 50 ~1of a 1:300 dilution of the

CYTOKINE, Vol. 1, No. 1 (November1989:56-61) sheep anti-IL-3 antiserum in PBS skim milk was added to all the wells. Bound sheepanti-IL-3 antibody was detected by the addition of peroxidaselabeled anti-sheep antibody and incubation for 1 h before washing nine times in double distilled water. Bound peroxidase was assayedby H,O, mediated oxidation of 2,2’-azinodi-(3-ethylbenzthiazoline sulfonic acid), the reaction product being detected by absorption at 405 in a Bio-Tek ELISA reader. The end point was defined as the reciprocal value of the last dilution which gave an absorption reading three standard deviations greater than the background reading. Several parameters were tested in the attempt to optimize reproducibility and sensitivity of the assay. It was found that the above conditions were optimal. An increase in the concentration of the first antibody, used to coat the trays, an increase in the incubation volume, or the use of more concentrated sheep anti-IL-3 serum did not significantly increase the signal over the background ratio (data not shown). The binding of IL-3 to immobilized first antibody was not influenced when incubation temperatures of 4°C or 37OC were tried; incubations were therefore carried out at room temperature.

Bioassay of IL-3 IL-3 bioactivity was assayedas described (Clark-Lewis et al., 1984) using the uptake of [‘Hlthymidine as an index of stimulation of growth of an IL-3 dependent cell line (R6-X. E4). Units of activity were calculated using a computer program which fitted a straight line to the descending part of the titration curve, the slope of the line being matched to that obtained by parallel titration of a standard preparation of IL-3 (Fazekas de St. Groth et al., 1986). One unit of biological activity was defined as the concentration of IL-3/ml required to stimulate 50% maximal responsein this assay. Acknowledgments

The authors wish to thank Dr. John Schrader for helpful discussions and the critical reading of the manuscript. We further gratefully acknowledge the expert technical assistance of J. Babcook, P. Owen, and L. Hasiuk for assistance in preparation of the manuscript.

REFERENCES Budd RC, Smith KA (1986): Interleukin-2 Immunoassay using monoclonal antibodies. Biotechnology 4983-986 Cebon J, Dempsey P, Fox R, Kannourakis G, Bonnem E, Burgess AW, Morstyn G (1988): Pharmacokinetics of human granulocytemacrophage colony-stimulating factor using a sensitive immunoassay. Blood 72:1340-1347 Chretien I, Van Kimmenade A, Pearce MK, Banchereau J, Abrams JS (1989): Development of polyclonal and monoclonal antibodies for immunoassay and neutralization of human interleukin-4. J Immuno1 Methods 117:67-8 1 Clark-Lewis I, Aebersold R, Ziltener HJ, Schrader JW, Hood LE, Kent SBH (1986): Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3. Science 231:134139 Clark-Lewis I, Kent SBH, Schrader JW (1984): Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells. J Biol Chem 259:7488-7494

Immunoassay for murine IL-3 / 61 Crapper RM, Clark-Lewis I, Schrader JW (1984): The in vivo functions and properties of persisting cell stimulating factor. Immunology 53:33-42 Cutler RL, Metcalf D, Nicola NA, Johnson GR (1985): Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium. J Biol Chem 2606519-6587 Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D (1980): Growth of factor-dependent hemopoietic precursor cell lines. J Exp Med 152:1036-1042 Dy M, Lebel B, Kamoun P, Hamburger J (1981): Histamine production during the anti-allograft response: Demonstration of a new lymphokine enhancing histamine synthesis. J Exp Med 153:293309 Fazekas de St. Groth B, Thomas WR, M&mm-Breschkin JL, Clark-Lewis I, Schrader JW, Miller JFAP (1986): P cell stimulating factor release: a useful assay of T cell activation in vitro. Int Arch Allergy Appl Immuno179: 169- 177 Ferrua B, Becker P, Schaffar L, Shaw A, Fehlmann M (1988): Detection of human IL-la and IL-la at the subpicomolar level by calorimetric sandwich enzyme immunoassay. J Immunol Methods 114:41-48 Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RJ (1983): Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. Proc Nat1 Acad Sci USA g&2931-2935

Ihle JN, Keller J, Henderson L, Klein F, Palazynski EW (1982): Procedures for the purification of interleukin-3 to homogeneity. J Immunoll29:2431-2436 Schrader JW, Clark-Lewis I, Crapper RM, Leslie KB, Schrader S, Varigos G, Ziltener H (1988): The panspecific hemopoietin “interleukin-3,” Physiology and Pathology. In: Lymphokines 15, JW Schrader (ed). Academic Press, New York, p 282-3 11 Schrader JW, Nossal GJV (1980): Strategies for the analysis of accessory function: the in vitro cloning and characterization of the P cell. Immunol Rev 53:6166 Watari K, Asano S, Shirafuji N, Kodo H, Ozawa K, Takaku F, Kamachi S-I (1989): Serum granulocyte colony-stimulating factor levels in healthy volunteers and patients with various disorders as estimated by enzyme immunoassay. Blood 73:117-122 Ziltener HJ, Clark-Lewis I, Hood LE, Kent SBH, Schrader JW (1987): Antipeptide antibodies of predetermined specificity recognize and neutralize the bioactivity of the pan-specific hemopoietin Interleukin-3. J Immunol 138:1099 Ziltener HJ, Clark-Lewis I, Fazekas de St. Groth B, Orban PC, Hood LE, Kent SBH, Schrader JW (1988): Monoclonal antibodies recognize Interleukin-3 and neutralize its bioactivity in vivo. J 1mmuno1140:1182-1187 Ziltener HJ, Fazekas de St. Groth B, Leslie KB, Schrader JW (1988): Multiple glycosylated forms of T-cell derived IL-3. Heterogeneity of IL-3 from physiological and non-physical sources. J Biol Chem 263:14511-14517