Crude Conditioned Medium Research Articles

Identification of sex pheromone of red swamp crayfish Procambarus clarkii and exploration of the chemosensory mechanism of their antennae

Red swamp crayfish, Procambarus clarkii, is a globally invasive species, which has caused great damage to biodiversity, agriculture, and fishing. Therefore, the development of effective management methods, such as pheromone control, is necessary for biological control and biodiversity protection. However, the components of P. clarkii sex pheromones have not yet been explored, and the chemosensory mechanism of the P. clarkii antennae after stimulation by sex pheromone also remains unknown. In this study, we isolated and identified the candidate bioactive component of the female P. clarkii sex pheromone using ultrafiltration centrifugation, semi-preparative liquid phase separation and omics technologies and conducted bioassays to determine its attraction ability. Meanwhile, RNA-Seq technology was used to analyze the potential chemosensory mechanism of antennae. Our results indicated that the male P. clarkii were uniaxially attracted to the female crude conditioned water (FCW), medium fraction (MF, isolated by ultrafiltration centrifugation), and preparative fragment 6 of females (PFF6, isolated by semi-preparative liquid phase separation). Metabolomic analysis revealed the presence of 18 differential metabolites between the PFF6 and PFM6 samples, among which 15 were significantly upregulated in the PFF6 sample. Bioassay test also showed that mestranol, especially at concentrations of 10−5–10−2 mol∙l−1, could significantly attract P. clarkii males; therefore, mestranol was identified as the candidate sex pheromone component of P. clarkii females. Furthermore, RNA-Seq results showed that most differentially expressed genes (DEGs) enriched in lipid metabolism and signal transduction pathways were up-regulated in P. clarkii males. In addition, high expressions of Ca2+-binding protein and ion transporting ATPases may enhance the sensitivity of the antennae of P. clarkii males towards sex pheromones. Our study provides data on P. clarkii sex pheromone composition and reveals the molecular mechanism of sex pheromone response in P. clarkii. Moreover, our study provides a referable method for the isolation of candidate bioactive molecules from the P. clarkii sex pheromone.

Asymmetrical Fc Engineering Greatly Enhances Antibody-dependent Cellular Cytotoxicity (ADCC) Effector Function and Stability of the Modified Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

Expression and secretion of Bluetongue virus serotype 8 (BTV-8)VP2 outer capsid protein by mammalian cells

VP2 is the outermost Bluetongue virus (BTV) antigenic protein, forming triskelion motifs on the virion surface. Although VP2 has been expressed successfully through many systems, its paracrine expression as a soluble form by mammalian cells represents a difficult task. In the present paper two fragments of VP2 have been expressed successfully into the medium of transiently transfected mammalian cells through a fusion peptides strategy. The crude conditioned medium containing the secreted peptide could be employed for immunodiagnostic assay development or vaccine purposes.

Expression of Bovine Viral Diarrhea Virus Glycoprotein E2 as a Soluble Secreted Form in a Mammalian Cell Line

Bovine viral diarrhea virus (BVDV) membrane-anchored type I glycoprotein E2 is an approximately 53-kDa immunodominant glycoprotein inducing the production of neutralizing antibodies in the animal host after natural infection or following immunization with live or killed vaccines. The E2 coding region lacking the transmembrane domain was constructed in a soluble secreted form (secE2) and expressed in the medium of a transiently transfected human cell line. The crude conditioned medium containing secE2 can be potentially employed to develop an enzyme-linked immunosorbent assay antigen for the diagnosis of BVDV infection or for vaccine purposes.

Prostate cancer mediates osteoclastogenesis through two different pathways

The present study was undertaken to test the effects of prostate cancer cell lines (LNCaP, DU145, PC3, and MDA PCa 2b) on osteoclastogenesis. Crude conditioned medium (CM) from all four prostate cancer cell lines enhanced expression of the mRNA for receptor activator of NF-κB ligand (RANKL) in a mouse osteoblast cell line, MC3T3-E1; however, CM had no effect on expression of osteoprotegerin (OPG) mRNA. Coculture of MC3T3-E1 with prostate cancer cells yielded similar results. The number of mature osteoclasts induced by soluble RANKL increased significantly when osteoclast precursor cells were cultured with CM from LNCaP and DU145 cells. CM from LNCaP and DU145 cells also induced maturation from precursor in the absence of soluble RANKL, and this effect was not blocked by OPG. Addition of CM from DU145 cells increased expression of MMP-9 mRNA by osteoclast precursors. Our findings indicate that prostate cancer mediates osteoclastogenesis through induction of RANKL expression by osteoblasts and through direct actions on osteoclast precursors mediated by some factors other than RANKL.

Characterization and determination of the relative abundance of two types of insulin-like growth factor binding protein-5 proteases that are secreted by human fibroblasts.

We previously reported that human fibroblasts secrete a protease into their conditioned medium that cleaves insulin-like growth factor binding protein-5 (IGFBP-5) into non-IGF-I binding fragments. Because the protease activity in the fibroblast medium has characteristics of both serine and metalloproteases, the activity was purified and analyzed to determine whether it retained serine or metalloprotease properties. The protease was purified by heparin Sepharose affinity chromatography followed by alpha1 antichymotrypsin affinity or gelatin agarose chromatography. The heparin Sepharose purified material degraded IGFBP-5 into 22-, 17-, and 16-kDa fragments. Amino acid sequencing showed that the 22-kDa fragment contained the amino-terminus of the protein. The protease activity in the fibroblast conditioned medium that was purified by heparin Sepharose was inhibited by both serine and metalloprotease inhibitors. To attempt to separate these activities, the heparin Sepharose purified activity was further purified by gelatin agarose chromatography. The IGFBP-5 protease activity that did not bind to gelatin agarose was inhibited by serine protease inhibitors, such as 3,4 dichloroisocoumain (3,4 DCI), whereas tissue inhibitor metalloprotease-1 (TIMP-1) had minimal activity. When this same pool of protease activity that had been eluted from heparin Sepharose was applied to an alpha1 antichymotrypsin peptide affinity column, the protease activity that bound to the column was inhibited by 3,4 dichloroisocoumain, but was not inhibited by TIMP-1. In contrast, the activity that did not adhere to this column was inhibited by TIMP-1. IGFBP-5 zymography showed that the Mr estimate of the protease that was inhibited by serine protease inhibitors was 92 kDa, whereas gelatin zymography showed that the metalloproteases had Mr estimates of 72, 69, and 55 kDa. When the protease activity in the crude conditioned medium was analyzed by zymography, almost all of the detectable protease had an Mr estimate of 92 kDa, suggesting that the metalloproteases that were detected in the partially purified fractions were inactive in the medium. In summary, fibroblasts secrete a 92-kDa protease that cleaves IGFBP-5 into 22-, 17-, and 16-kDa fragments. The protease inhibitor specificity results, chromatographic characteristics, and zymographic analyses suggest that this is a serine protease. Although metalloproteases are secreted by these cells, the 92-kDa serine protease is the predominate form of activity in the conditioned medium that cleaves IGFBP-5.

Purification of a Secreted Form of Recombinant Rabies Virus Glycoprotein: Comparison of Two Affinity Tags

Expression of recombinant eukaryotic proteins in transfected mammalian cell lines has become an important approach for the characterization of the structure and function of these proteins. However, it is often difficult to recover and purify the recombinant proteins. Therefore, the use of fusion proteins incorporating epitope or affinity tags has become more widespread. In this paper, we directly compare two affinity tags, the hexahistidyl tag and the biotin peptide mimetic,Strep-tag,for use in purification of a recombinant soluble form of rabies virus glycoprotein secreted by transfected Chinese hamster ovary fibroblasts. The recombinant rabies virus gly- coproteins are denoted RGP(WT)T441his and RGP(WT)T443s-tag, respectively. These affinity tags were chosen because the chromatographic matrices (Ni(II)-NTA-agarose and recombinant core streptavidin–agarose, respectively) were readily available and these methods offered the possibility of a one-step purification using mild elution conditions. However, in our hands, neither method allowed for a one-step purification protocol. Nonetheless, it was possible to purify RGP(WT)T441his to homogeneity from crude conditioned medium using a combination of metal–chelate affinity chromatography and immunoaffinity chromatography. In contrast, although theStrep-taghas been useful for purifying recombinant proteins expressed in bacteria, we were not able to effectively purify RGP(WT)T443s-tag from conditioned medium using chromatography on recombinant core streptavidin–agarose.

IL-10, T lymphocyte inhibitor of human blood cell production of IL-1 and tumor necrosis factor.

We have identified and purified a factor that inhibits the production of IL-1 beta and TNF by stimulated human mononuclear cells. The activity is produced by the T cell lines Hut-78 and Mo constitutively under serum-free conditions. Crude conditioned media have titers of up to 100 U/ml (one unit defined as the reciprocal of the dilution producing 50% inhibition). The activity resides mainly in a single size peak of 30 to 35 kDa and an isoelectric point around 8. Other cytokines in this size range that have been reported to be inhibitory for IL-1 and TNF production include TGF-beta, IL-4, and IL-6; these factors were excluded by lack of detection, neutralizing antibody, and low activity compared with our factor. Another factor with these size and charge properties is IL-10, which inhibits T cell cytokine production. By polymerase chain reaction analysis, Mo and HuT-78 lines contain IL-10 transcripts whereas JURKAT is negative; this correlates with inhibitor bioactivity from the three lines. Use of mAb specifically showed the inhibitor to be IL-10.

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor. Cultivation of human hepatoma Hep G2 in hollow fiber bioreactor.

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acusyst-P (Endotronic) with a total fiber surface area of 7.2 m2 6 x 1.2m2) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics- and serum-free IMDM medium, supplemented with 50 micrograms/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20-40 micrograms protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.

Immunomodulation with low doses of cyclophosphamide, thymostimulin and echinacin in patients with advanced gastrointestinal malignancies

A simple two-step method involving ammonium sulfate precipitation followed by hydrophobic chromatography is described for the separation of T cell growth factor (TCGF) from a number of other factors contained in medium conditioned by concanavalin A-stimulated spleen cells. Thus, granulocyte-macrophage colony-stimulating factor, P cell-stimulating activity, pluripotential stem cell-supporting activity and interferon activity were not detected in TCGF partially purified by these steps. T cell-replacing factor co-purified with TCGF. Macrophage activity factor (MAF) co-purified with TCGF, but the ratio of MAF to TCGF activities was reduced more than 20-fold relative to that in crude conditioned medium. All of the factors were present in the 50–80% saturated ammonium sulfate fraction, however, levels of concanavalin A were reduced by 98% in this step. TCGF, separated in this way from these other regulatory factors will be useful in experiments analyzing the actions of TCGF on mixed populations of cells.

Interleukin‐1β‐ and tumor necrosis factor‐α‐independent monocyte stimulation of fibroblast collagenase activity

To investigate the mechanism of cyclosporine (Cs)-induced fibrous gingival enlargement, the indirect effects of Cs on fibroblast collagenolysis via the drug's effect on the synthesis of the fibroblast regulatory monokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) have been studied. Peripheral blood monocytes stimulated with lipopolysaccharide (LPS) for 48 h produced conditioned media (MCM-LPS) that contained 665 pg/ml IL-1 beta and 16 pg/ml TNF alpha and significantly (P less than 0.001) enhanced the collagenase activity of a fibroblast strain (GN 23) derived from a healthy individual with clinically normal gingiva. The concurrent addition of Cs (50, 100, or 150 ng/ml) with LPS to the monocytes (MCM-LPS-Cs) significantly diminished their ability to enhance GN 23 collagenase activity in a dose-dependent manner, with MCM-LPS-Cs (150 ng/ml) causing the greatest effect. Cs also significantly inhibited IL-1 beta and TNF alpha production. Although the greatest inhibition of both cytokines was at 50 ng/ml Cs, the corresponding MCM-LPS-Cs caused the least diminution (16%) of the collagenase stimulation caused by MCM-LPS (no Cs). This suggested that factor(s) other than or in addition to IL-1 beta and TNF alpha might be responsible for the stimulation of GN 23 collagenase activity. MCM-LPS depleted of IL-1 beta by affinity chromatography retained its stimulatory effect on GN 23 collagenolysis, and human recombinant IL-1 beta and TNF alpha, when tested alone or together at levels found in the stimulatory MCM-LPS and MCM-LPS-Cs, did not stimulate GN 23 collagenase activity as did the crude conditioned media. This evidence suggested that the conditioned media contained the complex mixture of cytokines necessary to stimulate collagenase activity of this fibroblast strain and that IL-1 beta and TNF alpha were not necessarily involved. Cs may alter the synthesis of other collagenase-stimulating cytokines, accounting for the diminished ability of Cs-treated monocytes to enhance collagenase activity of susceptible fibroblast strains. Decreased collagenase activity, therefore, resulting from Cs suppression of monokine production, may be an important factor in the development of fibrous gingival enlargement seen in some susceptible patients treated with Cs.

A novel autoregulatory cytokine is required for the regulation of autoaggressive responses

Limiting dilution studies indicate that cells with the potential to lyse autologous target cells exist in the peripheral blood of all normal individuals. In contrast to allocytotoxic cells, autocytolytic cells are down-regulated by a second less frequent cell population. When recombinant interleukin 2 is substituted for crude lymphocyte conditioned medium in these limiting dilution experiments, autocytotoxicity develops normally. Under these conditions, however, the autocytotoxic response is not down-regulated. Mixing crude lymphocyte-conditioned medium together with recombinant interleukin 2 restores the regulation of autocytotoxicity normally seen at high responder cell dose. These findings indicate that a second soluble factor present in the conditioned medium is necessary either for the activation, growth, or differentiation of the regulatory cell population or alternatively, to render the cytotoxic population responsive to the activity of regulatory cells. Gel filtration studies indicate that the molecular weight of this factor is between 60 and 80 kd. This factor appears to be distinct from known immunologically active cytokines. It is conceivable that deficiencies of this cytokine may be relevant to the pathogenesis of autoimmune diseases or graft-versus-host reactions.

Tumor necrosis factor alpha stimulates the growth of the clonogenic cells of acute myeloblastic leukemia in synergy with granulocyte/macrophage colony-stimulating factor.

TNF-alpha has been shown to antagonize the proliferative effects of growth factors present in crude conditioned media from PHA-stimulated leukocytes or cell lines on the clonogenic cells of acute myeloblastic leukemia (AML) (19,21). In the present study, we investigated the responses of AML blasts to TNF-alpha in the presence of defined growth factors (recombinant granulocyte/macrophage-CSF [rGM-CSF], recombinant granulocyte-CSF [rG-CSF], rIL-3, and rIL-1) and under conditions described for autocrine stimulation (32). While TNF-alpha antagonized the stimulatory effects of G-CSF and IL-3 on blast progenitors, TNF-alpha did not affect blast colony formation in the presence of IL-1. Unexpectedly, TNF-alpha significantly enhanced blast proliferation in the presence of GM-CSF. Further, TNF-alpha also acted synergistically with an endogenous source of growth stimulatory signal to promote proliferation of blast clonogenic cells. Thus, on human leukemic cells, TNF-alpha appears to be a molecule that is at least bifunctional, having the ability to either support or inhibit cell proliferation, depending on the other growth factors present. It is postulated that the proliferative response of blast progenitors to TNF-alpha under conditions that favor autocrine stimulation may represent one property that allows the cells to escape from negative regulation and proliferate in AML.

High yield purification of melanoma growth stimulatory activity

Tumor cells produce a variety of hormones and growth factors that are associated with modulation of the growth pattern of malignant cells. Hs294T human malignant melanoma cells produce a monolayer mitogen, melanoma growth stimulatory activity (MGSA), that stimulates the growth of Hs294T cultures in serum-free medium. MGSA has been purified to homogeneity from conditioned medium of Hs294T human malignant melanoma cells using acetic acid extraction of the crude conditioned medium followed by three Chromatographie processes, including gel-filtration, heparin-Sepharose, and reverse-phase HPLC. MGSA was eluted from the heparin-Sepharose resin with 0.1–0.3 M NaCl. The binding affinity is similar to that of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) but much less than many endothelial cell-derived growth factors which require significantly higher salt concentrations for elution. These procedures resulted in a final yield of purified MGSA that was significantly greater than yields obtained using previously reported procedures. The homogeneous 16000 and 13000 molecular weight moieties obtained by means of these procedures exhibited similar bioactivities (stimulating a 2- to 3-fold increase in Hs294T cell growth) over a 0.06–6 ng concentration range. This bioactivity was progressively inactivated during storage at −80°C. These results indicate that the combination of heparin-Sepharose chromatography and reverse phase-HPLC provides a more efficient means of purification of MGSA.

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N, N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an M r 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This M r, 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor β (TGF-β) receptor. Therefore, this M r, 20,000 inhibitory polypeptide induced by DMF is probably TGF-β. TGF-β was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.

Regulation of lipoprotein lipase synthesis in 3T3-L1 adipocytes by cachectin. Further proof for identity with tumour necrosis factor.

We investigated the mechanism by which the endotoxin-induced macrophage secretory protein cachectin is able to suppress the activity of lipoprotein lipase in 3T3-L1 adipocytes. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. The results were nearly identical whether crude conditioned medium or a highly purified preparation was utilized as a source of cachectin. [35S]Methionine incorporation into acid-precipitable protein was minimally affected by purified cachectin, suggesting that the suppression of the lipoprotein lipase was not due to a general suppression of protein synthesis. These results, taken together with our previous work, provide additional evidence that cachectin and tumour necrosis factor are functionally identical.

Chondrocyte activation in response to factor(s) produced by a continuous line of lapine synovial fibroblasts

We have established a permanent line of lapine synovial fibroblasts called HIG-82. Upon appropriate stimulation, these cells mimicked primary cultures of lapine synovial cells in producing substances which activated primary cultures of lapine articular chondrocytes. Activated chondrocytes secreted prostaglandin E 2 (PGE 2) and latent neutral collagenase, gelatinase, and caseinase, but not acid hydrolases, into their culture media. PGE 2 itself did not activate the chondrocytes. Heating the crude, synovial-conditioned media at 70 °C for 30 min reduced their activating activity by 49.3 ± 20.5% ( n = 7). Production of PGE 2 by chondrocytes was maximal during the first day of exposure to synovial conditioned media, whereas the production of neutral proteinases peaked during the second day. All the chondrocyte-stimulating activity was present in a fraction of M r 10 000–25 000. Unlike the crude conditioned medium, this partially-purified material retained full activity following heating to 70 °C for 30 min. These data indicate that synovial fibroblasts (type B synoviocytes) are a source of chondrocyte activator(s) and that neutral, but not acid, proteinases may be involved in extracellular proteolysis which leads to the resorption of the cartilaginous matrix seen in bioassays of catabolin.

Functional properties of two human B cell growth factor species separated by lectin affinity column.

The supernatants from PHA-activated normal human T cells (conditioned medium) were fractionated by differential adsorption on Con A-Sepharose columns. The effluent contained both IL 2 activity (tested on the CTL-L2 cell line) and BCGF activity (tested on anti-mu-activated normal B-enriched cells). Absorption of such effluent on PHA-activated T cell blasts removed the IL 2 activity without affecting the BCGF activity. The eluate also displayed BCGF activity (tested on anti-mu-activated B-enriched cells) without detectable IL 2 activity. The two BCGF species isolated by this criteria synergize with semipurified IL 1 to support the anti-mu-induced proliferation of highly monocyte-depleted B cells. Both BCGF species required the presence of anti-mu Ab to support the proliferation of small B cells (at the G0 stage). However, in contrast to crude conditioned medium and to the BCGF present in the effluent, the BCGF present in the eluate was unable to induce the proliferation of unfractionated or large (at the G1 stage) B cells in the absence of anti-mu Ab. These results support the existence of two functionally different human BCGF species, differing at least in their relative sugar content and distinct from IL 2.

Macrophage-derived growth factor for osteoblast-like cells and chondrocytes.

Rat resident peritoneal macrophages in primary culture were found to elaborate a mitogenic factor (or factors) for rat osteoblast-like cells and chondrocytes but not for skin fibroblasts. Growth-promoting activity appeared in the incubation medium within the first 20 hr of macrophage culture and was released in amounts that paralleled the number of macrophages per culture. After their proliferative response, as judged by increases in DNA synthesis and cell number, the osteoblast-like cells became enriched in alkaline phosphatase, an index of osteoblast specialization. The macrophage-derived activity was nondialyzable and heat-stable, and it was eliminated by exposure to trypsin. Inhibition of prostaglandin cyclooxygenase failed to modify its generation. Partial purification (Amicon filter concentration, gel filtration) disclosed principal peaks of activity corresponding to Mr of 43,000 and 10,000. The crude conditioned medium and the Mr 43,000-peak, but not the low-molecular-weight peak, exhibited interleukin 1 activity, as judged by the ability to stimulate the proliferation of mouse thymic lymphocytes. The macrophage-derived growth factor described herein may participate in bone remodeling and repair and in primary bone and cartilage growth.

Production and characterization of monoclonal antibodies to human interleukin 2: strategy and tactics.

The unique hormonal characteristics of human interleukin 2 (IL 2), primarily the high affinity of the IL 2-receptor interaction, created several impediments to the generation of monoclonal antibodies to this lymphokine. Because normal cell sources produce only a few micrograms of IL 2 per liter, it was necessary to utilize high producer clones and subclones of a human T leukemia cell line to obtain immunogenic amounts of IL 2 protein. Moreover, assays that required antibody-mediated intervention of the high affinity IL 2-receptor binding were ineffectual in the identification of anti-IL 2-producing hybridomas, thus necessitating the development of immunoassays. Two of three initially derived antibodies detected by enzyme-linked immunoassay were found to react specifically with IL 2 as demonstrated by antibody concentration-dependent neutralization of IL 2 activity. The neutralization of cellular proliferation was specific for IL 2-reactive cells, coincided with an inhibition of IL 2 receptor binding, could be completely overcome by affinity-purified IL 2 and was species-specific; human and murine IL 2 were neutralized, whereas rat IL 2 activity remained unaffected. A third antibody, although much less effective in neutralizing IL 2 activity, bound to IL 2 more avidly and functioned as an efficient immunoabsorbent. IL 2 could be concentrated and purified by immunoabsorption from crude conditioned medium in a single step. The purified product, which retained biologic activity, was made up of a single protein (Mr = 15,500) as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase liquid chromatography, and amino-terminal amino acid sequence analysis. These results indicate that the distinctive biochemical and functional properties of individual lymphokines may well determine the efficiency with which antibodies may be elicited and detected. However, once produced, anti-lymphokines are uniquely suited for the exploration of the molecular and biologic properties of these immunoregulatory molecules.