Philippe Pradelles

Negative regulator of pluripotent hematopoietic stem cell proliferation in human white blood cells and plasma as analysed by enzyme immunoassay

This paper describes the analysis, by a highly sensitive and specific enzyme immunoassay (EIA), of AcSDKP, a tetrapeptide recently isolated from fetal calf bone marrow and subsequently purified and identified which substantially inhibits entry into cycle of hematopoietic pluripotent stem cells (CFU-S). This molecule has a marked protective effect in mice during anticancer chemotherapy with phase-specific drugs and plays an essential role in maintaining CFU-S out of cycle in normal mice. Using acetylcholinesterase-AcSDKP conjugate as tracer, rabbit specific antiserum and 96-well microtiter plates coated with a mouse monoclonal anti-rabbit IgG antibody, this EIA allows detection of AcSDKP at 15 fmol levels with a coefficient of variation less than 10% in the 50-500 fmol range. When combined with high-performance liquid chromatography, this assay clearly reveals the presence of this peptide in normal human white blood cells whereas in supernatant from cultured lymphocytes and in plasma the immunoreactive material is distinct from standard AcSDKP.

Distribution of a negative regulator of haematopoietic stem cell proliferation (AcSDKP) and thymosin, β4 in mouse tissues

A competitive enzyme immunoassay using acetylcholinesterase as tracer for thymosin β4, has been developed. Using this assay and a previously described EIA for AcSDKP, a negative regulator of pluripotent haematopoietic stem cell proliferation, the levels of these two peptides were determined in mouse tissue extracts. The combination of EIAs with different HPLC procedures validated these methods and clearly demonstrated the ubiquity of these peptides in mouse tissues. Similar results are reported for rabbit thymus which suggest different hypotheses for AcSDKP biosynthesis.

Determination of ILlα, ILlβ and IL2 in Biological Media using Specific Enzyme Immunometric Assays

Interleukins (ILs) are polypeptides which play a critical role in the inunune response by regulating leukocyte interactions (Mizel 1989). In addition, ILls (ILla and ILl/?) have been shown to be intimately involved in inflammatory reactions (Dinarello 1988). A clear understanding of IL synthesis and release both in vitro and in vivo requires very sensitive and specific assays. Initially, ILs were measured by means of various bioassays (Gearing & Thorpe 1989) which often proved very sensitive but suffered from a lack of specificity (the assays are sensitive lo different ILs), and were influenced by non-IL substances (activators or inhibitors). In addition, these assays are time-consuming, cumbersome and not ideal for routine analysis. For all these reasons, immunoiogical methods appear as very promising alternatives which obviate these drawbacks., and a great number of immunoassays (mainly radioimmunoassays and enzyme immunoassays) have been described during recent years (Lisi et al. 1987, Lonnemann et al. 1988, Ferrua et al. 1987, 1988, Tanaka et al. 1988, Kitamura et al. 1989, Poole et al. 1989). Our approach was to develop two-site immunometric assays based on the use of tandem monoclonal antibodies (mAb). In these assays, a 1st mAb is immobilized on a solid phase (wells of microtiter plates) while a 2nd mAb is covalentiy labelled with the enzyme acetylcholinesterase (AChE). We chose AChE as labelling enzyme because we have previously shown that this enzyme has

An enzyme immunoassay for synthetic thymulin

Thymulin, a metallononapeptide with the following aminoacid sequence: pyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-AsnOH is a thymic hormone involved in T cell differentiation requiring zinc to express biological activity as measured by the rosette assay. We established an enzyme immunoassay (EIA) for synthetic zinc-free thymulin with a thymulin-acetylcholinesterase conjugate as tracer and specific polyclonal rabbit antithymulin antibodies. The assay is performed as a classical competition assay in microtiter plates previously coated with mouse monoclonal IgG to rabbit IgG. A quantitative thymulin assay more sensitive than radioimmunoassays (RIAs) previously described was obtained with a sensitivity (IC50) of 32.5 +/- 5 pg/ml and a detection limit of 5 pg/ml. Analysis in the EIA of synthetic thymulin analogs showed that the minimal peptidic structure necessary for enzymatic tracer competition is the C-terminal part Lys3 to Asn9. It was also shown that the biologically active form of thymulin (zinc-bound) has the same immunoreactivity as zinc-free thymulin and that other thymic hormones, thymosin alpha 1 and thymopoietin II (or TP5) and unrelated short peptides do not cross-react with thymulin. These data demonstrate the specificity of this EIA for thymulin and show its suitability for application in biological fluids.

Enzyme immunometric assay for leukotriene C4

An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

Enzyme immunometric assay of thyroliberin (TRH).

An enzyme immunometric assay of thyroliberin (TRH) using monoclonal antibodies and a derivatization procedure is described. This assay, named SPIE-IA, involves a four step procedure after chemical derivatization of TRH and biological samples by diazotized APEA. Step 1: derivatized TRH was immunocaptured by a monoclonal anti-TRH antibody coated on a 96-well microtiter plate. Step 2: after washing, derivatized TRH was cross-linked via its amino group to the wells using glutaraldehyde. Step 3: washing and treatment with NaOH. Step 4: measurement of bound TRH using a monoclonal anti-TRH antibody labeled with acetylcholinesterase. The minimal detectable concentration was 0.1 pmol/ml: with a coefficient of variation less than 10% in the 0.156-10 pmol/ml range. This assay is 26-fold more sensitive and more specific than the competitive enzyme immunoassay using the same monoclonal capture antibody, derivatized TRH and TRH-acetylcholinesterase conjugate as tracer. Good correlation was observed between SPIE-IA and a sensitive competitive enzyme immunoassay using polyclonal antibodies.

AcSDKP serum concentrations vary during chemotherapy in patients with acute myeloid leukaemia

AcSDKP is a physiological negative regulator of cell proliferation in mammals. In Ara‐C‐treated mice its plasmatic concentrations decrease while the CFU‐S start cycling. Infusion of AcSDKP protects these animals from death by blocking the proliferation of primitive haemopoietic cells. We measured AcSDKP serum concentrations in 20 AML patients during the course of high‐dose cytoreductive treatment. We observed an early and sharp increase of AcSDKP during the induction treatment in 12 patients, reaching a peak during the initial 3 d of treatment in nine of them. These results are contrary to those observed in mice treated with high doses of Ara‐C. They encourage further clinical investigation, and suggest that treatments with synthetic AcSDKP (Seraspenide) will perhaps have to be adjusted to the type of disease and the schedule of chemotherapy in order to optimize its myeloprotective effect.

Enzyme immunoassays for Thyroliberin (TRH) and TRH-elongated peptides in mouse and rat hypothalamus

Enzyme immunoassays (EIAs) for Thyroliberin (TRH) and TRH-elongated peptides were developed. Three haptens less than E-H-P-NH2 (TRH). Less than E-H-P-OH (TRH-OH), and S-K-R-Q-H-P-G-K-R-F (P10) were conjugated by the use of different heterobifunctional cross-linking agents either to sun-flower globulin as carrier or to acetylcholinesterase as tracer. For a same hapten, the same chemical group in the peptide was used to prepare the immunogen and the enzyme conjugate. These EIAs were performed with a second antibody solid phase technique using acetylcholinesterase as label. They permitted the measurement of TRH and TRH-elongated peptides with a sensitivity threshold of 10 fmol/well for TRH and 2 fmol/well for P10. TRH EIA only detected authentic TRH whereas TRH-OH EIA detected TRH and TRH peptides elongated on C terminal part. Anti-P10 serum was specific of TRH peptides elongated both on C and N terminal parts and no cross reactivity was observed with TRH. Using these assays, TRH and TRH-elongated peptides were determined in crude or chromatographed mouse and rat hypothalamus tissular extracts. Several TRH extended forms were identified by P10 EIA, whereas TRH-OH EIA permitted detection of both TRH and TRH-elongated peptides in chromatographed extracts. Authentic TRH was measured by TRH EIA both in crude and chromatographed hypothalamic extracts. These assays can permit the study of the processing and maturation of TRH.

Development of an enzyme immunoassay for arginine-vasopressin (AVP)-like insect diuretic hormone

1. The AVP-like insect diuretic hormone is a biologically active antiparallel dimer present, along with its non-active monomeric form (Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-GlyNH2), in the African locust. 2. It exhibits diuretic activity by increasing fluid excretion at the level of the Malpighian tubules. 3. To date, both monomer and dimer have been assayed using a radioimmunoassay originally prepared for mammalian AVP. 4. We have developed here an original enzyme immunoassay based on the use of antibodies to insect AVP-like raised in rabbits against synthetic monomers and dimers, using acetylcholinesterase conjugate as an enzymatic tracer. 5. This enzyme immunoassay enables measurement of the dimer to be made with adequate sensitivity (0.3 nmol/l, i.e. 21 pg/well) and reproducibility while sensitivity of the monomer is somewhat lower (14 nmol/l, i.e. 480 pg/well). 6. The assay was validated by assaying native dimer and monomer throughout the different steps of purification (from a crude extract to reversed-phase liquid chromatographic fractions). 7. A good correlation was observed between radioimmunoassays and enzyme immunoassays. 8. The enzyme immunoassay was also used to measure the level of AVP-like peptides in several insect tissues not explored to date.