Use of monoclonal antibodies to human apo A-I in the determination of antigenic sites present on HDL surface.
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Antibodies, Monoclonal↗
Biomedical subjects
B Pau
Publications and source records attributed to B Pau.
Antigenic domains on the human thyroglobulin molecule recognized by autoantibodies in patients' sera and by natural autoantibodies isolated from the sera of healthy subjects.
We determined the regions on the human thyroglobulin (hTg) molecule recognized by anti-hTg autoantibodies (aAbs) in the sera of patients with Hashimoto's thyroiditis, Graves' disease, and thyroid carcinoma and by anti-hTg natural aAbs isolated from the sera of healthy subjects. Fifteen anti-hTg monoclonal antibodies (MAbs) directed against six distinct antigenic regions were used for this study. The anti-hTg aAbs in the patients' sera recognized mainly region II and occasionally region IV. The natural aAbs were present in the serum at low concentrations; consequently, we isolated and concentrated them for this investigation. The isolated natural aAbs inhibited the interaction of the anti-hTg MAbs with the majority of the antigenic regions identified. Region II was not well recognized, however, by these natural aAbs. This difference in specificity between the anti-hTg aAbs and the anti-hTg natural aAbs may have diagnostic significance.
Autoantibodies↗
Production and characterization of monoclonal antibodies to human growth hormone.
Three BIOZZI-HR mice were immunized with human growth hormone (hGH). From the determination of the titer, the average equilibrium association constant and the heterogeneity index of the antisera, it was possible to select the most suitable mouse for production of monoclonal antibodies (Mabs). Resulting from a single fusion, eight Mabs were produced, purified and characterized. The equilibrium association constant of the Mabs ranged from 5.10(8) M-1 to 9.109 M-1 at physiological pH. Four areas on hGH are recognized by the Mabs (the topology of the Mabs was investigated by two-site immunoradiometric assays). The Mabs, which recognize a same area, show similar cross-reactivities between hGH and human Placental Lactogen (hPL). No selected Mabs bound human Prolactin (hPRL), equine Growth Hormone (eGH) and porcine Growth Hormone (pGH). Two complementary Mabs enable a two-site immunometric assay of pituitary and E. Coli derived hGH.
Animals↗
Production and characterization of highly specific anti-methotrexate monoclonal antibodies.
Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.
Animals↗
Production and partial characterization of monoclonal antibodies against 3,3',5-triiodo-L-thyronine.
Spleen cells of Biozzi HL mouse (selection V) immunized with bovine albumin-triiodothyronine conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Thirteen monoclonal antibodies, selected by an enzyme-linked immunosorbent assay and a radioimmunoassay, were produced in mouse ascites fluid, purified and analyzed. All of them were IgG1 (kappa). The cross-reactivity of all these monoclonal antibodies with thyroxine (T4) was less than 0.2%. The association constant determined by Scatchard analysis ranged from 1.5 x 10(9) M-1 to 2.7 x 10(10) M-1.
Animals↗
Measurement of active renin by the 4G1 anti-human renin monoclonal antibody.
Three monoclonal human renin antibodies have been selected to settle two immunoradiometric assays of human plasma renin (IRMA). The first pair of antibodies 3E8-4G1 recognizes exclusively active renin (AR), as demonstrated by the lack of increase either of the number of AR molecules or the renin enzymatic activity, when plasma is set free of inactive renin (IR) by immunoaffinity chromatography with human renin prosegment monoclonal antibody. The second pair of monoclonal antibodies 4E1-3E8 gives results which are very significantly correlated to those obtained with the 3E8-4G1 pair after trypsin activation. The standardization of renin measurements with use of monoclonal antibodies, and the standardization of renin will allow the exchange of comparable informations between the various laboratories involved in the investigation of the renin-angiotensin system in humans.
Antibodies, Monoclonal↗
[Monoclonal antibodies and new markers in breast oncology].
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Antibodies, Monoclonal↗
Analysis of inactive renin by renin profragment monoclonal antibodies.
Two peptides were synthesized, corresponding to the sequences (-19 to -7) and (-26 to -17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (-19 to -7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.
Antibodies, Monoclonal↗
Distribution of the Mr 52,000 estrogen-regulated protein in benign breast diseases and other tissues by immunohistochemistry.
A secreted glycoprotein with a molecular weight of 52,000 is induced by estrogen in breast cancer cells and has been purified to prepare monoclonal antibodies. The protein has been detected in some breast cancers but not in normal breast and uterus. In order to study its potential value as a marker, we have tested by immunohistochemistry frozen sections of several normal and malignant tissues and of benign mastopathies. Among different tissues tested, the Mr 52,000 protein was detected only in liver, sweat glands, and some sebaceous glands, and in malignant melanomas and some breast tumors. Other estrogen-responsive tissues (ovary, placenta, endometrium, etc.) gave negative results. Immunoradiometric assay of the Mr 52,000 protein in biological fluid revealed an elevated concentration in cyst fluid (0.5 to 7.4 micrograms/ml), pleural effusions of certain metastatic breast cancer, and sweat. By immunohistochemistry, the Mr 52,000 antigen was also detected in 42% of 129 benign mastopathies. Gynecomastia, fibrous disease, fibroadenoma, and adenosis were mainly negative, whereas ductal hyperplasia and cysts were positive. The Mr 52,000 protein was found mostly in proliferative ducts and in cysts but not in lobular hyperplasia and nonproliferative lesions without cyst. More Mr 52,000 protein was found in postmenopausal patients than in premenopausal patients. We conclude that the Mr 52,000 protein is a marker associated with mammary cysts and proliferative ducts. On the basis of the increased risk of breast cancer in proliferative mastopathies, we suggest that the Mr 52,000 protein is useful for predicting high-risk mastopathies acting as a marker associated with the proliferation of ductal tissue.
Age Factors↗
Production and characterization of monoclonal antibodies against human thyroglobulin.
Spleen cells of Biozzi-HR mice immunized with human thyroglobulin (hTg) were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty monoclonal antibodies (MAbs) selected by an enzyme immunoassay (indirect ELISA) were produced, purified and characterized. The equilibrium association constant (Ka) of one of the MAbs, determined by Scatchard analysis of the ELISA data, was found to be 2 X 10(9) M-1; the Ka of the other MAb, estimated from titration curves by comparison with the aforementioned MAb, ranged from 8 X 10(9) M-1 to 6 X 10(7) M-1. The reaction between the MAb and hTg was not inhibited by thyroxin (T4), triiodothyronine (T3) and triiodothyropropionic acid (DT3). Species specificity of the MAb was studied using bovine and porcine Tgb. The topology of the MAb was investigated by competitive inhibition immunoassays. Seven distinct antigenic regions were identified.
Antibodies, Monoclonal↗
Levels of ventricular myosin fragments in human sera after myocardial infarction, determined with monoclonal antibodies to myosin heavy chains.
Serum levels of ventricular myosin heavy chains were quantitated in patients with acute myocardial infarction using a competitive radioimmunoassay involving monoclonal antibodies to the b-type myosin heavy chains of a human ventricle. Among the seven antibodies selected for their higher affinity for ventricular myosin heavy chains, only four antibodies detected significant and variable myosin amounts in the serum samples of nineteen patients with acute myocardial infarction; the same antibodies occasionally detected, if at all, low myosin amounts in the sera of patients with no clinical sign of myocardial infarction, and no myosin in the serum of the healthy control subjects. The peak levels of myosin release were observed 4.6 +/- 0.5 days (n = 13, P less than 0.01) after myocardial infarction and correlated rather well with the measured creatine kinase peak levels (the correlation coefficients were between 0.75 and 0.81, P less than 0.01, depending on the monoclonal antibody used for myosin determination). The time courses of myosin release varied according to the complexity of the heart attack observed. It was concluded that the titration of serum myosin was probably of little clinical value for therapeutic intervention during the acute phase of myocardial infarction; it could, however be an effective tool for retroactive detection of an infarct and for late estimation of infarct size.
Adult↗
Direct immunometric assay of active renin in human plasma.
None of the methods currently available can detect the small numbers of active renin (AR) molecules present in plasma. Among seven monoclonal antibodies (Ab), two Abs were selected which did not recognize the same epitope and could be used in a sandwich assay. The first monoclonal Ab, 3E8, binds soluble renin (B 50% = 1 x 10(-10) mol/l) and does not inhibit its enzymatic activity. It was coupled to magnetic beads (Magnogel) and was used to trap both active and inactive renin from 250 microliters plasma. The second Ab, 4G1, binds renin (B 50% = 3.5 x 10(-10) mol/l), inhibits its enzymatic activity, and recognizes inactive renin less than AR. It was iodinated and used to detect AR trapped on Magnogel by the first Ab during a 4-h incubation. The assay can detect 16 pg/ml in human plasma and is highly reproducible. The AR level of 15 normotensive subjects, aged 20-45 years, in an upright posture and on a normal sodium intake, was found to be 41 +/- 18 pg/ml (MRC renin standard). The plasmas were trypsin-activated and their total renin levels were measured with the same pair of monoclonal Abs. The mean value of 286 +/- 142 pg/ml is similar to the value obtained by other assay systems which measure total renin with Abs recognizing both active and inactive renin. The direct measurement of AR provides a convenient and standardized method, since the production of the two monoclonal Abs is unlimited.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Characterization of monoclonal antibodies to the estrogen-regulated Mr 52,000 glycoprotein and their use in MCF7 cells.
The Mr 52,000 glycoprotein is regulated by estrogen and released by breast cancer cells in culture (B. Westley and H. Rochefort, Cell, 20: 352-362, 1980). This rare protein was partially purified from 25 liters of medium conditioned by MCF7 cells and injected into Biozzi's selected mice. The spleen lymphocytes of one immunized mouse was fused with the murine myeloma P3-X63-Ag8-653. Sixteen hybridomas producing monoclonal antibodies to the Mr 52,000 protein were isolated, and seven of them were cloned and purified. The seven monoclonal antibodies were all of the immunoglobulin G1 isotype, and their dissociation constants ranged from 0.35 to 2.3 nM. The antibodies specifically recognized the secreted Mr 52,000 protein as evidenced by double immunoprecipitation and by immunoblotting after electrophoretic separation and transfer. Double-determinant immunoradiometric assay indicated that the seven purified monoclonal antibodies recognized three distinct regions of the Mr 52,000 protein, and it was used to assay the Mr 52,000 protein in biological fluids. These antibodies did not react with the external plasma membrane of MCF7 cells, as shown by immunofluorescence analysis. By contrast, the cytoplasm of MCF7 cells (but not T47D and RBA cells) was stained by the peroxidase-immunoperoxidase complex after plasma membrane permeation, indicating that the protein is secreted by exocytosis rather than shed from the plasma membrane.
Animals↗
Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in "ELISA" and immunoprecipitation technics.
Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.
Antibodies, Monoclonal↗
Immunohistochemical detection of the estrogen-regulated 52,000 mol wt protein in primary breast cancers but not in normal breast and uterus.
An estrogen regulated glycoprotein of molecular weight 52,000 is released by metastatic human breast cancer cells in culture. In order to detect this protein directly in human tissues, several high affinity monoclonal antibodies were produced against the 52,000 mol wt protein. Frozen sections of human breast cancer samples were stained by the peroxidase-anti-peroxidase method using these antibodies. In 20 of 25 samples, specific immunoperoxidase staining was observed in the cytoplasm of epithelial cells with six monoclonal antibodies to the 52,000 mol wt protein. The 5 samples that were not stained contained no detectable estrogen receptor. Epithelial cells were not stained in 6 normal mammary glands collected during reduction mammoplasties and in 9 normal uteri, whether tissues were collected during the follicular or luteal phase. The demonstration that the 52,000 mol wt estrogen regulated protein is present in the cytoplasm of some primary breast cancers but absent in normal mammary tissue and uterus indicates its possible use as a tumor marker.
Antibodies, Monoclonal↗
The purification of a high-molecular-weight, enzymatically inactive renin precursor from human kidney.
A high-molecular-weight enzymatically inactive form of renin has been purified to homogeneity from human kidney. It has an Mr of 48 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and an Mr of 51 000 by gel filtration on Sephadex G100. It is activated by treatment with trypsin and reversibly activated by exposure to acid. We conclude that this material represents a human prorenin.
Chromatography, Affinity↗
Centrifugal analyzer used for enzyme immunoassay of progesterone and choriomammotropin.
Recently we developed an enzyme immunoassay involving the use of steroid delta-isomerase (EC 5.3.3.1) as enzyme label and exclusion-affinity chromatography for rapid separation of free antigen-enzyme conjugate that bound to antibodies (J. Immunol. Methods 35: 267-284, 1980). Here we describe an automated version of this procedure, for immunoassay of progesterone and choriomammotropin (human placental lactogen) in serum with the use of a centrifugal analyzer. After incubation, suitable dilutions of sera or extract plus antiserum, conjugate, and double antibody were filtered on an estradiol affinity gel-filtration column; the enzyme activity of the filtrates was determined with the centrifugal analyzer. Results correlate well with those obtained by radioimmunoassay: r (progesterone) = 0.980, r (choriomammotropin) = 0.940. The within-run and between-run precision, specificity, sensitivity, accuracy, and speed of this system make it a useful tool for immunoassay.
Autoanalysis↗
[Immunotoxins].
Immunotoxins are conjugates between antibodies especially directed against cancer cells and a subunit of a powerful toxin. We used the A-chain of ricin. These conjugates are specifically cytotoxic when used at very low concentrations in vitro and can destroy more than 99.99% of clonogenic cells. The efficacy of immunotoxins was also demonstrated in vivo but is inferior to its in vitro potency. For this reason the first use of immunotoxins in man can be the cleaning up of bone marrow from leukemic cells in the near future.
Animals↗