Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules

Definitions

• This invention relates to monoclonal antibodies to the H-Y antigen and to their uses in differentiating between males and females.
• the monoclonal antibodies of the invention are of special interest in determining the presence of the H-Y antigen on the cell surface or in serum of mammals or in growth media of embryos.
• the invention also relates to hybridoma cell lines which produce antibody to the H-Y antigen and the monoclonal anti H-Y antibodies which are raised by said cell lines.
• the invention also relates to methods of use of these H-Y antibodies to differentiate and separate embryos by sex, to determine the presence of H-Y antigen in serum of vertebrates including various species of mammals and birds, to assign H-Y phenotype and to diagnose and clarify the nature of aberrant sexual development.
• a particular noteworthy aspect of the invention is that viable (live) male and female embryos are identified rather than embryos which are not viable anymore because of the selection process to which they have been subjected.
• the invention also relates to live embryos which are identified by an indicia (indicator) which provides information or is gender-specific, and to compositions comprising such embryos.
• Another particular noteworthy aspect of the invention is the unusually high activity of the H-Y antibodies (as measured by traditionally accepted tests) used in this invention.
• Embryo transfer and especially bovine embryo transfer has become an important commercial technique in the food industry. Embryo transfer refers to placing an embryo into the lumen of the oviduct or uterus. In a broad sense, however, embryo transfer has come to mean the sequence of steps for moving embryos from one female to another, including superovulation, embryo recovery and storage of embryos in vitro.
• the donor is the genetic mother from which embryos are recovered; the host, or recipient, is the surrogate mother that receives the embryos after storage.
• An informative discussion of bovine embryo transfer is found in Superovulation and Embryo Transfer in Cattle, by George E. Seidel Jr., 211, Science 351 (1981) which is herein incorporated by reference.
• Sex identification of embryos before transfer has several applications, especially in the cattle industry. Because of artificial insemination, the number of bulls required for breeding is greatly reduced and the value of the few very good bulls is greatly increased. Thus male calves will be more valuable than female calves in a few instances. In the great majority of cases, however, females are considerably more valuable than males. It is financially risky if one must count on at least five females from ten pregnancies for profitability. For example, in seven natural births, the probability of seven offspring of one sex is 0.0078125 if the true sex ratio is 50:50.
• sex is determined by the sex chromosomes.
• XY embryos with an X and a Y chromosome
• XX embryos with two X chromosomes
• sex-reversal can be explained by abnormalitites of Y chromosome function, as in XY females, or abnormal retention of Y chromosome function, as in XX males.
• H-Y antigen The Y chromosome is responsible for production of a cell surface molecule or group of molecules called H-Y antigen. Since there is no Y chromosome in normal females, H-Y is a male specific antigen, and can be used to produce a state of immunity in females. Thus, H-Y, meaning histocompatibility-Y, antigen was discovered with the observation that among highly inbred populations of mice, skin grafts from males (H-Y + ) are rejected by females (H-Y-), whereas skin grafts exchanged among the other three sex combinations (male-to-male; female-to-female; female-to-male) are accepted. Therefore H-Y antigen can be used as a marker for identification of male cells.
• H-Y antibodies Female mice that have been exposed to male tissues, as in skin grafts or inoculations of cells in suspension, produce antibodies (called H-Y antibodies) that can be used to identify male cells in serological systems.
• the antibodies specifically recognize and physically combine with H-Y antigen.
• H-Y antigen shall refer to the male-specific cell surface antigen or antigens identified by and capable of combining with H-Y antibody.
• Male cells can be identified by any of several assays. The following procedures are illustrative.
• H-Y antibody is used for applications of H-Y serology in the sperm cytotoxicity test.
• male cells are killed or labeled in such a way as to facilitate visual scoring or identification.
• An example of the killing technique is the complement-mediated epidermal cell cytotoxicity test, in which only male epidermal cells are killed. Scheid M, Boyse EA, Carswell EA, Old LJ, Serologically Demonstratable Alloantigens of Mouse Epidermal Cells, 135 J. Exp. Med. 938-955 (1972).
• H-Y antiserum which contains H-Y antibodies
• the white blood cells absorb the H-Y antibodies.
• the relevant antibodies the H-Y antibodies
• the antiserum loses its ability to react with H-Y target cells in cytotoxicity and labeling assays.
• Female cells do not absorb H-Y antibodies. See Wachtel SS, H-Y Antigen and the Biology of Sex Determination, Grune and Stratton, N. Y. 1983.
• H-Y + Other techniques for identification of male cells (H-Y + ) are reviewed in Wachtel, 1983, supra. They are useful techniques which can be used in the practice of this invention and such techniques are incorporated herein by reference.
• H-Y antibodies of the mouse recognize male cells of rat, guinea pig, rabbit and man. See Wachtel SS, Koo GC, Zuckerman EE et al., Serological Crossreactivity Between H-Y [Male] Antigens of Mouse and Man, 71 Proc. Nat. Acad Sci. U.S.A. 1215-1218 (1974). It has also been reported that H-Y antibodies of the mouse recognize male cells of the leopard frog and female cells of the chicken, Wachtel SS, Koo GC, Boyse EA, Evolutionary Conservation of H-Y ('Male') Antigen, 254 Nature 270-272 (1975).
• H-Y antibodies also recognize male cells of the goat (Wachtel SS, Basrur P, Koo GC, Recessive Male-Determining Genes, 15 Cell 279 (1978)), horse (Sharp AJ, Wachtel SS, Benirschke K, H-Y antigen in a fertile XY female horse, 58 J. Reprod. Fert. 157 (1980)), and bovine (Wachtel SS, Koo GC, Ohno s, "H-Y Antigen and Male Development", The Testis in Normal and Infertile Man, Troen P and Nankin HR, ed., Raven Press, New York 1977)).
• H-Y antigen is widespread among the vertebrates and it was concluded that H-Y antibodies of the mouse or rat can be used to distinguish between male and female cells of any vertebrate species.
• the invention is applicable by use of or with conventional or monoclonal H-Y antibodies produced in other vertebrate species, such as the rabbit or rat.
• Application involves the use of the same procedures as described in this specification.
• H-Y antigen The ontogeny of the appearance of H-Y antigen in embryos is not known.
• the presence of H-Y antigen in bovine embryos of 150 to 175 days has been demonstrated (Ohno S, Christian LC, Wachtel SS, Koo GC, Hormone-like role of H-Y antigen in bovine freemartin gonad, 261 Nature 597 (1976); Wachtel SS, Hall JL, Müller U, Chaganti RSK, Serum-Borne H-Y Antigen in the Fetal Bovine Freemartin, 21 Cell 917 (1980)).
• H-Y antigen in 8-cell embryos of the mouse has also been demonstrated (Krco CJ and Goldberg EH, Detection of H-Y (Male) Antigen On Eight-cell Mouse Embryos, 193 Science 1134-1135 (1976)).
• H-Y antigen is also helpful in the study of abnormal sexual development.
• H-Y antigen has been demonstrated in the cells of XX males (Wachtel SS, Ohno S, Koo GC, Boyse EA, Possible role for H-Y antigen in the primary determination of sex, 257 Nature 235 (1975)) and in XYY males (Wachtel SS, Koo GC, Breg WR, Elias S, Boyse EA and Miller OJ, Expression of H-Y Antigen in Human Males with Two Y Chromosomes, 293 New England J. Med. 1070 (1975)).
• the areas of H-Y research have been hampered by the lack of reliable H-Y antiserum.
• Antiserum is conventionally produced by injecting the antigen of interest into an immunologically responsive laboratory animal such as a mouse or rat, and subsequently preparing antiserum from the blood of the animal, which will contain a mixture of antibodies developed against the antigen together with other antibody substances.
• an immunologically responsive laboratory animal such as a mouse or rat
• Conventional mouse antibody not only is usually low-titered but contains naturally occurring heteroantibodies reactive with cell surface components of other species. The heteroantibodies would react indiscriminately with male and female embryos of other species.
• X-bearing sperm will elute out of a column in which Bryant's Immunoglobulin G antiserum is coupled with solid phase immunosorbent material, while the Y-bearing sperm attach to the antiserum on the column.
• the Y-bearing sperm are then eluted out of the column separately with more antiserum solution in accordance with the principles of competitive binding.
• Such conventionally-produced H-Y antiserum although it contains H-Y antibody substances, is usually low-titered and contaminated with heteroantibody and autoantibody which will react with male and female cells of other species due to species-specific cell surface components which are not related to the H-Y antigen.
• Hermaphroditic differentiation in XX subjects is believed to be caused by the presence of Y-chromosome material as an intact Y chromosome in a mosaic cell line, or as a minute part of extra Y- material attached to an X chromosome or an autosome.
• monoclonal antibody has the advantage of purity, specificity and potency, over conventional antibody, which consists, as discussed above, of a heterogeneous collection of relevant and irrelevant, or contaminant, antibodies. Characteristically, monoclonal antibodies can be used in highly dilute form.
• Spontaneous fusion of cells is usually rare with some exceptions, i.e., that of sperm and egg. Nevertheless two cells can be made to fuse by the addition of a fusing agent such as polyethylene glycol.
• a fusing agent such as polyethylene glycol.
• the fusion of the plasma membranes results in the formation of heterokaryons which possess two or more nuclei. At the next division the nuclei fuse and a hybrid cell results.
• the hybrid cells produce a single antibody and all descendants (the monoclone) of the hybrid cell produce the original, single antibody.
• Each monoclonal antibody is a single chemical entity, a protein with an amino acid sequence that can generally be determined.
• the cells that produce the antibody may be permanently maintained.
• the hybrid cells give pure antibody in response to not only a pure antigen but also in response to a complex stimulus such as a whole virus or a whole cell.
• Lymphocytes removed from the spleen of an animal previously injected with the selected antigen of interest are allowed to fuse with myeloma cells in the presence of polyethylene glycol or equivalent known fusogen.
• Thousands of "hybrid" myeloma cells are produced from the fusion.
• the supernatant from growth of each "hybridoma" cell culture is tested for the presence of the desired antibody activity. When such activity is found in the supernatant of one cell culture, it is cloned by limiting dilutions, and the clones produced are individually assayed for supernatant activity. Once the active cells have been identified they are cloned to reduce the chance of being overgrown by irrelevant cells. Once the hybridoma cells have been successfully cloned, they may be grown in bulk.
• the antibodies formed by these cells may then be purified using a variety of techniques including affinity chromatography on protein A-Sepharose. However, not all immunoglobulins bind to protein A since binding depends on the species and subclass of the antibody. In these cases the antibody may be purified by ion exchange on DEAE columns or affinity chromatography on anti-Ig-agarose. A discussion of hybridoma production and purification is contained in Goding JW, Antibody Production by Hybridomas, 39 J. Immun. Meth. 285-308 (1980), incorporated herein by reference.
• H-Y antibody methodologies have produced monoclonal anti-H-Y antibodies of widely varying specificities and physiological characteristics.
• An H-Y antibody less than highly specific will not provide an accurate detection of solely the H-Y antigen cell surface molecule (or component) which is necessary to enable identification and separation of male from female cells and accurate prediction of the results of sex immunoselection techniques, or locate fragments of Y chromosomes in cells of patients with abnormal sex chromosome constitutions, which are objects of this invention.
• monoclonal antibodies of the invention were found not to be too specific to the H-Y antigen inasmuch as they recognized the antigen, whereas they could also have failed to detect it for undue specificity.
• the production of a pure H-Y antibody which recognizes the H-Y antigen is thus highly desirable. Antibodies with higher titers would thereby be obtained and unwanted reaction caused by contaminants eliminated.
• Another object of the invention is a method for sex identification of bovine embryos and the products of such identification and selection.
• Another object of the invention is a method for sex identification of bovine embryos and the separation of live male embryos from live female embryos or vice versa.
• Another object of the invention is to provide a method for the assignment of H-Y phenotype in male, female and intersexual patients with chromosomal, genital, or endocrinological disorders.
• the invention provides pure, high-titer H-Y antibodies for various uses in the medical and veterinary fields of particular interest for use in testing and differentiation between sexes of embryos, for instance in cattle, and in other applications where such recognition is necessary.
• the invention provides for monoclonal antibodies to H-Y antigen and for methods and applications by which these antibodies are used in the medical and veterinary fields.
• H-Y antigen is present in all vertebrate XY embryos from the 8 cell stage to the late blastocyst stage. These are the stages at which the embryos can be used in accordance with the invention for identification of gender.
• H-Y antigen in bovine embryos is present at the time of embryos transfer. Many antigens have a transient expression and it is known that proteins which are involved in the biological cycle will appear and disappear. (M. Polackova, Ontogenetic Development of the Male-Specific Antigen in Mice, 16 Folia Biologica (Praha) 12-19 (1970)).
• the H-Y antigen is not present at the 2-4 cell stage, though it appears later.
• the timely presence of the H-Y antigen when the embryos is transferred is an unexpected and yet very important aspect of the invention.
• H-Y antigen is present in male bovine embryos during the entire period of the embryos' development during which a surgical or nonsurgical embryonic transfer can be performed without loss of viability of the embryo.
• Figure 1 shows the Inhibition of Reaction between TS, testis supernatant containing soluble H-Y antigen, and anti H-Y antibody ( ⁇ ) by supernatant fluid of male (O) and female (X) lymphocytes.
• Figure 2 shows Optical Density Scores which show the ratio of reading in the presence of lymphocyte supernatant to reading in presence of Hanks buffer alone. Each point represents the mean of duplicate scores from a single donor.
• Figure 3 shows Inhibition of uptake of labeled H-Y antigen by male and female serum.
• Figure 4 is a flow chart showing methodology for two-dimensional gel electrophoresis for H-Y antigen.
• Figure 5 shows a computer graphic printout of immunoprecipitation of H-Y antigen with H-Y antiserum.
• novel hybridomas producing novel antibodies to the H-Y antigen the antibodies themselves and diagnostic and therapeutic methods which employ the antibodies.
• the hybridomas were prepared generally by the immunization of highly inbred female mice with spleen cells from male mice of the same highly inbred population.
• the spleen cells from the immunized female mice were fused with cells from a mouse myeloma line and the resultant hybridomas were screened for supernatants containing antibody to the H-Y antigen.
• the desired hybridomas were subsequently cloned and characterized.
• the antibodies are used to identify the sex of individual embryos and as a diagnostic tool where aberrant sexual development is known or to be determined.
• the embryos of the selected mammal are exposed to the H-Y antibodies when the mammalian embryo has reached the stage at which there has developed H-Y antigen.
• bovine species this stage is reached when the cell division has adequately progressed and prior to the fetus stage.
• the embryos can be approximately 6 to 12 days old if best results are to be obtained. It may vary from mammalian species to mammalian species.
• the monoclonal antibodies thus produced in accordance with the invention are then used in the identification of male and female embryos.
• the identification of each embryo may be directly or indirectly accomplished.
• a direct method see Examples 1 and 2, infra
• the monoclonal antibody binds to the male embryo but not the female embryo.
• a secondary reagent such as Protein A or Protein A plus a visual or, when appropriate, a radioactive label
• an indirect method see Example 10, infra
• a single embryo is cultured for a suitable amount of time in a microtiter plate. The embryo is then removed to a separate nutrient medium and the first supernatant liquid is tested for the presence or absence of soluble H-Y antigen, thereby identifying the gender of the embryo.
• the secondary reagent added should be of the IgG type which can in turn react with Protein A or an antibody which itself has a label.
• the embryo from either means whose sex has been determined is then transferred to a pseudo-pregnant female (a female that has been set up hormonally) by standard procedures, for example but not limited to those described in Seidel, supra, for bovines. In this manner, up to about 85% of calves whose sex had been correctly identified are born at term and healthy.
• methods may be used to identify and provide live embryos of predetermined sex of those mammal vertebrates which produce H-Y antigen, more especially presexed embryos of bovines, equines, porcines, ovines and caprines.
• hybridoma clones and H-Y antibodies.
• the hybridoma clones are designated as murine hybridoma clones No. 2-4/3; gw 9/8; 16-3/6; 109-6/2; and 113. Specifically, these were produced by the following procedure. Panels of C57BL/6J female mice were immunized with five weekly injections of spleen cells from C57BL/6J males. The females were bled and the serum was separated and evaluated for the presence of H-Y antibodies.
• Epididymalsperm cell suspensions were prepared from male mice of the highly inbred BALB strain essentially according to Goldberg et al, Nature (1971), supra. The epididymis was removed and cut into several pieces in phosphate-buffered saline (PBS) (pH 7.0) containing 0.5% fructose and 2% fetal calf serum (previously heat-inactivated at 56°C for 30 minutes). After 5 minutes the sperm cells were pipetted off and placed in the PBS, and suspension maintained at room temperature until use.
• PBS phosphate-buffered saline
• Equal volumes of 0.05 ml of (a) putative H-Y antibody to be tested (serially diluted 1/2, 1/4 and 1/8); (b) sperm suspension (about 5 x 10 6 cells/ml); and (c) selected absorbed rabbit complement (diluted 1/20) were incubated at 37°C for 40 minutes in a rocking waterbath. A freshly prepared solution of trypan blue dye was added during the last 7 minutes of incubation to stain dead sperm. At the end of the incubation period, the suspensions were placed on ice and live and dead sperm enumerated in a hemacytometer.
• Table 1 shows an example of the sperm cytotoxicity test with ascites fluid using monoclonal H-Y antibody 16-3/6.
• Table 2 shows the results of two sperm cytotoxicity tests with male-absorbed and female-absorbed monoclonal H-Y antibody gw 9/8.
• Antiserum diluted and divided into 3 parts (A, B, C) .
• Parts B and C absorbed with 20 x 10 6 spleen cells from inbred female and male mice.
• Two ml of myeloma cells were placed in a tissue culture flask and 23 ml RPMI added together with 1% Azaguane.
• PEG polyethylene glycol
• HAT hypoxanthine aminopterin thymidine
• the suspensions were then plated onto 96-well feeder layers. After 4 days RPMI + HAT were added as necessary to each well and the medium changed twice weekly thereafter. After 2 weeks the medium was changed to RPMI + TH. Feeder layers were then prepared in 24 well plates.
• the supernatant was screened again, and positive clones transferred to 24 well feeder layer plates and then, after growth, to 96 well feeder layer plates.
• intial screening was repeated and positive clones transferred to 24 well plates. The clones were then dispersed into 4 wells each, and then to flasks.
• Selected clones were now injected into female C57BL/6J mice.
• the clones yielded ascites tumors that secreted malespecific H-Y antibodies.
• Secondary screening was now performed by absorbing the serum and ascites fluid with male or female spleen cells and scoring the serum in the sperm cytotoxicity and epidermal cell cytotoxicity tests (the latter being performed in essentially the same way as the sperm cell cytotoxicity test except that male and female epidermal cells are used as targets); male epidermal cells are killed; female cells are not; male cells absorbed the antibodies in inhibition tests; female cells did not.
• clones 16-3/6; 109-6/2; 2-4/3, gw 9/8 and 113 Five clones producing male-specific H-Y antibodies were thereby identified. These were clones 16-3/6; 109-6/2; 2-4/3, gw 9/8 and 113. These can be reproduced and generated using the procedure described above. The specific titers of the antibodies ranged from 1/10,000 to 1/100,000 in the two assays (Table 3). It was further found that clone 109-6/2 produces IgG antibodies, 16-3/6 produces IgG antibodies and clones 2-4/3, gw 9/8 and 113 produce IgM antibodies. The ascites fluid in which the IgG was found contained the prominant fractions IgG 2 a and IgG 1 .
• H-Y antibodies of the invention are remarkable in their significantly higher titer. This suggests that the instant antibodies are constituted of a unique sequence of amino acids. Clones 16-3/6 and 109-6/2 are further identified in that they produce antibodies of the IgG type rather than of the IgM type. Another feature is that they also show affinity for Protein A. These specific attributes of the clones further identify and distinguish them from others.
• antibodies from clones gw 9/8 and 16-3/6 are capable of immunoprecipitating molecules of 15,000 MW and 30,000 MW in sodium dodecyl sulfate polyacrylamide gel electrophoresis (see Fig. 4).
• the procedure followed was that of Hall JL and Wachtel SS, Primary Sex Determination: Genetics and Biochemistry, 33 Molec. Cell Biochem. 49-66 (1980).
• Monoclonal H-Y antibodies of the invention may be purified and concentrated by the following method.
• the serum proteins are precipitated with 43% ammonium sulfate. Then they are purified by using PA-Sepharose 4B (Pharmacia).
• PA-Sepharose 4B Puracia
• the IgG molecules are adsorbed to the PA Sepharose at room temperature for 10-15 minutes.
• the reaction mixture is washed thoroughly in PBS and the IgG molecules (H-Y antibodies) are then eluted off the PA Sepharose with 0.1M glycine-HCl acidic buffer (pH 3.5).
• the solubilized IgG antibody is dialyzed against PBS or borate buffer.
• the purified IgG antibody is then ready for use and may be preserved in 0.1% azide.
• One of the important applications of the monoclonal antibodies of the invention is in the identification, selection and separation of a live embryo of an undetermined gender, i.e. male or female.
• the monoclonal antibodies to the H-Y antigen may be used in several ways to select and obtain embryos of a desired sex. Illustrative examples, which are not to be construed as limiting the invention in any manner whatever, are given below.
• the monoclonal antiserum is diluted in PBS with a 2.5% concentrationof IPT. After incubation, the embryos are washed 3 times in PBS with 5% IPT, and transferred to a solution of FITC-PA in PBS at a 1:20 dilution for 30 minutes.
• FITC fluorescein-isothiocyanate
• PA is a component of the cell wall of Staphylocoecus aureus that binds to the Fc portion of antibody molecules, especially IgG antibody molecules.
• Monoclonal H-Y antibodies of IgG type stick to male (H-Y + ) cells. FITC-PA selectively adheres to the H-Y antigen-antibody complexes. Thus cells of male embryos but not female embryos fluoresce under UV light.
• the embryo may be implanted immediately or it may be stored in any of the ways well known to those skilled in the art until transfer implantation can take place.
• Examples of the storage procedures include but are not limited to embryo culture at ambient temperature, storage at 0 to 10°C and storage at -196°C in liquid nitrogen.
• the live embryo of the desired gender is then implanted in the uterus of a pseudo-pregnant bovine by a nonsurgical procedure known to one skilled in the art of embryo transfer. After the necessary gestation period the calf of preselected gender is born. In this manner, female and male calves can be obtained as are preselected.
• the desired embryo is implanted by a surgical means wherein a midline incision is made in the flank of a locally anesthetized pseudo-pregnant bovine and one of the uterine tubes is exposed.
• the live embryo is inserted directly into the uterus.
• a calf of predetermined sex is born full term and healthy. This procedure is followed for the male and female calf except that in the selection of the type of embryo, whether it be male or female, the procedure is different as described above.
• Plating is another suitable method of selection. Male and female embryos (6-12 days old) are reacted for 20 minutes with monoclonal H-Y antibody. The antibody is fixed on the H-Y antigen as follows.
• Bacteriological polystyrene plates are coated with rabbit anti-mouse affinity purified antibodies at a dilution of 1:10 to facilitate embryo removal after selection (1 rabbit antimouse:10 normal rabbit IgG). Bacteriological plates are used instead of tissue culture quality plates because treatment of the plate in this system could lead to non-specific adherence of the embryo; this does not occur with bacteriological plates.
• the antibodies are diluted in 0.05 M Tris pH 9.5 buffer, poured onto the plate, swirled and kept at room temperature for 40 minutes for attachment. Next the plates are shed with PBS followed by PBS containing 1.0% FCS. The embryos are then added to the plates and the plates incubated for 2 hours at room temperature. The fluid is gently poured off to remove female embryos. Five washes are performed. For positive selection, PBS with 1% FCS is added and vigorous pipetting (Pasteur) performed to dislodge the male embryos.
• Protein-A batch settling method Twenty embryos (male and female bovine, 6 to 12 days old) are exposed to antibodies as above. A suspension of PA Sepharose (commercially available) is prepared in a bacteriological plate or Buchner funnel. The embryos are added to the PA Sepharose and gently swirled or rotated for 20-25 minutes and allowed to settle for two hours at room temperature. At the end of that period the supernatant, containing the female embryos, is gently aspirated A solution containing concentrated normal IgG is now added with gentle swirling of the plate or funnel. This competes with male-embryo-bound monoclonal H-Y antibody for available PA, thereby liberating the bound embryos.
• PA Sepharose commercially available
• the embryos of the desired sex in this case the male embryos, are implanted in the surrogate mother as in Example 1 to produce full term healthy male calves. With female calves the same procedure is followed.
• Cytotoxicity is another test for selecting the desired antibody. Embryos recovered as in Example 1 above are exposed to H-Y antibody and absorbed complement as in the cytotoxicity tests described above, under conditions that kill male (H-Y + ) cells. Dead embryos are identified visually by uptake of trypan blue dye, or by noting lysis of the cells.
• Rabbit serum is used as a source of complement, which is required for lysis in this system.
• the rabbit serum is previously selected for low toxicity to mouse thymocytes and for high complement content, indicated by its ability to support a cytotoxic reaction.
• the complement is absorbed to remove heteroantibody which might be expected to kill bovine cells indiscriminately. That procedure is accomplished by twice reacting the undiluted serum with suspensions of bovine fetal thymus, spleen, and testis cells in the presence of ethylenediamine tetraacetate (EDTA) which chelates divalent cations.
• EDTA ethylenediamine tetraacetate
• Naturally occurring anti-bovine antibodies are thereby absorbed from the serum, but the complement system is not absorbed, being inactivated in the absence of Ca ++ . After the absorption, free Ca ++ is restored by addition of calcium chloride.
• the procedure allows use of a more concentrated complement source without the complicating effects of heteroantibody.
• complement refers to a complex group of proteins in body fluids that, working together with antibodies or other factors, play an important role as mediators of immune, allergic, immunochemical and/or immunopathological reactions. The reactions in which complement participates take place in blood serum or in other body fluids, and hence are considered to be humoral reactions.
• Embryos obtained from a naturally or artificially inseminated female (or females) can each be physically dissociated into single living cells.
• One or more of the separated cells can be taken from each embryo, and the sex of that cell (or cells) determined by (i) visual examination of chromosomes; (ii) serological assay for H-Y antigen; and (iii) physical methods such as sedimentation at unit gravity, gradient density centrifugation, and migration in an electric field; or (iv) by other methods deemed necessary or appropriate as, for example, labeling of male cells with the radioactive Bkm probe. Satellite DNA from the sex-determining W chromosome of the banded krait, a venomous snake of southern Asia, contains nucleotide sequences common to the Y chromosome of the mammal.
• the snake DNA (called Bkm for branded krait minor satellite DNA) thus can be used as a probe for Y chromosome-specific DNA by allowing it to hybridize in situ with enzyme dissociated DNA in male cells according to the techniques outlined by Jones FW and Singh L, in conserveed Repeated DNA Sequences in Vertebrate Sex Chromosomes, 58 Hum Genet 46-53 (1981) (see also Singh L, Jones KW, Sex Reversal in the Mouse [Mus musculus] is Caused by a Recurrent Nonreciprocal Crossover Involving the X and an Aberrant Y Chromosome, 28 Cell 205-216 (1982)).
• DNA molecules from male and female cells are digested with the restriction endonucleases Alu I and Hae III (these enzymes cause the DNA strands to "unwind").
• the Bkm probe which has been radiolabeled by replication in medium containing tritiated thymidine ( 3 H) ( for example) is added to the preparation.
• 3 H-Bkm hybridizes in situ with complementary DNA and thereby preferentially labels male cells (which carry the corresponding sequences in DNA of the Y chromosome).
• DNA can be scored visually by allowing the chromosome preparation to expose a photographic plate (autoradiography). The resulting concentration of silver grains over the small Y chromosome readily identifies male cells.
• the polynucleotides can be transferred to nitrocellulose and then hybridized with radioactive Bkm probes and run in a one-dimensional gel electrophoresis for detailed analysis of male specific banding patterns.
• the gels are used to expose photographic plates.
• the different DNA fragments migrate in the gels according to mass and charge, and thus form a series of distinct bands. Of the various hybridization bands that result, several are male-specific. In this manner male cells are identifiable by comparison with banding patterns obtained with DNA from adult male and female cells of the same species.
• Biotin-labeled deoxyuridine triphosphate (Bio-dUTP) complexes are produced according to the method of Ward (Longer et al, 78 PNAS 6633 (1981)), and these are incorporated into cDNA by nick-translation using E. coli polymerase I (Rigby et al, 113 J.M.B. 237 (1972)) and the Bkm probe (Singh and Jones, 28 Cell 208 (1982)) as template. Then cells dissociated from bovine embryos are disrupted, and in situ hybridization performed with the biotinylated DNA probe. After reaction with enzyme (HRP), or fluorescin-conjugated avidin, or anti-biotin antibody, the ELISA test can be performed, or fluorescence determined by mucroscopy.
• HRP enzyme
• H-Y + (XY) cells This selection can be accomplished by fluorescence as in Example 1 above, or by any technique which attaches a visual marker to a cell bearing H-Y antigen.
• H-Y antibody is allowed to engage H-Y antigen at the cell surface; and a marker is allowed to engage the antibody.
• markers which recognize IgG monoclonal antibody are protein A and goat antimouse IgG, the latter being a goat antibody directed against mouse IgG antibody, which is, in this case, an antigen.
• selective identification of XY cells can be accomplished by the following tests: IFA; MHA-HA; PA-SRBC; PAP; indirect radiobinding with 125 I-PA; ELISA; and other similar assays. Typical assays are described hereinbelow.
• Target cells are first exposed to H-Y antiserum (IgG) and then to a fluorescein isothiocyanate (FITC) labeled goat mouse IgG conjugate, or alternatively to FITC-PA as described in Example 1 above. Cells are incubated for 30 minutes with the FITC-goatanti-mouse or FITC-PA and then washed gently in PBS (pH 7.0) before fluorescence microscopy (Galbraith et al. Transplantation, 1978, supra).
• IgG H-Y antiserum
• FITC fluorescein isothiocyanate
• MHA-HA Mixed hemadsorption-hybrid antibody
• H-Y antibody H-Y antibody
• SRBC antisheep red blood cells
• hybrid antibody for such a purpose, ferritin or another visual electron microscopic marker being bound to cell surface antigen by the linkage: antigen - antibody - hybrid antibody (anti-Ig:: anti marker) - marker.
• the cells are reacted with SRBC, which bind to the H-Y antigen-positive (male) cell, forming a rosette.
• the specificities of the anti Ig arm of the hybrid reagent include at least anti , anti , and anti .
• Test cells are processed in the MHA-HA test by centrifugation through a discontinuous density gradient containing the reagents in appropriate sequence, a method developed to obviate the need for repeated centrifugation (see Koo GC, Boyse EA, Wachtel SS, "Immunogenetic Techniques and Approaches in the Study of Sperm and Testicular Cell Antigens", in Immunobiology of Gametes, M. Edidin and MH Johnson ed., Alden Press, Oxford, 73-84 (1977)).
• Gradients of the immune reagents, interspersed with wash solution are established in narrow tubes made from 1 ml disposable pipettes (internal diameter, 2.5 mm).
• Heat-inactivated fetal bovine serum is added to each layer in concentrations which prevent intermixing of layers during subsequent centrifugations of the cells (range: 20% top layer, to 45% bottom layer).
• the wash layers may be visually distinguished by phenol red.
• Gradients are prepared and maintained at 4°C, and used within one to two hours after preparation.
• the gradients comprise (from top to bottom): (a) sensitized test cells, (b) wash layer, (c) hybrid antibody, (d) wash layer, (e) SRBC. After centrifugation, the tubes are allowed to stand for a minimum of 30 minutes. Each tube is cut just above the pellet, which is then gently resuspended and the labeled and non labeled cells (embryos) scored microscopically.
• PA-SRBC Protein A - sheep red blood cell test
• This test described by Koo GC and Goldberg CL (A Simplified Technique for H-Y Typing, 23 J. Immunol. Meth. 197-201 (1978)) is based on the observation of Goldberg EH, Arrington T, Tokuda S (Detection of H-Y antigen on human male leukocytes, 23 J. Immunol. Meth. 23 203-206, (1978)) that Staphylococcus can be used to label human male leukocytes.
• Target cells are exposed to H-Y antiserum and then reacted with a suspension of sheep red blood cells that have been coated with Protein-A, a cell wall polypeptide which binds immunoglobulins of the IgG class. Male cells thus form rosettes.
• PA-SRBC are prepared according to the method of Goding JW (The Chromic Chloride Method of Coupling Antigens to Erythrocytes: Definition of Some Important Parameters, 10 J. Immunol. Meth. 61-66 (1976)): A solution of Protein-A purified from the cell wall of SA (Pharmacia Co.) is added to a suspension of SRBC. To this mixture a solution of 0.01% of CrCl 3 is added. The suspension is allowed to stand for five minutes at room temperature. The PA-SRBC are washed and stored as a 10% stock solution containing 0.01% sodium azide.
• the assay for H-Y antigen is a modification of the MHA-HA test.
• Target cells are reacted with H-Y antiserum and then washed and resuspended in PA-SRBC. The mixture is spun slowly and the pellet allowed to stand at room temperature for 30 minutes before reading in a hemacytometer. Any cell with three or more SRBC is scored as a 'rosette'.
• PAP Peroxidase anti perioxidase method
• Hsu 29 J. Histochem. Cytochem. 557 (1981)
• the technique may be used to stain H-Y + embryos (males).
• Our variation consists of a primary monoclonal H-Y antibody, a biotinylated goat (affinity purified), secondary antibody and an avidin-biotinylated horseradish peroxidase complex (which consists of many biotinylated HRP molecules crosslinked by avidin into a 3-dimensional array (commercially available from Vector Labs, Burlingame, CA as
• Vectastain TM ABC kit Vectastain TM ABC kit
• the primary monoclonal antibody is reacted with the embryo or with male cells for 30 minutes. Then the second biotinylated antibody is reacted for 30 minutes. Finally the avidin DH:Biotinylated HPR H complex is added; the substrate is added; and color is produced which can be scored.
• the radiobinding assay exploits the specific reaction of protein A with the Fc portion of mouse IgG. Accordingly radio-iodinated protein A (I-PA) can be used to label H-Y antibody, thereby identifying antigen-positive cells.
• Soluble H-Y antigen from the supernatant of the mouse testicular cell preparations of Daudi cell cultures (see Wachtel supra, 1983) is diluted with phosphate buffered saline (PBS); 0.05 ml of the dilution is placed in each of several wells in a microtiter plate. The plate is incubated for several hours in dry heat to evaporate the PBS; the wells are thereby coated with a residue of H-Y antigen.
• PBS phosphate buffered saline
• the plate is washed several times and radioactivity scored in a gamma counter.
• the techniques can be used as an indirect assay by absorbing portions of H-Y antisera with male and female cells and then using the absorbed portions with soluble H-Y antigen, or as a direct assay, by using unabsorbed H-Y antisera directly with target cells in place of soluble H-Y antigen.
• Radio-iodination of PA is performed according to the techniques of Dorval G, Welsh KI, Wigzell H (Labeled Staphylococcal Protein A as an Immunological probe in the Analysis of Cell Surface Markers, 3 Scand. J. Immunol.
• Enzyme-linked immunosorbent assay This is described in detail in Example 11 below.
• the ELISA can be used to detect cell bound or soluble H-Y antigen, and as a direct or indirect test.
• the indirect test is accomplished by first absorbing H-Y antibody with male, female or test cells, and then applying the antibody as in a direct test.
• the separation is accomplished as in Example 2 above.
• the means of separation may also include sedimentation at unit gravity, gradient density centrifugation, and migration in electric field.
• H-Y antigen Absorption.
• the cells of a tissue or organism to be typed for H-Y antigen are suspended in aliquots of H-Y antibody, which is then used in cytotoxicity tests with sperm or epidermal cells, or in any assay for H-Y.
• the loss of reactivity indicates absorption of H-Y antibody and thus expression of H-Y antigen in the absorbing cells.
• H-Y antibody itself is labeled with a radioactive isotope such as 125 I. Attachment of the radiolabeled H-Y antibody to a particular (H-Y + ) cell is monitored by scoring cell bound radioactive counts per minute (cpm) in a standard scintillation counter.
• a radioactive isotope such as 125 I.
• H-Y antigen is shed or secreted by male cells in culture, and this occurs as a soluble constituent of the nutrient medium in which male cells are cultivated.
• Shalev et al (1980) reported that 'free' H-Y was present in the medium of cultured skin fibroblasts of the goat. It has been reported (Wachtel et al, 1980) that H-Y may occur as a soluble component of (male) fetal calf serum.
• H-Y antiserum is reacted with H-Y + target cells; H-Y antiserum that has been first exposed to soluble H-Y is inhibited from reacting with H-Y + target cells, because available molecules of H-Y antibody which are bound to soluble H-Y antigen have been specifically removed by male supernatant.
• the culture fluid is reacted with monoclonal H-Y antiserum in any of several assays including (i) the enzyme-linked immunosorbent assay (ELISA), (ii) the immune fluorescent assay (IFA), (iii) cytotoxicity, (iv) the Protein-A sheep red blood cell (PA-SRBC) assay, or (v) the radiobinding assay.
• ELISA enzyme-linked immunosorbent assay
• IFA immune fluorescent assay
• cytotoxicity cytotoxicity
• PA-SRBC Protein-A sheep red blood cell
• Soluble H-Y can also be detected in the enzyme-linked immunosorbent assay (ELISA).
• ELISA enzyme-linked immunosorbent assay
• Polystyrene tubes or microtiter plates are coated with H-Y monoclonal antibody in mouse ascites fluid.
• the antibody is diluted in 0.1 M bicarbonate buffer.
• the mixture is incubated for 3 hours at 37°C and then stored at 4°C until use.
• the tubes or plates are washed several times with NaCl solution.
• the antigen source is added in PBS containing 0.05% Tween 20, and the mixture is agitated gently for several hours at room temperature to promote formation of H-Y antigen-antibody complexes.
• the mixture is washed again with NaCl-Tween 20 to remove non-adherent molecules.
• Monoclonal H-Y antibody conjugated with alkaline phosphatase (diluted in PBS-Tween) is then added, and the mixture is allowed to stand overnight at room temperature. After washing, nitrophenylphosphate (NPP) is added. This reacts with alkaline phosphatase giving a yellow color to the solution. The optical density of the solution is determined with a spectrophotometer at 400nm. The amount of yellow color present is a function of the amount of alkaline phosphate present. That is a function of the amount of H-Y antigen that has complexed to the solid phase.
• NPP nitrophenylphosphate
• ELISA An alternative procedure of ELISA consists in measuring the inhibition of the reaction between monoclonal H-Y antibody and mouse testis supernatant brought about by addition of supernatants from male or female lymphocytes.
• Mouse testis supernatant (TS) a known source of soluble H-Y, is prepared according to Wachtel SS, Hall JL (H-Y Binding in the Gonad: Inhibition by a Supernatant of the Fetal Ovary, 17 Cell 327-329
• the lymphocyte supernatant is prepared by incubating 10 x 10 6 lymphocytes of each subject at 4°C overnight in Hank's buffer. Serial dilutions of TS are prepared in 0.1 M bicarbonate buffer, pH 9.6, at an estimated total protein concentration of 100 g/ml to 0.12 g/ml. One hundred fifty lambdas of each dilution are placed in wells of a polystyrene microtiter plate, and the plate is incubated at 37°C for 3 hours. After incubation, the plate is washed in 0.15 M NaCl containing 0.02% Tween 80; 90 of a 1:60 dilution of the monoclonal conjugate are added to each well.
• lymphocytes from 10 normal men and 10 normal women were isolated from the blood, the concentration was adjusted to 10 x 10 6 cells/ml Hank's, and the assay run as above using the TS at 1:30 dilution. In this case introduction of male lymphocyte supernatant gave readings 69-78 percent of those obtained with TS, (mean 75 percent). The results are shown in Figure 2. The scores are expressed as a ratio of the reading obtained when TS alone was allowed to react with H-Y antibody. In this manner it was possible to identify the male lymphocytes from the female ones.
• H-Y antigen is a component of the serum.
• the serum-borne molecule or family of molecules can be detected and assigned the proper H-Y phenotype using the following procedure.
• Monoclonal H-Y antibody is diluted in 0.1 M bicarbonated buffer, and portions of the diluted serum are placed in each of several wells of a microtiter plate. Plating efficiency is improved by addition of poly-L-lysine. The plate is incubated for three hours in dry heat (37°C) to coat the wells with antibody. After refrigeration overnight, the plate is washed several times, and human sera from male or female controls or an unknown sample are added to each well. The plate is allowed to stand at 37°C, 22°C and 4°C for twenty minutes at each temperature. After repeated washing, a small portion of triple-labeled ( 3 H-leu, 3 H-ala, 3 H-val) H-Y antigen is added to each well.
• the H-Y antigen is prepared and partially purified according to Hall JL and Wachtel SS, Primary Sex Determination: Genetics and Biochemistry, 33 Mol. Cell Biochem. 49-66 (1980). After incubation, the plate is washed again and allowed to dry. The bottoms of the wells are punched out with a manual cam punch press and dissolved in scintillation fluid for enumeration of cpm.
• soluble H-Y antigen in male serum reacts with the plated antibody.
• the reaction blocks subsequent binding of the labeled antigen, and the cpm is decreased relative to the cpm scored for wells containing female serum (H-Y).
• Figure 3 shows the results of a preliminary assay run with conventional H-Y antibody. At the optimal dilutions of 1/8 and 1/16, human male serum readily blocked solid phase uptake of labeled H-Y antigen, but human female serum did not.
• the horizontal dotted line represents standard cpm in plate containing buffer, antibody and labeled antigen. The same procedure is applicable to monoclonal H-Y antibody.
• mutant forms of H-Y antigen may also be detected.
• Biochemical systems that detect H-Y antigen offer the advantage that information about the nature of the molecule itself is gained in the same operation that determines its presence or absence. Thus mutant H-Y antigens could be detected as shown in the schematic Figure 4.
• Solubilized membrane fragments from cells of the subject under study, are dispersed in cylindrical gels according to the technique of iso-electric focusing. See Anderson NG, Anderson HL, Analytical Techniques for Cell Fractions. XXI. Two-dimensional Analysis of Serum and Tissue Proteins: Multiple Isolectric Focusing, 85 Analyt. Biochem. 331-340 (1979), which is incorporated herein by reference.
• Ampholines are added to a polyacrylamide matrix prepared in a glass cylinder. Under the influence of an electric charge, the ampholines distribute themselves so as to establish pH gradient in the gel. The solubilized membrane preparation is then added, and a current of 10,000-12,000 volt hours applied to induce migration of individual peptides. Under those conditions, the peptides migrate to their isoelectric points, where they attain a zero net charge. To insure accurate estimation of isoelectric point, proteins of known isoelectric point are focused in parallel gels. The cylindrical gels are removed and equilibrated in a second SDS buffer. Secondary application of SDS is necessary because SDS, which is negatively charged, focuses on its own, causing dissociation of SDS-peptide complexes.
• the cylindrical gels, containing the focused peptides, are now run at right angles to the initial migration, according to the technique of SDS slab gel electrophoresis, in a polyacrylamide gradient of 10-20% acrylamide.
• the cylindrical gels are placed on top of slab gels and electrophoresis carried out at 100 ma per gel until the bromophenol blue tracking dye migrates to the end of the preparation.
• the gels are fixed by soaking in a series of diluted alcohol and acetic acid washes.
• Polymers such as polyacrylamide provide a molecular sieve through which protein molecules migrate at a rate proportional to the size of the SDS-polypeptide complex. Relative molecular weight is determined by reference to migration patterns of standard protein markers.
• the peptides are thus distributed according to isoelectric point and molecular weight, and it remains to visualize them. This can be accomplished by staining with Coomassie Blue dye or by radioactive labeling of the proteins followed by auto radiography.
• visualization can be accomplished by using the commercially available silver staining technique developed by Sammons DW, Adams LD, Nishizawa EE, Ultrasensitive Silver-Based Color Staining of Polypeptides in Polyacrylamide Gels, 2 Electrophoresis 135-140 (1981) incorporated herein by reference. This method allows a third dimension of resolution based on the formation of colored polypeptide-silver complexes.
• Membranes from male and female cells are prepared by solubilization in urea or detergent as above, and the membrane fraction subjected to immunoprecipitation with H-Y antibody and PA-Sepharose according to the two step procedure.
• monoclonal H-Y antibody of class IgG is reacted with protein A-coated Sepharose beads.
• the protein A binds the Fc portion of IgG; those molecules are attached to the beads so that their antigen reactive sites are exposed.
• super natants containing H-Y antigen are added, and the mixture of beads and supernatant is agitated for one hour. This is designed to promote formation of antibodyantigen complexes at the surfaces of the beads.
• the method described here in accordance with the invention permits ready identification of a certain percentage of H-Y mutants incorporating single amino acid substitutions or simple deletions, in addition to providing typing information.
• the H-Y receptor phenotype is assigned in the following manner.
• the gonadal H-Y receptor is present in the sexually indifferent embryonic gonad in XX and XY embryos. Usually H-Y is present only in the XY gonad? and so it is usually the XY gonad that becomes a testis. Presence or absence of the H-Y receptor can be tested by adding soluble H-Y antigen to cells of the gonad in question, and then by determining whether or not the H-Y has reacted positively, i.e. is attached.
• Labeled H-Y is prepared as follows: 150 x 10 6 exponentially growing Daudi cells in RPMI 1640 with 10% FCS are suspended in RPMI 1640 with 2% FCS in which 3 H-leucine,
• 3 H-alanine and 3 H-valine (100 uci/ml each) have been substituted for "cold" leucine, alanine and valine.
• the cells are, pelleted and resuspended in RPMI 1640 with 10% FCS for a recovery period of 24 hours. During the next 16 hours the cells are incubated in RPMI 1640 with 2% FCS, containing cold leucine, alanine and valine.
• H-Y serology Other applications contemplated by the invention include several areas in H-Y serology.
• an area contemplated by the invention is in gene mapping.
• analysis of H-Y phenotype in cases of XY gonadal dysgenesis may provide valuable clues as to the risk of gonadal malignancy in that condition, with implications for etiology, as discussed further below.
• H-Y antigen can be useful in diagnosis of the existence of such genes. Another area of interest is in the diagnosis of sexual abnormality. Detection of H-Y in a female is almost always a sign of abnormality, and H-Y + phenotypes in white blood cells or cultured skin fibroblasts from phenotypic females are correlated in general with aberrant development of the gonad. Thus, it is contemplated that H-Y antigen serology be used in conjunction with the endocrinologic assays in the diagnosis of XX true hermaphroditism, i.e. sexual ambiguity caused by concurrent development of testis and ovary in the same individual.
• Clinical H-Y typing is expected to clarify the nature of aberrant sexual development. Absence of H-Y in an XY female is expected to indicate loss of testis determining genes, for instance. The presence of H-Y in an XY female is expected to indicate failure of engagement of the inducer and its gonadal receptor. It is within the contemplation of the invention to differentiate between that alternative and that failure attributable to mutation of the receptor or of the H-Y inducer.
• Another area of application of the invention is in the prediction of risk of malignancy.
• the syndrome of XY gonadal dysgenesis is characterized by retarded puberty and amenorrhea in females whose ovaries fail to develop properly.
• the gonad is usually represented by a fibrous streak of ovarian-like tissue, but germ cells and follicles are absent and endocrine function is abnormal.
• the invention therefore contemplates an early diagnosis of gonal malignancy.
• any embryo of a vertebrate e.g. warm-blooded mammal in which there is present H-Y antigen can be identified or typed for gender.
• a vertebrate e.g. warm-blooded mammal
• H-Y antigen e.g. human fet al.
• bovine selection and for highest yield of live embryos, it has been found desirable to test the embryos when they are from about 6 to 12 days old. A lesser number of viable embryos are likely to be obtained. Identification of gender can nonetheless be obtained when the treatment is performed earlier or later.
• Figure 1 shows the inhibition of reaction between TS and anti H-Y antibody (_) by supernatant fluid of male (0) and female (X) lymphocytes.
• Figure 2 is the optical density scores showing ratio of reading in presence of lymphocyte supernatant to reading in presence of Hanks buffer alone. Each point represents the mean of duplicate scores from a single donor.
• Figure 3 shows the inhibition of uptake of labeled H-Y antigen by male and female serum.
• Figure 4 is a diagram of the two-dimensional gel electrophoresis of H-Y antigen.
• Figure 5 is a computer graphic printout of immunoprecipitation of H-Y antigen with H-Y antiserum.

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