GB2223226A - A monoclonal antibody against HIV peptide - Google Patents

Description

2223226 "A MONOCLONAL ANTIBODY AGAINST A SYNTHETIC PEPTIDE" The present

invention relates to a monoclonal antibody for detecting human immunodeficient virus (HIV), etc., particularly to establishment of monoclonal antibodies which can be utilized as positive standard of high safety for various AIDS detection kits and which are applicable to basic study of HIV.

Acquired immune deficiency syndrom (AIDS) was firstly reported in America in 1981 (See, Gottlieb, M.S. et al., N. Eng. J. Med., 305. 1425(1981)). This disease is recognized to be caused by retro virus which is called human immunodeficiency virus (HIV) at present (See, Coffin. J. et al., Science, 232, 697 (1986)). For final test for detecting antibodies against HIV in serum, western blotting method has been conventionally utilized. This method comprises subjecting HIV to electrophoresis to be fractionated into constitutive proteins such as gp120, gp4l, p24, and p18, transferring the resulting fractions onto a nitrocellulose membrane to form a strip, and conducting enzyme immunoassay (EIA) by making the obtained strip to react with human serum. This method uses inactivated serum derived from AIDS patients who are known to have anti- HIV antibodies as positive standard (See an instruction manual, Bio-Rad Company, (1987)).

As noted above, in detecting methods such as EIA and Western blotting method, anti-HIV antibodies derived from AIDS patients are mainly utilized as positive standard. These anti- HIV antibodies are inactivated, but still leave infectious risks to examiners. Moreover, the absolute amount of serum from AIDS patients is not enough and that the large amount of the antibodies cannot be stably available.

Therefore, the following method has been recently used in many trials to obtain the large amount of monoclonal antibodies which specifically conjugate with HIV. In this method, a mouse is firstly immuned with HIV itself, lymphocytes are extracted from the immuned mouse, the lymphocytes are fused with a suitable myeloma cells to form hybridoma, and finally the hybridoma are cultured to obtain the objective monoclonal antibodies.

For example, monoclonal antibodies against one of constitutive proteins for HIV, gp120 has been reported by Michael S.C. Fung et al. (Biotechnology, 5, 940, (1987)). Monoclonal antibodies against p24 has been also reported.

As noted above, several reports disclose the monoclonal antibodies against gp120 and p24. However, monoclonal antibodies against gp4l which is embedded in virus membranes have not yet been obtained. Although gp41 has much importance in the study of HIV infection and the manifestation, the study of gp41 has been lastly improved among the studies of other HIV constitutive proteins.

3 - U.S. Patent No. 4520113 discloses that reactivity and specificity of antibodies which are detected in serum from AIDS or Pre-AIDS patients are mainly caused from the protein gp4l which constitutes envelope antigens of HIV. Taking this into consideration, James et al. studied amino acid sequence which is present in gp4l-constituting protein and which has high immunogenicity against antibodies present in serum from AIDS patients (James J.G. Wang, et al., Proc. Natl. Acad.

Sci. USA, 83, 6159 (1986)). Consequently, a peptide was specified comprising the following 21 amino acids which correspond to 584- 604th amino acid sequence of the envelope antigen.

Arg-Ite-Leu-AZa-VaZ-Gku-Arg-Try-Leu-Lys-AspGkn-Gkn-Leu-Leu-Gty-Ite-Trp-Gty-Csy-Ser wherein, Ata is alanine, Arg is arginine, Asp is asparagine acid, Csy is cysteine, Gkn is glutamine, GZu is glutamic acid, My is glycine, Ite is isoleucine, Leu is leucine, Lys is lysine, Ser is serine, Trp is tryptophan, Tyr is tyrosine, and VaZ is valine.

James et al. also disclose in this report that anti-HIV antibodies in serum can be specifically detected at high sensitivity by directly coating the synthetic peptide of said amino acid sequence on an internal surface of a reaction container, etc.

The object of the invention is to provide a monoclonal antibody which can be used as positive standard for Western blotting method, EIA etc. and which is specifically reactive with HIV constitutive protein, gp4l.

This object can be attained by a monoclonal antibody which is specifically reactive with HIVconstitutive protein, gp41 and which is not reactive with gp120, gp65, gp55, p32, p24 and p18.

A monoclonal antibody of the present invention can be suitably utilized as positive standard for Western blotting method, EIA, etc. Since the monoclonal antibody is not reactive with HIV itself, they can be effectively used for detection of destroyed viruses and exposed gp4l, etc. and analysis of their conditions.

Also, such monoclonal antibody is also included in the invention, as produced from hybridoma of lymphocytes of animals other than human beings and a myeloma, wherein said animal is immuned with synthetic peptide which contains an amino acid sequence corresponding to a part of that of membrane protein gp4l of human immunodeficiancy virus, and as not being reactive with said virus itself and reactive with the membrane protein gp4l only of said virus. These monoclonal antibodies do not cause biohazard risks, because they are derived from hybridoma.

This invention can be more fully understood from the following detailed description when taken in

1 conjunction with the accompanying drawings, in which:

Fig. 1 is a graph showing a fraction pattern obtained by fractionating the ascites derived from the, mouse immuned by the hybridoma 2E6 among hybridomas shown in Table I by means of DEAE column chromatography; Fig. 2 is a photograph a SDS-PAGE (polyacrylamide gel electrophoresis) image of each peak region of the fraction pattern in Fig. 1; Fig. 3 is a graph showing a result of ELISA of a culture supernatant solution of each hybridoma with synthetic peptide; Fig. 4 is a graph showing a result of ELISA of the culture supernatant solution of each hybridoma with diluted solutions of the synthetic peptide; Fig. 5 is a photograph of results of Western blotting method of a cultured supernatant solution of each hybridoma, ascites and serum from a AIDS patient; Fig. 6 is a graph showing the inhibiting activity of antibodies produced by hybridoma 2E6 against the reaction of the synthetic peptide and serum from a AIDS patient; and Fig. 7 is a schematic view showing a conjugation of an anti-HIV antibody with a complex of the synthetic peptide and a monoclonal antibody according to the present invention in assay for anti-HIV antibody wherein the synthetic peptides are coated by means of the monoclonal antibodies.

monoclonal antibodies of the present invention are specifically reactive with HIV constitutive protein, gp4l. Particularly, the monoclonal antibodies are preferably produced by utilizing a synthetic peptide which has an amino acid sequence corresponding to a part of that of gp41 as immunogen. Since the monoclonal antibodies thus produced contain no natural HIV which has infection risk, examination utilizing these antibodies can be free from biohazard possibilities.

The following are detailed descriptions of the present invention according to one embodiment of the invention.

Immunogen A synthetic peptide comprising the following amino acid sequence was prepared as immunogen; Arg-Ite-Leu-AZa-VaZ-GZu-Arg-Try-Leu-LysAsp-Gtn-GZn-Leu-Leu-GZy-IZe-Trp-GZy-Cys-Ser wherein, Ma is alanine, Arg is arginine, Asp is asparagine acid, Cys is cysteine, GZn is glutamine, GZu is glutamic acid, My is glycine, Ise is isoleucine, Leu is leucine, Lys is lysine, Ser is serine, Trp is tryptophan, Tyr is tyrosine, and VaZ is valine. Preparation of Lymphocytes First, 100 pg of the above synthetic peptide were dissolved in 100 Vg of polyvinylpyrrolidone (PVP). The c 1 7 resultant solution was intraperitoneality injected to a six week-female BALB/C mouse (available from The Charles River Inc.) to immunize it. one month later, the same solution as said was intraperitoneally injected to the mouse for additional immunization. Further, the same solution was injected again one month later from the second injection. This injection was divided into the back (subcutaneously), the tail-vein, the eye, etc. Four days later from the last injection, the spleen was enucleated from the mouse.

The spleen enucleated was firstly put into Dulbecco's Minimal Essential Medium (D-MEM Medium) and shaken several times to wash and remove excessive fat mass, etc. Next, the spleen was transferred to a Petri dish containing 10 mZ of D-MEM medium and cut into about four parts. Then lymphocytes were extruded with tweezers. The D-MEM medium containing the extruded lymphocytes was filtered by a stainless mesh to remove tissue masses and a lymphocytes suspension was obtained. Preparation of cells to be fused and PEG Solution The lymphocytes as obtained above and mouse myeloma cells P3X63-Ag8.653 (ATCC No., CRL-1580) were fused as follows.

First, 5 x 107 of myeloma cells which were cultured in a large culture bottle (150 cm2) were transferred to a centrifuge tube and centrifuged at 1200 rpm for five - 8 minutes to collect cells. The cells collected were resuspended in 10 mZ of D-MEM medium containing no serum. This suspension was mixed with the said lymphocyte suspension containing 2 x 108 lymphocytes as said above and centrifuged again at 1200 rpm for five minutes.

Polyethylene glycol (PEG) solution was prepared is for utilizing in fusion as follows. First, 1 g of PEG (Merck #9727) was poured into a glass test tube. The tube was covered with an aluminium cap and heated to 1200C at 1.2 atm for 15 minutes in an autoclave. After the tube was cooled to 600C, 1 mZ of D-MEM medium which was heated to 450C was added into the tube. The resultant mixture was then stirred and kept in a water bus at 450C. Fusion The supernatant solution of centrifuged mixtures of myeloma cells and lymphocytes was removed as much as possible. one milliliter of PEG solution as obtained above was then dripped with a pipett over the time period of one minute. During the dripping of PEG solution, the centrifuge tube was heavily shaken to loose the myeloma mass. The tube was further shaken for one minute even after dripping to facilitate fusion. Next, 1 mZ of D-MEM medium was dripped into the tube for one minute, and 1 mZ of the medium was further dripped with a new pipett. After adding of D-MEM medium to gradually dilute PEG as said, 8 M of D-MEM medium was lastly dripped over the time period of two minutes to straightly dilute PEG. Fusion was thus finished. Culture of Hybridoma The suspension containing the fused cells in the centrifuge tube was centrifuged at 1200 rpm for five minutes. The supernatant solution obtained was then abolished, and the fused cells were suspended in 30 mt of HAT growth medium for hybridoma. This HAT growth medium contains 13.61 pg/mZ of hypoxanthine, 3.88 pm/mZ of thymidine, and 0.176 pg/mZ of aminopterin in a mixture solution of 70% of D-MEM medium, 20% of fetal bovine serum and 10% of NCTC medium. one hundred microliters of the obtained medium were dispensed in wells of three 96 well-microplates. The medium were then cultured in an incubator under 5 C02 at 370C. on the fourth day from culturing, 100 VZ of HAT medium was added into each well. on the seventh and tenth day, 100 p9, of the medium in each well was further replaced with the same amount of fresh HAT medium. on the ca. 15th day of culture, grown hybridoma begin to form a colony in each well. At this time, specificity of the colonies obtained to the synthetic peptide (immunogen) was examined by screening.

Screening A 96 well-flat bottom plate (Nucn: 4-68667) was prepared. one microgram of said synthetic peptide were is dispensed in each well, and the plate was allowed to stand at 370C for one hour to coat the synthetic peptide. Next, each well was washed with 0. 01M phosphate buffered saline (PBS) three times, and 100 p of 0.01M PBS containing 3% bovine serum albumin (BSA) were further added into each well, followed by standing at 370C for one hour. After that, the plate was washed three times with 0.01M PBS (pH 7.5) containing 0.05% Tween 20.

To each well of the plate having the synthetic peptide coated as said above, 100 pt of the supernatant solution were transferred from each well in which the culture of hybridoma was performed. The plate was then allowed to stand at room temperature for one hour. Iach well was then washed with Tween 20-PBS solution four times. Next, peroxidase labelled goat anti-mouse IgG was dispensed into each well as second antibodies, and the plate was allowed to stand at room temperature for one hour. At this time, the peroxidase labelled goat anti-mouse IgG used (manufactured by KPL Company) had been diluted by 1000-fold with 0.01M PBS (pH 7.2) containing 2% of BSA and 5% of Tween 20, and 100 pZ of the diluted solution was dispensed in each well. After that, each well was washed with Tween 20-PBS solution four times, and 100 IA of a solution which was prepared by dissolving 1.5 pk/mZ of orthopheny1diamine (OPD) in 0.1M citrate buffer solution (pH 5.5) and adding 1 pVm 11 - is of 30% hydrogen peroxide. This plate was allowed to stand in the dark at room temperature for 15-30 minutes for reaction. Then 50 pt of 2N sulfuric acid was added into each well to terminate the reaction. Next, absorbance at 490 nm in each well was measured by a microplate reader (Biotech Company, EL-310).

From the wells which are indirectly confirmed by this ELISA method to contain antibodies reactive to the synthetic peptide, hybridomas were picked up in the order of higher absorbance. These hybridomas were transferred to a 24 well-culture plate and further cultured in the same way as said above. Cloninq Single cell of positive hybridomas selected by the screenina was cloned bv culturina the sinale cell in a 24 well-plate according to a general double layer soft agar cloning for two weeks, and taking out grown clones. These clones were subjected to the same screening as said above several times to establish hybridomas which produce monoclonal antibodies specifically reactive with antigen peptide. Strain names of the established hybridomas and immunoglobulin classes of the antibodies produced from the hybridomas were shown in Table 1. The immunoglobulin classes were determined by Oucheterlony method.

Table 1 is Strain Ig Class Strain Ig Class 1B5 Igm 3E3 Igm 1E9 Igm 3Ell I9G1 2B2 Igm 3F5 I9M 2E6 IgG1 3H10 Igm 3A2 I9M 4C10 I9M 3C11 Igm 4H10 Igm 3D7 Igm 4H11 IgG2a Production and Purification of Monoclonal Antibodies To the same series mice as immuned with said antigen peptide, 107 of each established hybridoma were intraperitoneally administered. These hybridomas formed ascites in 15-20 days after administration. From the abdominal cavity of the each mouse, a large amount of ascites was obtained containing antibodies of high antibody activity. The ascites obtained was subjected to cation exchange chromatography to separate antibodies.

Fig. 1 is a fraction pattern which are obtained by measuring absorbance at 280 nm of a fraction obtained by subjecting ascites from a hybridoma 2E6-administered mouse to the chromatography and eluting. In Fig. 1, the abscissa axis indicates time which passed from the time when ascites begins to flow in a column, and the ordinate axis indicates absorbance. Numbers in Fig. 1 indicate peaks corresponding to each fraction.

Fractions showing each peaks 1-6 of the fraction' pattern of Fig. 1 was analyzed by SDS-PAGE polyacryl- amide gel electrophoresis, respectively. The result was shown in Fig. 2. Two left lines in Fig. 2 show results obtained by utilizing molecule weight standard substances and pre-chromatographed ascites. It is apparent that objective antibodies are present in peak 3. Properties of Monoclonal Antibodies Media containing at least I x 106/mZ of each grown hybridoma shown in Table 1 was taken out, and diluted solutions of these media were prepared at every 3-fold by utilizing a diluent obtained by adding 5% FBS in D-MEM medium. Hundred microliters of each diluted solution were transferred into the same ELISA plate as used in said screening and allowed to react. The result of the reaction was shown in Fig. 3. In Fig. 3, the abscissa axis in each graph indicates the multiple of dilution, and the ordinate axis indicates.absorbance at 490 nm. The mark -9- in Fig. 3 shows the results when ELISA plates coated with synthetic peptides was used, and -o- shows the results of control cases when ELISA plates were used, which were made by removing a step of adding synthetic peptides and therefore coated with no synthetic peptides. Strain names of hybridomas used - 14 were shown in the upper right of each graph.

As apparent from Fig. 3, for plates coated with synthetic peptides, all strains show reactivity to the' synthetic peptides. IgG produced by three hybridomas of 2E6, 3E11 and 4H11 are not found to react with BSA and show no non-specific adsorption. while, IgM produced by hybridomas other than 2E6, 3E11 and 4H11 show weak adsorption to BSA.

Next, 10 pg/mZ of synthetic peptides were diluted at the very multiple of 2-fold up to 26 to prepare diluted solutions. Hundred microliters of each diluted solution were utilized to coat peptides to each plate. The amount of peptides coated in each well was 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 pg/well. To each well, 100 pZ of each hybridoma supernatant solution was added for reaction, and absorbance at 490 nm was measured. The results are shown in Fig. 4. In Fig. the abscissa axis indicates the amount of synthetic peptides, and the ordinate axis indicates absorbance at 490 nm. The mark in the ordinate axis indicates detection limit over which level detection cannot be performed.

As apparent from Fig. 4, hybridoma supernatant solutions of all seven strains measured quantitatively react with coated synthetic peptides. Particularly, IgG produced by 3E11, 2E6 and 4H11 show high sensitivity.

Next, specificity of each monoclonal antibody to HIV-constitutive protein was examined by the known Western blotting method. Measurement was implemented by utilizing Immunoblot Assay (Bio- rad Company, catalog No. 197-1001) as a kit and making band patterns to correspond to each sample. The kit used include strips of a nitrocelullose membrane on which images obtained by fractionating HIV- constitutive proteins in the order of its molecule weight by electrophoresis was transferred. Agents and their use method were o beyed to the kit, except that goat anti-mouse IgG or IgM was used as second antibody against the antibodies derived from a mouse of the present invention. The results was shown in Fig. 5. In Fig. 5, six bands from the left are samples in the order of left to right; supernatant solution of cultured 2E6, 3E11 and 4H11, ascites derived from each mouse administered with 2E6, 3E11, 4H11, and 3F5 which is an example of hybridoma producing IgM. Three bands from the right show fresh medium (a mixture of 90% D-MEM and 10% of FBS) as negative standard, a specimen of serum as positive standard derived from a AIDS patient who shows high reactivity, and a specimen from low reactivity-AIDS patient. The ordinate axis indicates molecule weight.

Fig. 5 apparently shows that both supernatant solution and ascites of 2E6 conjugate with a fraction of 41-43000 molecule weight (gp4l). While, for 3E11, only ascites weakly conjugate with gp4l, and 4H11 was not detected to conjugate with fractions. IgM strain of 3ES showed overall non-specific adsorption, and no specificity to gp4l. Consequently, among hybridomas which equally produce IgG, only antibodies produced by 2E6 are strongly conjugated with gp4l. It is also apparent that IgG strains of 2E6,. 3E11 and 4H11 show no reactivity with other HIV-constitutive proteins such as gp160, gp120, gp65, gp55, p32, p24, p18.

Moreover, for a synthetic peptide, the site recognized by antibodies of 2E6 and that by antibodies of AIDS patient serum were measured by utilizing reaction-inhibiting activity.

First, diluted solutions of AIDS-patient serum and normal human serum were prepared at the multiple of every 3-fold by utilizing PBS (pH 7.2) containing 20% of goat serum. Next, a microplate coated with synthetic peptide in 1 pg/well was prepared in the same manner as said ELISA plate, and 100 VZ/well of the diluted solution of said AIDS-patient serum and the same amount of the diluted solution of normal human serum was dispensed in each well. Specifically, one sample was dispensed in two wells of one line of the plate, and two line are used for one sample; one sample was dispensed in four wells. After the sample was dispensed and allowed to react for one hour, the solution in each well was abolished and the well was washed with 0.01M PBS three times. Next, the supernatant solution of 2E6 was 17 - equally added to each well in 100 pZ/well and allowed to react for one hour. At this time, each two lines of each plate were under the same condition for both diluted solutions of AIDS-patient serum and normal human serum. After reaction, the solution in each well was abolished again and the well was washed in the same manner as said three times. Next, for said two lines equally treated with AIDS-patient serum or normal human serum, one line was added with goat anti-mous"e I9G which reacts with antibodies produced by 2E6, and another line was added with goat anti-human IgG which reacts with antibodies in AIDS-patient serum as peroxidase-labelled second antibodies. The solution in each well was allowed to react at room temperature for one hour.

is After reaction, each well was washed in the same manner as said ELISA. Substrate reaction was then allowed to made and absorbance at 490 nm was measured. The results are shown in Fig. 6.

in Fig. 6, the abscissa axis indicates the multiple of dilution of the patient serum and normal human serum, and the ordinate axis indicates absorbance. The mark -- shows the results when the solution in the well of said ELISA plate was made to react with antibodies produced by 2E6 after reaction with the patient serum. The mark -o- shows the results when the solution in the well of the plate was made to react with antibodies produced by 2E6 after reacting with normal human serum.

Real lines in Fig. 6 indicate the results of the wells which are added with goat anti-mouse IgG as second antibodies, and dot lines indicate the results of the wells added with goat anti-human IgG.

Fig. 6 shows that for both cases that an epitope of the synthetic peptide was blocked with anti-HIV antibody in the patient serum (real line a) and that the epitope was not blocked by adding with normal human serum (real line o), antibodies produced by 2E6 are conjugated with the synthetic peptides. This indicates that the epitope of anti-HIV antibody in the patient serum is different from that of antibody produced by 2E6 to conjugate with the synthetic peptides. In other words, monoclonal antibodies of the present invention do not inhibit reaction of anti-HIV antibodies and the synthetic peptide.

Finally, reactivity of monoclonal antibody groups produced by the hybridomas established in this example with inactivated HIV. For measurement, commercially available ELISA kit for human serum screening (DIAGNOSTICS PASTEUR, product No. 7D224) was used. The kit comprises a 96-well microplate which is composed of two kinds of wells; one is directly coated with AIDS virus, and another with no AIDS virus. To these wells of two kinds, each monoclonal antibody of this example, AIDS patient sera of different reactivities, and D-MEM medium (containing 10% Fetal Bovine z Z is Serum) as control were added respectively, and the solution in each well was allowed to react. For second antibody and subsequent agents, the same agent for a mouse as used in said ELISA was used instead of agents for human serum attached to the kit. The results are show in Table 2.

Table 2

HIV(+) HIV(-) 1B5 + + normal human 2B2 ++ + serum 2E6 patient serum ++ 3C11 ++ + (high reactivity) 3E11 patient serum + 3F5 + + (low reactivity) 3H10 ++ + D-MEM 4H10 4H11 Absorbance (490 nm) 0 to 0.10 0.10 to 1.00 + 1.00 to 2.00 + 2.00 to - ++ As shown in Table 2, every patient serum are reactive with virus-positive well, and are not reactive with negative wells. This indicates that patient serum - has specificity to the virus. on the other hand, IgGs produced by 2E6, 3E11 and 4H11 are negative to both wells as said above. This shows that monoclonal antibodies of these IgG strains do not conjugate with HIV itself. While, IgMs produced by other hydridomas are positive to said wells of two kinds, and do not show specificity to HIV.

As apparent from said results, the monoclonal antibodies of the invention can be used as positive standard in Western blotting method and EIA, which specifically recognize with HIV constitutive-protein of gp4l. The monoclonal antibodies of the invention are derived from mouse hybridomas, and that they are free from biohazard risk which is concerned when patient serum is used in a test room. Moreover, the ability of mass production of the present monoclonal antibodies will cancel the chronic deficit of standard samples.

one immunoassay embodiment utilizing monoclonal antibodies of the invention is shown in Fig. 7. As note above, the monoclonal antibodies conjugate with immunogens of synthetic peptide at the site which is different from that when anti-HIV antibodies conjugate with the peptide to form complexes. Therefore, as shown in Fig. 7, by coating the complexes by fixing monoclonal antibodies of the invention to a container, etc., the epitope of the synthetic peptide against anti-HIV antibody can be still exposed. on the other is - 21 hand, when the synthetic peptides are directly coated to a container, adsorption sites of the peptides to a container are randomly selected, and that some epitopes of the peptides to anti-HIV antibody are possibly hidden. Therefore, by applying the present method to known agglutination, EIA or the like, anti-HIV antibodies can be detected more effectively and stably.

The present invention is not limited to above-said example, and various modifications can be possible.

For example, instead of said synthetic peptide used as immunogen, a peptide comprising additional amino acid sequence to the synthetic peptide can be used. Instead of specific carrier such as PVP used in immunogen administration, suitable adjuvant such as Freund's complete adjuvant can be used. In order to fuse with lymphocytes, known myeloma cells can be used instead of mouse myeloma cells P3X63-Ag8.653. Further, for cloning, limiting dilution method can be used instead of double layer soft agar cloning.

Claims (3)

Claims:

1. A monoclonal antibody which is specifically reactive with HIV constitutive protein of gp41 and which is not reactive with gp160, gp120, gp65, gp55, p32, p24 or p18.

2. A monoclonal antibody which is produced by hybridoma of lymphocytes of an animal other than a human being and myeloma cells, wherein said animal is immuned with synthetic peptide which comprises an amino acid sequence corresponding to a part of that of membrane protein gp4l of human immunodeficient virus, and which is not reactive with said virus itself and reactive with the membrane protein gp4l of said virus only.

3. A monoclonal antibody against a synthetic peptide, substantially as hereinbefore described with reference to the accompanying drawings.

Published 1990 at The PatentOffice. State House. 66 71 High Holborn. London WClR4TP. Further copies maybe obtained from The Patent Office Sales Branch, St Mam, Cray, Orpington. Kent BR5 3RD. Printed by Multiplex techniques ltd. St Mary Cray. Kent, Con. 187 z