WO2019219154A1

WO2019219154A1 - Separation of antigens and hcv by diluted acids

Info

Publication number: WO2019219154A1
Application number: PCT/EG2019/000013
Authority: WO
Inventors: Mohammed Abd Allah Taha +ABD EL MOTALEB; Abd Allah Taha ABD EL MOTALEB; ALI Abd Allah Taha ABD EL MOTALEB
Original assignee: Abd El Motaleb Mohammed Abd Allah Taha; Abd El Motaleb Abd Allah Taha; Abd El Motaleb Ali Abd Allah Taha
Priority date: 2018-05-14
Filing date: 2019-05-09
Publication date: 2019-11-21

Abstract

Method was discovered for separation of antigen from antibodies after union, idealy by using diluted acids, this was applied on separation of Hepatitis C Virus by fixing the antibodies of the virus in a solid phase then passing serum positive for HCV virus on it and separation. Detection of the virus by E.M and positive PCR.

Description

Separation Of antigens and HCV by diluted acids

Technical Filed :

Medical

Background Art :

1- The antigen united with the antibodies was separated by using Raji cells. ( 1 ), by electrophoresis, then followed by immunological methods for separation of antigens ( 2 ), by electrophoresis and chromatography. ( 3 ) , by passing on a solid phase containing staphylococcus-aureus having protein A. ( 4 , 5 ) , by passing on a solid phase containing anti human globulins. ( 6 ) , by passing on ELP phase which is a union between staphylococcus-aureus having protein A and elastin like peptide then seperation of the antigen united with antibodies by heating pericipitation . ( 7)

2- Separation of antigen from the antibody after union by using high pressure and changes in

temperature. ( 8 )

3- The antibodies and antigens dissociated from the immunocomplexes extracted from ovarian

carcinoma ascetic fluid by 8 M urea and separation of antibodies and antigens by chromatography .

(9 )

4- Isolation of c DNA clone from the blood of patient infected with viral hepatitis which is non A non B , and using it for diagnosis of its antibodies in patient infected with viral hepatitis which is non A non B . (10, 11)

5- The single strand RNA from the nucleus of hepatitis C virus is containing open frame possible to read and we can able to transcript polypeptide which cleaved to structural proteins ( core and envelope A, B ) and non structural proteins (NS3 , NS4 , NS5A , NS5B ) (12 ) and this single strand was translated to proteins having antigenicity and was used for synthesis a serological testes for diagnosis of HCV . The non structural proteins ( NS ) were translated in insects by using of Baculo virus (13) . And structural proteins of the core were translated in E.coli cells (14) and insects by Baculo virus (15). And structural proteins of the envelope were translated in mammalians and insects ( 16)

6- Self-assembly of nucleocapsid like particles from recombinand Hepatits C virus core protein (17)

7- Non enveloped nucleocapsids of Hepatitis C Virus was seperated by density gradient centrifugation and seen by electron microscope. The viruses were be found in a variable size, its diameter between 38 to 43 or between 54 to 62 nanometer this is . by electron microscope. (18 )

8- Formation of native Hepatitis glycoprotein complexes like the envelope of the virus in function but without the core. ( 19)

9- Flavi viruses like particles were seen by immunoelectron microscopy. Its diameter between 55 to 65 nanometer and have spike-like projections its length is about 6 nanometer. This done from the serum of HCV patient by immunoelectron microscopy. ( 20 )

10- By electron microscope, a Precipitations of the viruses' antibodies complexes were founded in a variable size . Its diameter between 22 to 38 and between 48 to 56 nanometer . This Precipitation reaction was showed by adding positive HCV serum to another serum of HCV patient at late stage of the disease (21)

1 l-The HCV particles labeled with anti El and anti E2 antibodies were seen by the indirect Electron microscope. These particles obtained from the lowest fraction of the sucrose density gradient centrifugation after immunoprecipitation with D32.10 ( 22 ).

l2-The HCV particles labeled with anti El and anti E2 antibodies were seen by the indirect Electron microscope . These particles obtained from the lowest fraction of the sucrose density gradient centrifugation. (23 ,24 ) 13-From a human liver cells , The HCV particles were purified by density gradient centrifugation using iodixanol and by size using gel filtration to the lowest fractions , seen by monoclonal antibodies anti El glycoprotein . The results indicate the association between HCV and VLDL occur in the liver ( 25 )

14-The supernatant of the culture ( after separation by isopycnic sucrose density gradient centrifugation ) by negative staining by the electron microscope had been seen as spherical (40 - 75 nm in diameter ) bleomorphic and some of them were containing HCV E protein and Apo lipoprotein on their surface . ( 26 )

15- Infectious HCV genome was generated with an affinity tag fused to enveloped glycoprotein , using these affinity grids to isolate proteins and macromolecular complexes for single particle electron microscope and were used to purify enveloped particles from cell culture media , also to increase particle number for cryo electron microscope , more over it enabled ultra structural analysis of verions produced by primary human hepatocytes .HCV appears to be the most structurally irregular member of Flavi viridie family. The Particles were spherical with spike like projections and heterogeneous in size. ( 27 )

16-Newly discovered HCV microcores circulating in human blood were detected by monocolonal antibodies against C terminal region of P 21 core and microcore . The purification wase based on heparin and An++ precipitate ( 28 )

17- Visualization and analysis of Hepatitis C virus non - structural proteins using super - resolution microscopy . By florescence microscopy in culture immunoflorescente stained showing significant difference in size between NS protein (29 )

18-The HCV was showed for the first time by electron microscope. The inventors using several

protein specific antibodies and lipids , they were finally able to distinguish these lipoviro particles from single lipoprotein circulating in the serum of individuals. (30)

The Problem in the Background Art

- No.(l) , is the separation of the antigen united with the antibodies .

- No. (2) the separation was done by using high pressure and changes in temperature and this is difficult in practice .

- No. (3) The results were different as mentioned by the inventors

- No. (4) this is apart of the virus .

-No. (5) Is transcription of HCY like particles.

-No. (6) Is formation of HCV nucleocapsid like particles only in function by recombination Hepatits C virus core proteins.

-No. ( 7 ) Is showing of the HCV nucleocapsid in the serum by electron microscope without its envelope .

- No. ( 8 ) Is the formation of glycoprotein complexes like the envelope of the HCV virus only.

- No. ( 9, 10, 1 1, 12, 13 and 14 ) The HCV particles were not pure and contain antibodies and other contents of blood and the antibodies had been added to it during indirect electron microscopy .

- No. ( 15) The particles contains antibodies and not pure .

- No. ( 16 , 17 ) The HCV particles contains antibodies and not pure, also our invention reveals micro viruses have the shape of Flavi viruses even 7 micro meter in diameter.

- No. ( 18 ) The inventors mention that the virus was separated united with antibodies and the background is not clear Disclosure Of the invention :

The New In The Invention

1- A method for separation of antigens from antibodies ideally by using diluted acids .

2- Separation of HCY from antibodies and showing it by electron microscope an positive by PCR .

The Benefit of the invention

1- By this method, it is possible to synthesis Kits for diagnosis of viruses antibodies also Kits for diagnosis of viruses itself.

2- The separated viruses outside the body can be used preparing viruses vaccines .

3- The separated viruses outside the body in a pure form can be used experimentally for testing new dugs and other different studies .

4 - By this method , it is possible to separate another viruses and antibodies.

Detailed discription for separation of antigens from antibodies after union

The used reagents

- Blood sample containing anticoagulant ( as EDTA ) which is positive for RH.

- Anti RH antibodies Solution .

- Sulfuric acid with 0.5 molarity .

- Sterile NaCl 0.9%.

The method

1- The blood sample diluted 1 :20 by normal NaCl 0.9%.

2- sulfuric acid (0.5 molarity ) diluted ( 1/12.5 , 1/15 , 1/20 ,1/25, 1/30 and 1/40).

3- An agglutination was done on a slide by adding 20 microliter from the diluted blood and 20 micro liter Solution containing RH antibodies and shaked till

The agglutination had been seen by naked eye and detected under microscope.

4- To each agglutination , 40 microliter from each dilution of sulfuric acid had been added . and the slide was shaked for one minute and seen under the microscope . we noticed the following :

A. In dilution 1/12.5,1/15 and 1/20 the agglutination was disappeared

B. In dilution 1/25 few agglutination was still remained

C. In dilution 1/30 the agglutination was not completely disappeared

D. In dilution 1/40 the agglutination was not disappeared

So the dilution 1/20 of the sulfuric acid (0.5 molarity ) is suitable to separate the agglutination

The agglutination solution was doubled in volume i.e. the concentration of the used diluted acid is decresed near to half i.e. the total dilution of the suitable 1/20 of 0.5 molar sulfuric acid is 1/40

So this total dilution 1/40 of 0.5 molar sulfuric acid is suitable in the separation of Ag from Abs wich is used for separation of HCV from fixed Abs in microwell . The detailed discription of separation of HCV

The used reagents:

1 - HCV serum Positive by the PCR in a sample tube labeled ( A ).

2- HCV serum positive for HCV antibodies by ELISA in a sample tube labeled ( B ).

3 -Three sample tubes each one contain different serum negative for HCV antibodies and Ag.

4- four polystyrene microwells with flat button such as ELISA wells .

5- Sodium chloride 0.9 % solution well sterilized .

6- Sodium chloride ( 1.8 %) solution well sterilized

7- Wash solution containing Sodium chloride 0.9 % solution and Sodium thioersal 0.01 % solution..

8- Deionized DW .

9- Sulfuric acid (0.5 molar) dilute 1/40 with Sterile NaCl 0.9%. .

10-Carbonate-bicarbonat buffer solution PH 9.6 well sterilized

11 - Concentrated solution ( 1.5 g Na2Co3 + 2.93 g NaHCo3) / 500ml for dilution .

The method

The four polystyrene micro wells were washed 4 times by 350 Micro liters of solution (7)

Microwells coating

- 30 micro liter from serum (B) was added to a micro well and labeled ( B ).

- 20 micro liter from negative three sera were added to other wells and Labeled H, O, Z) respectively.

- 250 micro liter from solution (10) was added to all micro wells.

All wells were kept at (37 C) temperature for 90 Minute.

Step of separation and purification

- The micro wells (H, O, Z) were washed 4 times by solution (5)

- The micro wells were arranged in a series as following:H,0,B, and Z

- 200 micro liter from solution (8) was added to microwell (H)

- 30 micro liter from Positive serum (A) was added to it(H).

- The micro well was kept at room temperature ( 25 C ) for at least 30 Minutes

- The contents of micro well ( H ) were transferred to microwell ( O )

- The microwell was kept at room temperature ( 25 C ) for at least 30 minutes

- The contents of the microwell (O) were transferred to microwell (B).

- The micro well was kept at room temperature (25 C) for at least 60 minutes

- The microwell ( B ) was washed 4 times by solution ( 5 )

-150 micro liter from solution (9) was added to well (B), and was slowly shaked for 45 seconds.

- The contents of microwell (B) were transferred to sterile 2 ml tube and the PH was adjusted to 7.0 by using solution (6, 11).

- After that the contents of the 2ml tube were transferred to well (Z).

- The microwell was kept at room temperature (25 C) for at least 30 minutes

- At the end , the contents of microwell (Z) were transferred to three sterile 2 ml tube which where used for Confirmation .

N.B. large volumes from the separated solution can be obtained by using large wells and suitable volumes .

_.Confirmation step :

■ By direct Electron microscope :

- This step was done after separation of the virus. - The virus was seen by the electron microscope . In photos , The HCV was appeared circular in shape with its envelope and spike like projections . This viruses was similar in shape , pure , numerous , variable in size from 7 - 400 nanometer

- The staining was done by Urinyl acetate 1 % for the fig. ( 1 - 5). This stain is supposed to surround the virus and inside the virus appears unstained but in HCV photos appeared totally stained . This means that the virus absorb the stains .

- phospho tungstic acid staining was done for fig ( 6 - 9 ) this stain like the first stain and also it appeared the core

■ By HCV ELISA kit, The separated solution was tested for HCV Abs. It was assured to be free from HCV antibodies .

■ Molecular test:

RNA extraction was done for the separated solution and PCR magnification was positive for HCV by REAL TIME PCR

The discussion

- In separation step , The virus is nearly pure under electron microscope because The virus is separated from another serum containing HCV antibodies and by passing on other 3 negative HCV antibodies sera but contains different antibodies.

- In washing stage before separation , we removed all contents of the serum except the the HCV Ag united with HCV antibodies. And by separation the vims appears pure.

- In electron microscope pictures, the viruses were appeared with spike like projections and this agrees with picture of immune microscopy in the art No (9) in which the virus appear having spike like projection.

- also, it is possible to increase the accuracy of the virus by increasing the number of negative wells

- Now, the HCV was separated and seen directly by the electron microscope, free from antibodies and positive by real time PCR.

Breif Discription Of The Drawings :

Pictures for HCV by Electron microscope. The staining was done by Urinyl acetate 1 % for the fig. ( 1 - 5). .This stain is supposed to surround the virus and inside the virus appears un-stained but in HCV photos appeared totally stained this means that the virus absorb the stains . The HCV was appeared circular in shape , variable in size and with spike like projections as Flavi viruses to which HCV relates . The backgrond of the picture was appeared clear because we get rid of another contentes of the serum.

1- ln Fig. No. 1 ( 20000 power of magnification ) ,The virus was appeared variable in size and its diameter from ( 5 - 200 ) nano meter .

2- In Fig. No. 2 ( 40000 power of magnification ) , The virus was appeared variable in size and its diameter from ( 5 - 150 ) nano meter .

3 - In Fig. No. 3 ( 70000 power of magnification ) , The virus was appeared variable in size and its diameter from ( 5 - 150 ) nano meter .

4 - In Fig. No. 4 ( 70000 power of magnification ) , The virus was appeared variable in size and its diameter from ( 200 - 300 ) nano meter.

5- In Fig. No. 5 ( 70000 power of magnification ) , The virus was appeared big in shape and its diameter about (400 ) nano meter

6-ln Fig. No. 6 ( 160000 power of magnification ) , The virus was appeared variable in shape and its diameter about ( 130-200 ) nano meter

7- In Fig. No. 7 ( 160000 power of magnification ) , The virus was appeared variable in shape and its diameter about ( 130-200 ) nano meter

8-In Fig. No. 8 ( 91000 power of magnification ) , The virus was appeared variable in shape and its diameter and the core appear clearly

9-In Fig. No. 9 ( 52000 power of magnification ) , The virus was appeared variable in shape and its diameter about ( 130-200 ) nano meter Refrences

1- Theofilopoulos AN, Eisenberg RA, and Dixon FJ ( 1978 isolation of circulating immune complexes using Raji cells. Seperation of antigens from immuno complexes and production of antiserun. J. clin. Invest. (1978 ) ; 61 (6) : 1570 - 1581 .

2- Macintyre RJ, Dean MR And Batt G ( 1978 ) : Evolution of acid phosphatase- 1 in the genus Drosophilia Immunological studiesJ of molecular evolution. (1978) Dec. 29; 12 (2): 121 - 42.

3- Nielsen RG, Rickard EC, Santa PF, Sharknas DA and Sittampalam GS (1991) : Separation of antibody- antigen complexes by capillary zone electrophoresis isoelectric focusing and high-performance size-exclution chromatography J. of chromatography. ( 1991) ; 539 (1): 177- 185

4- Frigard M (1983) : Quantitation of a highly immunogenic leucocyte antigen L-l by radioimmunoassay methodolgical evaluation. J. of immunological-methods ( 1983 ) : 65 ( 1-2 ) : 245-256

5- Ying A And Guillemin R (1979 ) : Direct staphylococcus-aureus as a rapid immunological separating agent in rapid immunoassays. J. of clinical endocrinology and metabolism ( 1979 ) ; 48 (2): 360-362

6- Reichlin N, Copley DP And Klockel FJ (1980) : Rapid radioimmunoassay for serum myoglobulin. Americal J. of physiology ( 1980) 239(4) : H 565 -H 569

7- Kim JY, Malley S, Mulchandani A, Chen W ( 2005 ) Genetically engineered protein A fusion as a universal platform for homogenous phase separation immunoassy. Anal. Chem. Apr.l5;77(8):2318-22.

8- Gavalda E and Gegraeve P (1996) High pressure-induced modulation of the antigenic interactions between two beta-galactosidases and anti-beta-Galactosidase antibodies. Enzyme- and mictobial. Technology. 1996 ; 18 (1) : 10-17

9-Qian H, Feng J and Fu T (1985) The study of antibodies and antigens dissociated from the immune complexes extracted from overian carcinoma ascitic fluid. Gynecologic-oncology. 1985 ; 20 (1) : 100 - 108

10- Choo QL, Xuo G, Weiner AJ, et al ( 1989 ). Isolation of a cDNA clone derived from a blood-borne Non- A, Non-B viral Hepatitis genome. Science 244 : 359 : 359 -262.

1 1- Kuo G, Choo QL, Alter BJ, ef al ( 1989 ). An assay for circulating antibodies to a major etiologic virus of human Non A Non B hepatitis. Science 244 : 346 - 346

12- Van Doom LJ ( 1994 ) Review : Molecular biology of the hepatitis C vims. J. Med. Virology 43 ( 4 ) . 345 - 356

13- Hirowatari Y, Hijikata M, Tanji Y, Shimotohno K : Expression and processing of putative nonstructural proteins of Heptitis C virus in insect cells using baculovirus vector. Vims research ( 1995 ). 43 - 61 .

14- Yoshikawa A, Kishinoto S, Tsuda F, Akahane Y, Naito S, Tamaka T, Yoshikawa H, Yamasaki M,

Okamoto H, Miyakama Y, Mayumi M. : Serodiagnosis of Hepatitis C virus infection by ELISA for antibodies against the putative core protein ( P200 ) expressed in Escherichia coli. J. Immunol. Methods 1992 : 184 : 143 - 50

15- Chiba J, Ohba H, Matseura Y, Watanabe Y, Katayama T, Kikuchi S, Saito I, Miyamura T : Serodiagnosis of Hepatitis C virus ( HCV ) infection with an HCV core protein expressed by a recombinant baculovirus : Proc. Nat -Acd. Sci. USA ( 1991 ) 88 : 4641- 5.

16- Matsuura Y, Harad S, Suzuki R, Watanabe Y, Inoue Y, Saito I And Miyamurea T ( 1992 ) :

Expression of processed envelope protein of Hepatitis C virus in mammalian and insect cells J, Virol. 66 (3) : 1425 - 1431

17- Kunkel MM, Lorinczi R, Rijnbrand SM, Lemon and Watowich SJ (2001) : Self-assembly of nucleocapsid like particles from recombinand Hepatits C virus core protein J. Virol. 75 : 2119 - 2129

18- Maillard P, Krawczynski K, Nitkiewicz J, Bronnert C, Sidorkiewicz M, Gounon P, Dubuisson J, Faure G, Crainic R, and Budkowska A( 2001 ) : Non enveloped nucleocapsids of Hepatitis C Virus in the serum of infected patients. J. Virol. Sept. 200l( 8240 - 8250 ).

19- Deleersnyder V, Pillez A, Wychowski C, Beril B, Xu J, Hahn YS, Rice CM And Dubvisson J : formation of native Hepatitis glycoprotein complexes. J. Virol. ( Jan. 1997 ) P : 697 -704

20- Kaito M, Watanabe S, Tsukiyama-Kohara K, Yamaguchi K, Kobayashi Y, Konishi M, Yokoi M, Ishida S, Suzuki S And Kohara M ( 1994 ) Hepatitis C vims paticle detected by immunoelectron microscopy study. J. of ganeral virol. 75 - 1755 - 1760

21- Kim CY, Yoon JS, Kim YT, Jung HC, Lee H, Yoon YB And Song IS ( 1999 ) : A precipitation reaction found in patients with Hepatitis C as a marker for the purification of virus-like particles . J. intervirology 42 ( 4) : 263-70).

22-.Marie-Anne Petit , Marjory Lievre , Simone Peyrol , Sylvie De Sequeira , Pascale Berthillon , Rob W.H. Ruigrok , Christian Trepo ( 2005 ) : Enveloped particles in the semm of chronic hepatitis C patients. Virology 336 ( 2005 ) 144 - 153 . 23-Masahiko Kaito , Shozo Watanabe , Hideaki Tanaka , Naokifujita , Masayoshi Konishi , Motoh Iwasa , Yoshinao Kobayashi , Esteban Cesar Gabazza , Yukihiko Adachi , Kyoko Tsukiyam-Kohara And Michinori Kohara ( 2006 ) : Morphological identification of hepatitis C virus El and E2 envelope glycoproteins on thr virion surface using immumogold electron microscopy. International Journal of molecular medicine 18: 673- 678, 2006

24- Masahiko Kaito , satoshi Ishida , Hideaki Tanaka , Shinichiro Horiike , Naoki Fujita , Yukihiko Adachi Michinori Kohara , Masayoshi Konishi , Shozo Watanabe ( 2006 ) : Morphology of Hepatitis C and Hepatitis B virus particles as detected by immunogold electron microscopy . Med Mol Morphol (2006 ) 39:63-71 .

25 - Soren U Nielsen , Margaret F Bassendine , Caroline Martin , Daniel Lowther , Paul J Purcell , Barnabas J King , Dermot Neely and Geoffrey L Toms ( 2008) : : characterization of hepatitis C RNA - containing particles from human liver by density and size . Journal of virology ( 2008 ) 89 , 2507 -2517 .

26- Pablo Gastaminza , Kelly A Dryden , Bryan Boyd , Malcolm R Wood , Mansun Law , Mark Yeager and Francis V Chisari ( 2010 ) : Ultrastructural and Biophysical characterization of Hepatitis C Virus Particles Produced in cell culture . Journal of virology 2010 P. 10999 - 11009 .

27- Maria Teresa Catanese , Kunihiro Uryu Martina Kopp Thomas J Edwards Linda Andrus , William J Rice Mariena Silvestry , Richard J Kuhn and Charles M. Rice ( 2013 ) : Ultrastructural analysis of hepatitis C virus particles . PANS /june 4,2013 / vol. 110 / no. 23 / 9505 - 9510 .

28- Francis J. Eng , Ahmed El-Shamy , Erin H. Doyle , Arielle Klepper , A Scott Muerhoff And Andrea D Branch ( 2018 ) Newly Discovered Hepatitis C Virus Minicores Circulate in Human blood . Hepatology Communications Vol . 2 No. 1, 2018 .

29- Christopher Bartlett , Alistair Curd , Michelle Peckham and Mark Harris ( 2018 ) : Visualisation and analysis of Hepatitis C virus non - structural proteins using super - resolution microscpy . scientific reports / (2018) 8 : 13604 / DOL: 10.1038/s41598-018-31861-0 .

30- Inserm Press Office ( 2016 ): Hepatitis C virus observed under a microscpe for the first time.

Claims

' - Separation of antigens from the antibodies by using diluted acids as Sulfuric acid or other acids f - By this method , separation of HCV completely with its enveloped and core seeing by electron microscope .

f - The separated viruses can be used for synthesis of vaccines and testing new dugs on them.

The separated viruses can be used for synthesis Kits for diagnosis of viruses antibodies also Kits for diagnosis of viruses itself.

This method can be applied for separation of another viruses and antigens .

^"t- Any updates for this method.