JPH04503664A - Synthetic peptides and antibodies against Epstein-Barr virus nuclear antigen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] [Name of invention] Synthetic peptides and antibodies against Epstein-Barr virus nuclear antigen (Technical field) The present invention relates to Epstein-Barr virus, its nuclear antigen, and cytomegalovirus. Immunogens, antigens, inocula, and antibodies useful in the treatment and diagnosis of diseases associated with BODY, METHODS AND COMPOSITIONS.

[Background of the invention]

Epstein-Barr virus (EBV) is a member of the herpesvirus group. It is the causative agent of infectious mononucleosis (NM) in humans.

EBV also causes Burkjtt's lymphoma, which occurs in immunosuppressed patients. , nasopharyngeal tumors, and the pathogenesis of B lymphocyte neoplasms. Circumstantial evidence also in human autoimmune diseases such as rheumatoid arthritis and sigrain syndrome. This suggests a potential role for this virus.

EBV is a very common environmental agent that infects 80-100% of people worldwide. Ru. The initial or primary infection may be acute or subclinical. This state remains connected for a long time. During this period, EBV infection increases the amount of B-phosphorus present in the circulating blood, lymph nodes, and membranes. Hidden in the ball.

Latency is the process by which a virus resides within a cell in an unexpressed or partially expressed state.

This latency is reactivated. Few host factors control latency in vivo Although it is only known for There is some evidence that this is the case.

The cytotoxic and suppressive T-cell component of the immune response to EBV is associated with EBV in the IM. It has been reported that it is extremely important in suppressing acute infections caused by. These key points The compound also suppresses the uncontrolled natural growth of B lymphocytes latently infected with EBV. It is also important to control

Defects in T-cell suppressor mechanisms may be associated with immunosuppressive treatments used to prevent rejection of organ transplants. Resulting African Burkitt's lymphoma, nasopharyngeal tumor, B-cell lymphoma, important in allowing the development of lymphomas that occur during the treatment of autoimmune disorders and various autoimmune disorders. @Epstein and ^chong [, “The Epstein-Barr Virus', Spv of Berlin Paidelberg ing-Verleg (1970); and Crawford et al., Lanc. et.al., 1355 (1980), and further discusses the defects and consequences of these T-cell mechanisms. Overgrowth of EBV-infected lymphocytes plays a role in rheumatoid arthritis. Then, as taught by Slaughter et al., J. Exp, ? led, , 148: 1429 (1978); Depper et al., J, tw-uno logy, 1 2 7: 1 8 9 9 (1981); and Tosa to et al., N. Engl, J. Med, 305:123B (1981 ).

The serological and cell-mediated immune responses following primary EBV infection are well published. This reflects the host response to viral antigens expressed during infection. of these responses Detection of antigens externally and in tissues is becoming increasingly useful in the diagnosis of EBV-related diseases. ing.

Classically, secondary infection is caused by antibodies against viral capsid antigen (VCA). During the recovery period, antibodies against EBV-encoded nuclear antigen (EBNA) rise. (Henle et al., Hus+, Pathol, 5:551-565). (1974)), EBNA-1 (also referred to herein as EBNA) ), that is, the first nuclear antigen recognized was 65.0 by immunoplotting. 00~ss, ooo It has been identified as a chiadalton (KD) protein (S trnad et al., J. Virol-+38: 990 (1981) HHe nnessay et al., Proc, Natl, Acad, Scj, USA, 80: 5665 5669 (1983); and BiBllin et al., Pr. oc, Natl, Acad, Sci.

USA, 80 Near 104 (1983)).

The size of EBNA-proteins ranges from 65,000 to 85,000 in various B cell lines. EBV-DNA has a molecular size in the range of 000, with approximately 77 KD being typical. (Hennessay et al., Pro c, Natl, Acad, Sci, U S A% 80:5665- 5669 (1983)), the IR-3tri region is a major area of EBNA-proteins. Encodes a repeating glycine-alanine sequence that has been characterized as a pitope (Billings et al., Proc.

Natl, Acad, Sci, USA, 80 Near 104 (1983 )).

Although commonly used assays test VCA and then EBNA-1, , EBNA-1 is the first EBV-associated antigen that can be detected after infection. EBNA is It has been detected in the membranes of latently infected growing transformed B lymphocytes. EBNA is It was also detected in the membranes of African Burkitt tumor lymphoblasts and plastic nasopharyngeal carcinoma cells. It's being served.

The concentration of EBNA in the cell nucleus of EBV-infected B lymphocytes varies at various stages of the cell renewal cycle. It fluctuates in In other words, EBNA is thought to be periodically synthesized and degenerated. ing. As a result of such degeneration, protein fragments of EBNA (polymer peptide) is thought to traverse the cytoplasm and be present or expressed on the outer membrane. ing. However, no specific EBNA degenerate polypeptide has been identified to date. Not yet.

In or on the extracellular membrane, the EBNA degenerate polypeptide is transferred to host T lymphocytes. constitutes a significant stimulus to the immune system and initiates an immune response that results in the production of anti-EBNA antibodies. It is considered that In addition, B cells expressing EBNA degenerate polypeptides on their surface Specific T cell responses to cells limit the growth of EBNA-containing (EBV-infected) B lymphocytes It may also contribute to the production of cytotoxic and suppressive T cells that are important for .

That is, assaying for the presence of both EBNA and anti-EBNA antibodies is a common clinical practice. It is important in certain situations. Furthermore, vaccines against EBV-infected B lymphocytes are also available. may be clinically important.

Anti-EBNA antibodies are typically assayed using redundant anti-complement immunofluorescence (ACIF). Say. Reedman et al., Int. J. Cancer, 11.499- 520 (1973). This assay uses EBV-transformed human B cells in microscopic slides. Including fixing to the id. Various dilutions of patient serum were then added to the fixed cells. Ru. Anti-complementary sera can produce false negative reactions or prozones when mixed with complement (two-step method), test cell specimens are sequentially treated with serum, complement, and anti-complement-fluorescent solution. Supplementation with conjugate is essential (3-step method).

Several problems exist with this assay. It requires relatively little assays. It is sensitive and requires amplification by complement. Furthermore, this assay is completely elucidated in patients whose serum contains antibodies against mammalian cell nuclei rather than specific for Can not. Furthermore, the quantitative results obtained using the anti-complement immunofluorescence assay were difficult to express. For these and other reasons, anti-EBNA antibody assays B is generally limited to use in a few specific laboratories.

The above difficulties in assaying anti-EBNA antibodies are due to the lack of relatively pure EBNA. Due to existence. Purification of EBNA from mammalian cell tissue culture involves the use of low concentrations of the antigen. complex due to degree and polymorphism. If EBNA is not expressed as in the current law, Using whole cells is simple and inexpensive, but issues of specificity and reproducibility This is a direct result of using whole cells.

Synthetic peptide containing part of glycine-alanine E BNA-1ii region It has been suggested that Rho is reactive with serum from patients with EBV-IM (Rho Des et al., Herpesvirus % R, Rapp & i, New York. ^fan R, Li5s, 487 496 (1984); Rhodes et al., J , fnunology, 134:211-216 (1985); Sm1t H et al., J. Infec, Dis, 154: 885-889 (198 6) and Ge1tosky et al., J, Cl1n, Lab, Analysis s, 1: 15f-162 (1987>), U.S. Patent No. 4.654,419 and other subsequent publications, P62-based peptides The acute phase of EBV-IM was used in an ELISA assay to compare the convalescent and convalescent phases of IM. (Swith et al., J. Infec.

Dos, 154:885-889 (1986) and Geltsky et al. J, CIjn, LabAnalysis, 1: 153-162 ( 1987)).

The acute phase of the disease can be detected by the appearance of IgM antibodies against the peptide. Ru. During the recovery period, the IgM antibody titer decreases and IgG antibodies can be detected (Sm ith et al., J. Infec, Dis, 154. :885-889 (1 986)) e Patients with long-term infection are susceptible to the above peptides as the main immunoglobulin group. It has TgG antibodies.

Antibody production against EBNA-proteins is highly abnormal.

IgM antibodies that recognize glycine-alanine peptide P62 can be detected within 1 day of onset of the above disease. detected within (Sa+ith et al., J, I++4ec, Dis, +1.54 :885-889 (1986)), when analyzed by immunoblotting, These [gM antibodies can be used to target EBNA-proteins as well as more than one dozen healthy cell proteins. was found to recognize white matter (Rhodes et al., J. Exp, Med. , 165: 1026-1040 (1987)). EBNA-1 and self Antibody binding to antigen can be inhibited by peptide P62. That is, acute E BV infection is associated with the glycine-alanine repeat region of EBN A-1. It induces the synthesis of autoantibodies that appear to occupy the pitope.

In contrast, IgG antibodies against EBNA-1 remained active until several months after the onset of the above-mentioned disease. does not appear. IgG anti-peptide measured by ELISA assay P62 antibody (Smith et al., J. Infec, Dis.

is4:5ss-ssq (1986) and Ge1tosky et al., J., Cl. 1n, Lab Analysis, 1: 153-162 (1987)) , anti-EBNA-] antibodies measured by standard anti-complement immunofluorescence, and immunofluorescence. All IgG antibodies against BBNA and white matter measured by blotting were appear at the same time. These IgG antibodies can also be suppressed by peptide p62. EBNA is also protein-specific and no longer reacts with cellular autoantigens ( Rumpold et al., J, 1m@uno1. ．． 1 3 8: 5 9 3 - 5 9 9 (1987); Rhode S et al., J. Exp. Med. 165: 1026-1040 (1987); and Dillner et al., P. roc, Natl, Acad, Sci, USS, 82: 465 2-4656 (1984)).

Cytomegalovirus (CMV) is another member of the herpesvirus family. . CMV infection in immunocompetent humans is usually without significant complications and is typically caused by EBV. It is similar to karyonucleosis.

Diagnosis of CMV infection in patients with IM-like symptoms is by isolation of virus from vascular areas. can be established. Antibodies produced in response to CMV infection are neutralized (NT), corrected fixative (NT), CF), immunofluorescence and platelet aggregation (PA) methods*( ifeller, N., Engl.

J. Med., 285:203 (see >1,971).

CMV infection is also insidious, and the disease can be diagnosed in patients and opportunistically during immunosuppressive treatment. Occurs in infected patients. CMV infection is most common in patients receiving allogeneic pancreatic bone grafts. is also a common infection and is an important determinant of the success or failure of this transplant (Ne iman et al., J. Infe-ct. Dis, 136:754 (1977) ).

In adults receiving immunosuppressive therapy after renal allograft transplantation, more than 90% of adults receive immunosuppressive therapy. Express tomegalovirus antibodies. Approximately half of seronegative patients subsequently receive immunosuppressive treatment sometimes infected. Few seronegative recipients receive kidneys from seropositive donors. All of them appear to have post-operative infection and exhibit symptoms of infection.

Immunocompetent humans, i.e. those not receiving immunosuppressants and ARC and AI In humans without immunocomplicated diseases such as DS, CMV mononucleosis (CMV-I M) The infection affects young children (from birth to 2 years) and adults, i.e. people around 30-35 years old. It is typically found in Infectious mononucleosis caused by EBV (EBM-IM) ) is particularly prevalent among people between the ages of 10 and 25, especially those between the ages of 15 and 25. It is commonly found in

Patients with both diseases had atypical myosomal cysts, pharyngeal symptoms, abnormal liver function tests, Shows spleen and fever. Sera from CMV-IM patients are heterophilic, whereas Sera from most EBV-IM patients are heteropositive.

Genetic engineering and synthetic polypeptide technologies have recently enabled the production of large amounts of proteins and polypeptides. This provides a solution to the problem of producing peptide antigens.

However, both techniques only work when the amino acid residue sequence of a natural protein is known. It is effective in many cases.

The amino acid residue sequence of a natural protein can be determined from the protein itself; Often difficult. The nucleotide sequence of the gene encoding that protein is also The protein amino acid residue sequence can be revealed. However, all DNs The A sequence has three potential reading frames, each of which contributes to a different protein. Ru. Therefore, knowing the exact reading frame allows you to extract the exact amino acid residues from that gene. The base sequence must be estimated.

The exact reading frame of a DNA sequence encoding a protein, and therefore its The amino acid residue sequence of a protein can be determined using antibodies. This method uses amino acids A series of protein fragments whose residue sequences correspond to those obtained from three potential gene products. including producing a fragment or polypeptide. Natural protein production of genes Protein fragments or polypeptides that elicit antibodies that immunoreact with an organism are The exact reading frame of the gene is thereby identified. On the other hand, natural protein Recognizes protein fragments or polypeptides produced by antibodies against the protein In this case, a relationship between genes and proteins can also be established.

Helfer et al., J. Virol, 44:311-320 (1982), Consists of direct repeats of one hexanucleotide and two nonanucleotide sequences EBV genome protein DNA found to contain internal regions, named IRs The sequence was reported. They found that the sequence surrounding and containing IRs encodes EBNA. We showed evidence suggesting that it contains the gene. However, three potential He1l was not known to translate the target DNA sequence reading frame. er, etc. (The above mentioned clearly deduces the amino acid sequence of potential EBNA protein. I couldn't do it.

In September 1983, Hennessy and Kieff, Proc, Nat. l, Acad.

Sci., U.S.A., 80: 5665-5669 (1983), supra. The natural reading frame of the EBV DNA sequence reported by Hefer et al. It was reported that it was established. Essentially, they isolated the IR, DNA, and transferred this to a small These small pieces were inserted into the [acZ gene] of the E. coli expression vector. All three EBNA gene reading frames, each in a different clone. It was inserted so that it would be expressed. The IacZ gene is beta-galactosidase, That is, it encodes a microbial enzyme. IR3-1acZ gene cattle are E, c between amino acids 7 and 9 of the beta-galactosidase protein molecule (8 is deleted during the construction process). Expressed as protein.

Hennessy and Kieff expressed IR3 DNA in its natural reading frame. The expressed IR,-]acZ gene frame is recognized by anti-BBNA positive human serum. The fusion protein was identified by screening for fused proteins. Same as that The determined plasmid was named pKH182-44.

Proteins expressed by pKH182-44 contain EBNA-specific antigenic determinants. In order to confirm that the above-mentioned Hennessy and 1eff contain bromide silicon, CNBr-cleaved IR,-galactosidase fusion protein in rabbits antiserum was produced.

The CNBr fragment contains 53 amino acids homologous to EBNA and An immunogen containing 89 amino acids homologous to lactosidase was used. this Their antiserum recognized natural EBNA in EBV-infected cells using indirect immunofluorescence. Ta.

Hennessy and Kieff's results suggest that the repeatability of the EBNA I R3 domain It seems to depend on it. Fusion protein produced by pKH182-44 is a relatively long segment of homology with the IR, domain (e.g., 53 amino acids ). Therefore, the above fusion protein and its CNBr fragment are It is not surprising that it contains a primary determinant. Furthermore, Hennessy and Kjeff suggest that the repeat sequences in the above fragment act as antigenic determinants. and was not identified.

Hennessy and Kief4 identify substances recognized by anti-EBNA antibodies in human serum. could be produced genetically, but because of their design, they could not be used in clinical applications That would be difficult to handle. 5 of their fusion proteins that are homologous to EBNA The three amino acid residue segment is the physical component of the beta-galactosidase protein. and chemical part. Therefore, its immunological properties cannot be separated Affected by beta-galactosidase protein molecules. fact , all of the human serum used in their study reacted with beta-galactosidase and the above absorb this reactivity before testing specificity for genetically produced proteins. It required treatment with beta-galactosidase to remove it.

Determining the exact reading frame of genes and clinically producing large amounts of pathogen-related antigens (immunogens) Another approach to the related problem of producing synthetic polypeptides for diagnostic purposes is It's about using chemistry. The method for producing this antigen (immunogen) is based on the genetic engineering described above. It has advantages over the academic method. Synthetic polypeptide antigens are natural protein byproducts or does not contain that fragment and therefore its use may lead to undesirable cross-reactivity. potential and serum samples as in the Hennessy and Kieff study. Eliminates the need for pre-treatment of files.

Prepare a synthetic antigen (immunogen) and use this antigen to induce antibodies of a given specificity Although the general concept has been disclosed, the technology continues to resist predictability. There is a wide area. There are two reasons for this. First, the Adult antigens (immunogens) are intact proteins and antibodies that react immunologically in their natural environment. It does not necessarily induce the body. Second, naturally produced immunogens such as viral proteins The host's natural antibodies against it correspond to short linear portions of the natural immunogenic amino acid residue sequence. Rarely immunoreacts with polypeptides. This latter phenomenon is a necessary secondary or tertiary phenomenon. This results in short linear polypeptides lacking the following conformational structure: It is believed that there is.

Many studies on peptide-antibody binding to proteins have been conducted on Be njamini+ E, etc., Current Topics in Micro biol-ogyand Immology, 58: 85-134 (19 It is summarized in a commentary by 72). What is the role of peptide structure in antibody binding? Goodsan, J, L, I+mmunoche@, 6: l 39- 149 (1969).

Most studies on how changes in peptide sequence affect antibody binding We interpret this as indicating that the structure of the antibody binding site plays a major role. child The effects of sequence and structural changes in these studies were mixed and difficult to distinguish. It is. Some of these studies are equally well explained by structural changes in the antigen that performs the binding. I can explain.

Antibody responses at the molecular level have a limited sequence (-substructure) and a limited conformation. formation (secondary and tertiary structure) of the antigen. for protein antigens The immune response shall be specific to the primary, secondary and tertiary structure of the protein. It has been traditionally explained.

This classification scheme has a well-defined overall structure at physiological temperature and solution. It has a certain validity as a protein odor. However, its legitimacy is more dynamic. This is doubtful for peptide antigens with a similar structure.

Some groups include silk fibroin (Anderson et al., J. Mol.

Biol, 67:459-468 (1972)) and collagen (And erson et al., BBRC39:801808 (1970); Doyle et al., J , Mo1. Biol, 51: 47 59 (1970)) model. A polymer of repeating sequence of glycine and alanine or glycine and serine synthesized as a polymer. We report a structural study of Rimmer. The most systematic studies are based on a series of block homopolytes. Brack et al., who report the synthesis of remeric polypeptides, rs, 11: 563 586 (1972), and the polypeptide The homopolymeric block repeat unit in has the formula (AA'ax GiVr) , where when x=1, y=t, 2 and 3, and when x=2, y=1.2 and and 3, and when x=3, y=3.

The results reported in the latter study above indicate that in the solid state, almost no alanine The block homopolymer is α-helical and contains mostly glycine. The rock polymer was said to be messy. In solution, polyalani It has been reported that the ion is α-helical, but the poly-(Ala@-CI!y,) reported that it is θ-antiparallel. The polymer of glycine lynch is α-heli It is explained that it has another fixed structure that is neither cal nor β-structure. It has been made clear.

The homopolymer block of glycine and alanine reported by Brack et al. Dipeptides or hexapeptides with a carboxy-terminal glycyl residue in the form of a ster It is prepared by polycondensation of repeating units. Polymerization degree of 2 to 68 is poly(A) la-GI Yz).

The solvent used in these studies was physiologically acceptable, e.g. water or phosphate buffer. Even in the non-saline case, the results show two points: (1) Structural changes are (2) Structural changes can also occur from solution to solid state. It explains what happens during the conversion to .

[Contents of the invention]

The present invention contemplates CMV homologous polypeptides. This polypeptide is shown in Figure 1. about 6 to 40 having a sequence corresponding to that of the polypeptide encoding the CMV shown. preferably about 15 to about 40 amino acid residues, pharmaceutically acceptable salts thereof, and and antigenically related variants thereof, and the above sequence has the formula: -GLy -R' -Gly-R''　-Gly-, in which R1 and Rg may be the same or different when shown individually.Ala, Asn sArg % Gly 5Leu 5Pro 5ser and Thr However, both R' and R2 are not Gly.

In another embodiment, the present invention provides an anti-EBNA antibody antibody in a biological sample. related to the research method. This method involves converting a patient biological sample into a CMV homologous peptide of the invention. Sample the immunoreaction mixture thus prepared, including mixing with The anti-EBNA antibodies present in the peptide immunoreact with the peptide to produce an immune reaction product. The presence of the formed product is then measured and The presence of anti-EBNA antibodies in the sample is determined by: In a preferred embodiment In particular, this method is immunoglobulin-grade specific.

The present invention also provides diagnostic methods for assaying the presence of anti-EBNA antibodies in biological samples. system. This system provides fal CM V homologous polypeptides in separate packages. Indicating the presence of immunoreactivity between the peptide and Tol polypeptides and human anti-EBNA antibodies. and instruction means for the instruction.

Preferably, the indicating means is an enzyme-labeled human 1-1g specific antibody.

The invention also provides a plurality of linkages in which at least one repeating unit is a polypeptide as described above. The present invention relates to multimers containing repeating polypeptide units. This polypeptide repeats The units may be linked by amino bonds in a head-to-tail configuration. In addition, synthetic polypeptide monomers - such as through the use of intermolecular and interpolypeptide cysteine disulfide bonds. Polymer multimers can also be prepared by binding by methods other than mino binding.

The presence of anti-EBN^ antibodies using the above-mentioned polypeptides or multimers as antigens is Also contemplated by the invention are methods of assaying. In this method, liquid human A body sample is used and mixed with the polypeptide described above. Support the resulting immunoreaction mixture. The anti-EBNA antibody present in the sample immunoreacts with the above polypeptide to form an immunoreactant. for a predetermined period of time sufficient to generate . The presence of an immune response is then determined.

In particularly preferred implementations, the presence of an immune response is determined by anti-IgG, anti-1gM or anti-1gM. An anti-human long chain antibody such as gA is mixed with the above immunoreactant and this second immunoreactant mixture is The above-mentioned anti-human antibody immunoreacts with the anti-EBNA antibody present in the immunoreactant. for a predetermined period of time sufficient to generate a second immunoreactant; by measuring the presence of specific human anti-EBNA long chain antibodies present. Measure.

Most preferably, the above assay is carried out on a solid matrix forming a solid support. 8 Blood, serum, which is carried out using the above-mentioned polypeptide or multimer fixed , a biological sample such as serum, saliva or sputum is mixed with the above solid support to form a solid phase/ Prepare a liquid phase immunoreaction mixture. This mixture was maintained as above to prepare anti-EBNA. A primary step in which a solid-phase bound immunoreactant containing antibodies is prepared, and the solid and liquid phases are then separated. The presence of solid phase-bound immunoreactant is then determined.

Furthermore, diagnostics that assay the presence of antibody molecules against EBNA in human biological components system is intended. Such a system combines a polypeptide of the invention with anti-E Includes indicator means for immunoreactivity with the BNA antibody molecule. A more preferred embodiment In this case, the system also includes a solid matrix on which the polypeptide is immobilized. Contains a solid support. The system also includes means for identifying the isotype of immunoreactive antibody molecules. It can be done.

[Brief explanation of the drawing]

In the drawings forming part of the description of the present invention: Figure 1 shows polypeptide (F); (B) and (E) are plots of circular dichroism spectra. these pepti Also used herein are polypeptides F13, R62 and R12, respectively. It is called. Each spectrum contains 1 milligram/milliliter (■/, w/) of physiological solution. is the average of 10 successful scans of the polypeptide in solution (phosphate buffered saline). Turn Luminous intensity is expressed in milliradians and plotted against polarized waves in nanometers (nm). I did it. A relatively featureless plot of polypeptide F (R13) of this size A random conformation is shown, which is the usual result for peptides.

The dip and peak spectrum of polypeptide B (R62) are relatively stable secondary It is characterized by a structure or conformation, perhaps β-pleats. data Although not shown, polypeptides P27, R60, R14 and R15 are also included in the book. More preferred polypeptides of the invention have similar stable conformations in physiological solutions. It has a very similar spectrum indicating that it exists as Polypeptide E The spectrum of (R12) shows a partial assumption of such a conformation. Ru.

Figure 2 shows rabbit anti-peptides against synthetic polypeptides C (R60) and B (R62). Nitrocellulose analysis of whole cell extracts of EBV-transformed W[-L□ cells using antiserum - Photographic diagram of the swim-immunoplot. The front part was determined to be anti-EBNA positive. That is, human serum (from patient TJ) containing anti-EBN^ antibodies was added to the positive column in lane A. A dilution of 1:20 was used as a control. t: Rabbit anti-P60 in SO dilution (C) Serum (lane B) and rabbit anti-P62 (B) serum at 1:10 dilution were positive. These peptides recognized natural EBNA by immunoreacting with the same band as the sex control. I showed that I understand.

Lanes A-G are indicated at the bottom of the figure.

Recognition of EBNA by anti-P60 serum was 40 micrograms per milliliter (μg A 1:50 diluted anti-P60 serum containing polypeptide P60 of This was suppressed by incubating for 1 hour before washing. Similarly, anti-P62 Recognition of EBNA by serum containing 40 μg/− of polypeptide P62 1;10 Diluted anti-P62 serum was incubated for 1 hour before immunoblotting. Therefore, it was suppressed.

The antigen association of P2O and P62 is shown in lanes 6 and 7. Lane 6 is EBNA band In lane 7, showing 1:10 diluted anti-P62 serum immunoreacting with anti-P6 2 The reactivity of the serum with the EBNA band was determined by incubating polypeptide for 1 hour before immunoblotting. It was suppressed by inkjehate with TidoP60.

Figure 3 shows the antagonism of the anti-P62 serum activity of patient 1011's EBNA-positive serum in solution. It is a graph showing inhibition by polypeptide.

ELISA using polypeptide P62 as a solid phase target Used in EL I SA with either P27, P62, P2O, P89 or PI6. The serum from patient 1011 was pre-incubated for 1 hour before use.

Polypeptides P27, P62, P2O, P89 and PI3 are each defined herein as Also referred to as polypeptides A, B, C, D and G in . percent Antagonism of anti-polypeptide activity of micrograms per milliliter (8 g/M1) Plotted as ordinate versus polypeptide concentration.

Figure 4 shows antibodies against EBNA (dotted line, open circles) and polypeptide P62. Illustrative example of antibodies (solid lines, black circles) produced in the case of EBV-contagious mononucleosis (BBViM). It is a graph showing the current parallel time elapsed. Sequential sera were collected after clinical onset and anti-E Catalano et al., J. Cl1n, Invest, on BNA activity. 65: Right ordinate according to the procedure reported in 1238 1242 (1980) Titered above. Each serum sample also contains polypeptides in the ELISA of the invention. Optical density of 405 nanometers (OD4°, ) was assayed using the plot as shown on the left ordinate.

Figure 5 shows 1 (en+e, G, etc., J, Infect, Dis,, l 3 0:231 (1974) using the classical ACIF method for detection. Antibody against polypeptide P62 (solid line, black circle) [! BNA (dotted line , white circles) show two graphs (Figures 5A and 5B) showing relatively early detection. ), sequential sera were collected from two patients with clinically proven infectious mononucleosis (#1 #4 top panel, Figure 5A; #2 bottom panel, Figure 5B).

Each serum had anti-EBNA activity as reported by Catalano et al., supra. The titer was measured. Anti-polypeptide activity is achieved by targeting polypeptide P62 on a solid phase. The activity was measured using the ELISA of the present invention, which was used as a kit, and the activity is shown in Figure 4. There is.

Figure 6 shows acute EBV-1M sensitivity assayed by EBNAP62 ELISA. Figure 3 is a graph showing the analysis of sequential serum from patient B, who had an infection. acute stage IgM response is shown by a black circle, recovery phase ■gG response is shown by a white circle, IgG/IgM The ratio is shown by the white square.

The EBNA polypeptide P62 assay was performed as described in the Materials and Methods section below. I went there.

Figure 7 is a photograph of an immunoplot showing the analysis of sequential serum from patients A and B. Ru. EBV-immortalized Wi L2 (also referred to as WI Lx) cell lysate was added to each protocol. The F, BNA-BNA protein was also used as a protein source. On the left side of the plot (Figure 7A) In this case, a healthy (NORM) VCA-positive sample was used as a control. It showed 77KD EBNA protein white matter. Each plot is 77KD EBNA Delayed responses of IgG antibodies to proteins (left) and these during the acute phase of the disease. A rapid increase in IgM antibodies against the protein was shown (right side, Figure 7B). disease onset The number of days to come is indicated by numbers to be read vertically below each lane.

Figure 8 shows a first infection with CMV-IM followed by a second infection with EBM-IM. Figure 2 is a graph showing the analysis of sequential serum from patient R. Acute phase 1gM response is shown as a black circle, the convalescent 1gG response is shown as a white circle, and the IgG/IgM ratio is shown as a black square. vinegar.

Figure 9 shows the sequential immunoblotting analysis of serum from patients R, S. Figure 3 is a photograph of the immunoplot test shown. On the left side of the plot (Figure 9A) is the patient's serum. The right side of the plot (second section) shows the acute phase 1 gM response to EBNA-alpha white matter. Figure 9B) shows the IgG response from patient serum. Numbers below each lane and NOR The lanes labeled M are the same as in FIG. EBNA-Also white matter is later Prepared from W i-Lz cell extracts as described in the Materials and Methods section.

Figure 10 from two patients with CMV-1M infection who had a previous EBV infection. Two groups showing polypeptide P62EL I SA analysis of sequential serum samples of It's rough. In Figure 10A of patient R, S, the IgM antibody response is indicated by a white circle. The IgG response is shown as a black circle, and the IgG/IgM ratio is shown as a black square. ing.

In Figure 10B for patients S, G, the 1gM response is indicated by a white circle, and the IgG response is indicated by a black circle.

Figure 11 shows four patients with acute EBV-IM and six patients with acute CMV-IM. Contains two panels that are photographs of immunoplot analysis of four sera from human patients. In panel 8, each serum was collected from 562 cells (EBV-, CMV-'). IgM antibodies were specifically detected by immunoblotting on extracts of . panel In Group B, each serum was blotted on extracts from late CMV-infected human fibroblast cells. IgG antibodies were detected by blotting. All sera were used at a 1:20 dilution. Ta. The letter markings "E' and "C" above each lane indicate EBV-IM or Shown are sera from patients with either CMV-IM.

Figure 12 shows the acute stage of the patient's illness, B, D, S, and T, G. 57) IgM and IgG (7) from serum samples collected during the recovery phase. W i L* cells, im showing inhibition of binding to extracts from EBV” B cell line This is a photo of Noplot. Patient G is a healthy VCA", EBNA-1"" donor. - was. Lanes 1 and 2 are patient samples obtained 15 days after the onset of illness (pod). Lanes 3 and 4 contain serum samples from patients from the 12th pod. and lanes 5 and 6 are from patients T and G with acute IM taken on the 4-day pod. and lanes 7 and 8 are from patient B on day 514, lane IO Lanes 11 and 11 are from the 471-day pod patient, S, and lanes 11 and 12 are from the healthy control. It was from NaG. All sera were diluted 1:50. Odd lanes indicate serum Contains serum + polypeptide and is marked with a minus sign (-), and even lanes indicate serum + polypeptide. It contains P62 and is marked with a plus sign (+). Polypeptide P62-1 The concentration was 400 micrograms per milliliter (Ig /d) and was 50 μg/w1 in lanes 8.10 and 12. . [gM antibodies were specifically detected in lanes 1-6, while Igc antibodies were detected in lanes 7-6. 12 was specifically detected.

Figure 13 is from 5Trapraus et al., J. Virol, 57:5916. 02 (1986) of the CMV-encoded polypeptide antigen. The amino acid residue sequence is shown. The number position of each residue in the sequence is indicated by the number at the left end. present invention Preferred CMV homologous peptides range from about residue position 200 to about residue position indicated in the figure. It contains an amino acid residue sequence corresponding to the sequence of I220.

Figure 14 shows EBV homologous peptide P62 and CMV homologous peptides C9 and C2. Anti-EBNA Ig G (open symbols) or I in the serum of patient DB during EBV infection. It is a graph showing the detection ability of gM (no symbol) antibody. In the acute phase of infection is an anti-EBNA IgM antibody with EB homologous peptide P62 (triangle), CMV Using each ELISA of homologous peptide CI (circle) and CMV homologous C3 (square) The onset of the recovery phase, which was detected and confirmed the presence of infectious mononucleosis, was caused by anti-EBNA IgG. indicated by a rise in IgM antibodies and a concomitant drop in IgM antibodies.

Figure 15 shows EBV homologous peptide P62 and CMV homologous peptides C1 and C2. Anti-EBNA Tg G in the serum of patient DS during CMV infection followed by EBV infection (white symbols) or JgM (no symbols) is a graph showing the ability to detect antibodies. both During the acute phase of the disease, anti-EBNA IgM antibodies are combined with EBV homologous peptide P6. 2. ELISA of CMV homologous peptide C1 and CMV homologous peptide C3 was used to confirm the presence of infectious mononucleosis at the onset of each acute illness.

[Detailed description of the invention]

■Introduction Humans infected with Epstein-Barr virus (EBV) undergo viral transformation B. Express antibodies against viral nuclear antigen (EBNA) present in lymphocytes. Hi Traditional clinical techniques used to assay for EBNA and anti-EBNA antibodies are Moreover, current methods for purification of EBNA from cell cultures are difficult to produce in large quantities. It is not something that can be easily adapted to production.

The present invention uses synthetic polypeptide technology to overcome some of the problems of current methodologies. intend to. Short synthetic polypeptides immunologically mimic antigens on natural proteins may be a determinant and, therefore, may be a precursor to the recognition of natural proteins. It can be used to produce the body.

The term "immunologically mimetic" is used herein to describe the immunogenic polypeptides of the present invention. peptide binds to the inducing polypeptide and to the same sequence in the intact protein. It is meant to include the production of antibodies that This phenomenon has been demonstrated both experimentally and clinically. Can be used.

Experimentally, antibodies against synthetic polypeptides have been developed in the DNAR frame and and thereby the amino acid residue sequences of clinically important proteins such as EBNA. Can be used to establish. Clinically, the front end produced for synthetic polypeptides Antibodies of determined specificity can be used for diagnostic and therapeutic purposes.

Helfer et al., J. Virol, 44:311-320 (1982). A DNA nucleo having characteristics indicating that it may contain a gene encoding BNA reported the sequence. They found that if they literally translated this DNA into protein, there would be three latents. The current reading frame depends on the expressed DNA reading frame, but (i) Zon, arginine, and glycine; (ii>glycine and alanine; or (i ii) More than 200 molecules consisting only of glutamine, glutamate and glycine It was predicted that it would encode an IRi protein domain of amino acid residues.

The reported chemical properties of the EBNA molecule are similar to the potential stop codon of the EBNA gene. When taken together with the distribution, IRj mainly consists of glycine and alanine residues. It was shown that

To evaluate this suggestion, we determined that the amino acid residue sequence was substantially corresponds to the amino acid residue sequence of the EBNA protein, which is a random copolymer. We synthesized polypeptides of this type.

As will be seen below, these synthetic polypeptides, particularly one designated P62, was found to immunologically mimic EBNA. moreover , a group of particularly preferred polypeptides encompassing polypeptide P62 is human anti-EB It was also found that it binds NA antibodies. Various assay methods for these polypeptides The use in order will also be explained later.

In further detail with respect to such assays, specific preferences, preferably solid phase We developed an assay using a unique polypeptide.

This assay is used for infectious mononucleosis (IM) due to EBV and IM due to CMV ( CMV-IM), another disease associated with EBV, is the nasopharyngeal cavity. It has been found that it can be clinically reliable in cancer (NPC) tests. each The specific results of assaying in a region are generally described as assay methods.

■, Preferred embodiment A0 synthetic polypeptide ■、Array A series of small synthetic polypeptides (5 to 21 amino acids in length) used in this study. Acid residues) were synthesized using the solid phase method of Merrif 1eld. Merrifi eld et al., J, As, Chew, Soc, 85: 2149-21 54 (1963), the sequence is within and just outside the above proposed lR35l region of EBNA. The sides were chosen to represent different areas.

As used herein, "synthesis" refers to synthetically synthesized polypeptide molecules or polypeptides. If the repeat unit is prepared by biological means such as by genetic engineering techniques, Rather, it means to be constructed by chemical means, that is, to be chemically synthesized. . That is, synthetic polypeptides embodying the present invention are derived from natural proteins and their flags. Does not include ment.

Therefore, chemically synthesized polypeptides are prepared by the action of cyanogen bromide, a natural protein. It is different from the decomposition products of natural proteins such as

A well-known method for adding blocked amino acid residues in a sequential manner to obtain the desired polypeptide. Phase chemical synthesis is the preferred method of synthesis and is described in more detail below.

All amino acid residues shown herein are in the natural or L-form. standard polype According to the petid nomenclature, the abbreviations for amino acid residues are as follows: Correspondence table Name Amino acid 1-character 3-character F　Phe　L-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine L-order e L-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P Pro L-proline K Lys L-lysine HHis L-histidine Q Gin L-glutamine E Glu L-glutamic acid WTrpL-Tryptophan RArg L-Arginine D Asp L-aspartic acid N Asn L-asparagine CCys L-cystine One aspect of the invention is about 6 to about 40 amino acid residues, preferably about 15 to about 20 amino acid residues. contains amino acid residues, and from left to right, from the amino terminus to the carboxy terminus. Expression written in direction: % expression % (In the formula, R1 and R2 are individually the same or different, and Ala, Asn , Arg % Gly % Leu s Pro , , Ser and Thr However, R1 and Rt are both not GIV). Polymeric polypeptides are intended. The polypeptide also has at least 25 molar % glycine residues and when conjugated to a carrier and administered to a mammalian host in an effective amount. can induce the production of antibodies that immunoreact with EBNA.

In one preferred implementation T*, R1 and R1 are amino acid residues in the polypeptide. Contains base sequence: -GIVyArgGly-Arg-G11y- Both sea urchins are ^rg. In another preferred embodiment, R- is As n, R1 is Leu, and the polypeptide has the amino acid residue sequence; -GJ! y Contains -Asn-Gly-Leu-Gly. Yet another preference In some embodiments, R1 and R8 are both Sets and the polypeptide is an amino acid. Noic acid residue sequence: -GA! y　-5er　-Gj! y　-3er　-Gj! y- Contains.

Preferred amino acid residue sequences include the formula: %formula% The left-to-right hair amino-terminal to carboxy-terminal sequence represented by, its pharmacology. and antigenically related variants thereof.

In more preferred embodiments, R1 and R8 are AJa or Gly. example For example, R1 can be ^Ila and R1 can be ^2a; R1 can be Ala and R2 can be Gly. Possible; R1 can be Gly and RX can be Aja. Thus, preferable to these A preferred embodiment includes (i) -Gly -Aj2a -G11y -Ajla - (ly　-;(ii)-Glly-＾A!a　-G11y　-Gay　-Gly −; and (iii) −(ly −Gay −Gly−＾1a −Gj!y There is a sequence of five amino acid residues represented by the formula selected from the group consisting of -1.

The term "random copolymer" is used herein in its ordinary meaning.

That is, these polypeptides contain multiple repeat units of different amino acid residues. Therefore, it is a copolymer. These copolymers can be made into alternating or block copolymers. are relatively random because the individual amino acid residues of these polypeptides The group is an alternating copolymer, or as described in Anderson et al., Doyle et al., supra, or In the repeat unit of the homoblock copolymer prepared by Brack et al. This is because it is not present in the specific repeating unit as found.

That is, a continuous repeating sequence: -Ala -GAy-^1a -Gly-Gay It was named R62 containing -Gly -Affia -GJy -Gly- Even the polypeptide (Table 1) has -Al at the carboxy terminus. It further contains an a-GIV peptide.

As a result, there are no amino acid residue sequences that repeat throughout the polypeptide; P63 should be viewed as a random copolymer, with specific amino acid residues Prepared by Brack et al. in which the same blocks of sequence repeat throughout the polymer. Prepared by poly<AlaK-Glya) material or ^nderson et al. It is not a homoblock copolymer such as poly(Set-Gly).

Random copolymer polypeptides of the invention are often referred to herein simply as Referred to as a "polypeptide," "synthetic polypeptide," or "peptide." This angel The usage is for brevity.

The term "antigenically related variants" as used herein refers to fewer than one antigenic determinant. Amino acid residues as a whole that have at least one moiety and are therefore immunologically cross-reactive Used to refer to polypeptides that differ in sequence.

As used herein, the term "antigenic determinant" refers to the same or related antigen or Due to the specific reaction with the corresponding antibody (immunoglobulin) molecules elicited by the immunogen Refers to the structural component of a molecule that is responsive.

As used herein, the term "immunogenic determinant" when used as an antigen In the host, an antibody containing an antigen-binding site (ideotibe) that binds to the immunogen of Refers to the structural component of a molecule that is responsive to an inducement.

The term "antigen" as used herein refers to an entity that binds to an antibody. .

The term "immunogen" as used herein refers to an immunogen that induces antibody production in a host animal. means an entity that exists. In some cases, the antigen and immunogen are the same entity; In other cases, the two entities are different.

For example, as described below, polypeptide P62 can be used to stimulate antibody production in rabbits. induced and therefore used as an immunogen. The antibodies thus induced can be used as antigens. When used, it binds to polypeptide P62. Polypeptide P62 is therefore immunogenic. It is both a source and an antigen. Anti-EBNA antibodies target both EB as an immunogen and antigen. It binds to NA and also to the polypeptide p62 as an antigen.

A preferred embodiment of the present invention is a random copolymer polymer as shown in Table 1 below. Peptides P89, R12, R13, pharmaceutically acceptable salts thereof, and antigenic It is a mutant to reach the point. Each of these polypeptides is -cly-R'- cly-R”-Gj!y-amino acid residue sequence, R1 and R2 are as described above. each polypeptide has at least 25 mol% Gj! Contains y; Each can elicit antibodies that bind to EBNA, as described above.

Table 1 Sequence of synthetic polypeptide Peptide 9 Supervisor - 111 row P60(C) H-Gly-Gly-cly-Ala-Gly-Ala=Gly -Gly-8la-Gly-Ala-Gly-Gly-Gly-Gly-Arg -OH:R89(D) H-Arg-person 1a-Arg-Gly-Arg-Gly -Arg-Gly-Arg-Gly-Glu-Lys-Arg-Pro-Mat -07F12(E) H-Eng 1e-Mat-5er-85p-G1. u-Gly -Pro-Gly-Thr-Gly-Asn-Gly-Leu-Gly-Glu -○H:F13(F) H-Pro-Gly-Ala-Pro-Gly-Gly -5er-Gly-5er-Gly-R27(A))I-Lys-Gly-T hr-His-Gly-Gly-Thr-Gly-Ala-Gly-Human 1a-G ly-Ala-Gly-Gly-Ala-Gly-Ala-Gly-oHrP6 2(B) H-Ala-Gly-Ala-Gly-Gly-Gl, y-Ala- Gly-C1y-Ala-R14H-Gly-G1. y-Al　a-Gly-G ly-Al　a-G　y-Gly-Al　a-Gl　y-人1a-Gly -Gly-Gly-Ala-Gly-OH; R15H- to 1y-Ala-Gly -Gly-81a-Gly-Ala-Gly-Gly-Gly-人1a-Gly -Gly-Ala-Gly-Gay-OH; R16 (to) H-Gly-Gly -Ala-nily-person1a-Gly-Gly-Ala-ni my-Ala-Gly -Gly-Ala-Gly-Ala-Gly-Gly-Ala-nily-person 1a -Gly-OH; Capital letters in parentheses indicate the corresponding polypeptide in some of the figures and tables. used for

Results of polypeptide/anti-polypeptide receptor binding and binding inhibition tests are provided in Chapter H. This will be described later in D. These results are relative to the amount of sequence homology in the polypeptides. Examples of cross-reactivity and cross-inhibition effects are shown. For example, polypeptide P6 0 contains a 10 amino acid segment homologous to R62. Polypeptide P 27 has eight amino acids homologous to polypeptides P60, R62 and DI (Table 2). Contains acid segments. Polypeptide D2 (Table 2) is polypeptide P27, R 7 amino acid residue segments homologous to each segment of 60, R62 and Di. Contains

Polypeptide 89 that did not significantly cross-react in the test was polypeptide P27. , I) Contains no sequence homology with 62 and R62.

More importantly, the polypeptides P27, R62, R60 and DI share Eight amino acid residue sequences are common to all three random copolymer polypeptides contain at least one antigenic determinant that makes these three polypeptides It is considered an antigenically related variant. Furthermore, there are 6 shared segments. Amino acid residue sequence -cg-＾i? a-Gl! 'l-GIY-ia-Gly- and the formula: -Gi)y -R' -Gly -R'' -Gly (where R1 and R1 are both Afa), i.e. -Gi'y -Ajl'a -Gi! y-Ala-G It contains an overlapping sequence indicated by ly-.

“Duplicate” means that the sequence with the second name is contained in a part of the sequence with the first name. Taste. This duplication of amino acid residues results in a single amino acid residue in polypeptide P62, shown below. Illustrated by overlapping "box" sequence sections using letter amino acid residue codes.

G-G-G-A-G-G-A-G-A-G-G-G-A-G-G shared six axes Random containing both amino acid residue-containing sequences and overlapping 5 amino acid residues Copolymer peptides constitute a further particularly preferred embodiment of the invention. . In such particularly preferred embodiments, random copolymer polymers of the present invention The repeptide contains about 8 to about 40, preferably about 15 to about 20 amino acid residues. In addition to the -czy-R'-Gay-R''-Gly-amino acid residue, the left Formula written from amino terminus to carboxy terminus to the right: %formula% and contains at least about 50 mol% Gll, and (a) EBNA and immune reaction when bound to a carrier and introduced into a mammalian host in an effective amount; (b) inducing the production of corresponding antibodies; and (b) inducing the production of antibodies corresponding to Both are capable of immunoreacting with human antibodies.

Particularly preferred amino acid residue sequences include left-to-right hair amino-terminus to carboxy-terminus; Each sequence represented by the following formula written in the direction of its pharmaceutically acceptable salt and its antigen There are variants related to: (i) -Gly-Gly-Gly-Ala-Gl y-Ala-Gly-Gly-Ala　-Gly-人1a-Gly-Gly-G ly-Gly-Arg-; (ii) -Lys-Gly-Thr-His-Gl y-Gly-Thr-Gly-Ala-Gly-Ala-Gly-Ala-Gl y-Gly-Ala-Gly-Ala-Gly-; (iii) -Ala-Gl y-Ala-Gly-Gly-Gly-Ala-Gly-Gly-Ala-Gl y-Ala-Gly-Gly-Gly-81a-Gly-Gly-Ala-Gl y-; (iv) -Gly-Gly-Ala-Gly-Gly-Ala-Gly-Gl y-Ala-Gly-Ala-Gly-Gly-Gly-Ala-Gly-:( V) -Gly-person 1a-Gly-Gly-Ala-Gly-Ala-Gly- Gly-Gly-Ala-Gly-Gly-Ala-Gly-Gly-; (vi ) -Gly-Guy-Ala-Gly"Ala-Gly-Gly-person 1a-G ly-person 1a-Gly-G1. y-person 1a-Gly-Ala-Gly-Gly- Ala-Gly-Ala-Gly-; (vii) -Gly-Gly-Gly-Ala-Gly-Gly-Ala-G ly-Ala-Gly-Gly-Gly-Ala-cly-Ala-Gly-: (viii) -Person 1a-Gly-Gly-Person 1a-Gly-Person 1a-Gly- Gly-Gly-Ala-Gly-Ala-Gly-2 The first and last dashes in an amino acid residue sequence indicate the amino and carboxy termini. groups such as H and OH at each end or a total of 40 within the polypeptide chain was noted to indicate a bond to one or more amino acid residues up to the amino acid residue of Or rather, the corresponding polypeptide itself, i.e. the polypeptide P60 shown in Table 1. , R27, R62, R14, R15 and R16, and Dl and R16 shown in Table 2. and D2, their pharmaceutically acceptable salts, and antigenically related variants thereof. is particularly preferred, polypeptide P62 is herein particularly preferred. Used as an illustration of the example of putide. Polypeptide P62 is also often R62, Displayed as Petit P62, EBNAP62, etc., but all displays including 'P62' shall be understood to refer to this substance; the terms “peptide” and “polypeptide” shall be understood to refer to this substance; "Do" is used tautomerically herein.

As used herein, the term "pharmaceutically acceptable salts" is a term well known in the art. Sodium, potassium, lithium, calcium, magnesium prepared by the method of Non-toxic alkali gold used in the pharmaceutical industry such as salts such as ammonium and ammonium etc. genus, alkaline earth metal and ammonium salts. The term also refers to the present invention. Nontoxic acid addition salts are also commonly prepared by reacting a compound with a suitable organic or inorganic acid. Representative salts include hydrochloride, hydrobromide, sulfate. , bisulfate, acetate, oxylate, valerate, oleate, laure ate, porate, benzoate, lactate, phosphate, tosylate, silane There are trate, malate, fumarate, succinate, tartrate, etc.

In another preferred embodiment, the present invention provides EBNA or at least a sequence homologous (corresponding) to the sequence of the CMV-encoded polypeptide. also contains from about 15 to about 40 amino acid residues and has the aforementioned formula: -G7! y- Polypeptides comprising R'-Gly-R"-GIV- are contemplated. E of the present invention BN/', homology and CMV-homologous peptides were immunized with anti-EBNA antibodies. Further characterized as having the ability to react.

2. Size The ability of synthetic polypeptides to bind to anti-EBNA antibodies The effects of the changes were tested. When the size of a polypeptide is reduced, its antibody and It has generally been found that the ability to bind is also reduced.

Polypeptide P62 is shown in Table 2 using the one-letter code for amino acid residues. 20 amino acid residues in which the first 9 residues from the amino terminus are directly repeated It will be. The amino acid residue triazodine (3-fold axis of symmetry) is attached to the amino terminus of the P62 peptide. A series of polypeptides homologous to the P62 deleted from was synthesized and shown in Table 2. Eel polypeptides D1, D2 and D3 were obtained. Therefore, the increase in sequence symmetry of P62 The majority are absent in polypeptides DISD2 and D3.

Table 2 Polypeptide display Amino acid residue sequence “Di: GGGAGGAGAGGG GAGGAGD2: AGGAGAGGGAGGAGD3: AGAGGG AGGAG A5: GGGAG A6: AGGGAG A7: GAGGGAG A8: AGAGGG8G To A9: GAGAGGGAG #Polypeptide sequences using one-letter codes are left to right, \amino terminus to carboxyl. Shown at the boxy end.

* Three nonamer direct repeat junction group polypeptides in polypeptide P62 E to immunoreact with human anti-EBNA serum and rabbit anti-P62 serum as described below. Each polypeptide was tested on the solid phase of LISA. Antibodies that bind to DI Almost identical to the antibody that binds to the original polypeptide P62 in all tested sera. there were. In contrast, all sera except rabbit anti-P62 showed no binding to solid phase D3. There was no match. D2 results were intermediate and depended on the test serum. That is, the poly of this group Antibody recognition of peptides reduces polypeptide length from 20 to 11 amino acid residues It decreased as time went on.

The fact that antibody binding was reduced in all antisera indicates that the polypeptide was contains the original determinants, and only one of them was affected by the sequence deletion, so there is only one unique This indicates the opposite possibility that the target antigenic determinant is missing due to sequence deletion of amino acid residues. are doing. In addition, the sequence symmetry of P62 is due to the 4 to 8 amino acids present in P62. All of the sequences of acid residues are also present in D3 except for the residues at the repeat junction. Guaranteed.

EIjSA solid support - conformational change of bound polypeptide May contribute to suppression of antibody recognition. This possibility could be explained by some serum solid-phase bound P62. binding (immunoreactivity) is inhibited using various concentrations of antagonistic polypeptides in solution. Tested by controlling. Each antiserum was tested for polypeptide P62, DI, D2 or P62 is mixed in solution with D3 for a predetermined period of time sufficient for an immune reaction (binding) to occur. Maintain (incubate) before adding to coated microtiter plates. g) did. The results of this study with sera from five patients are summarized in Table 3 below. .

Table 3 Concentration of antagonistic polypeptide that gives 50% inhibition of antibody binding to peptide P62 1 Tsume 5 Yuzubutido (wll [I　P62　dan-上　dan-1dan-1 TJ　O,050,050,1200 VM O, 30, 30, 43 CV　O, 10, 10, 11O N6 0.6 0.6 0.6 3 JC], 1 1 500 *The EL I SA procedure used for these measurements is described in the following chapter [[E (2) and I[I/D].

The inhibitory effect on antibodies that bind to P62 of polypeptide D1 is due to the inhibition of P62 itself. It is considered indistinguishable from the control effect. True for all tested sera Met.

More interestingly, polypeptide D3 is approximately larger than polypeptide D3. As well as inhibiting some of the sera, namely VM and N6. This strong restraint What effect did any of these sera have on D3 bound to a solid support in an ELISA? This occurred even though they did not show any binding. These data are polypeptide maintains a defined secondary structure when bonded to the plastic surface of a microtiter should have a minimum length, i.e., at least about 15 amino acid residues, to This is considered to indicate that.

Thus, in solid phase assays, peptide lengths of about 15 to about 40 residues are preferred. and more preferably from about 15 to about 20 lengths.

The minimum antigen size required for recognition is polypeptide A5, A6, A7, A8 and A9. Further tests were conducted using As shown in Table 2, polypeptide A5 has five amino acids. each member of A6 to A9 has a polynucleotide residue up to the nine residues present in A9. It increases in length by one amino acid residue compared to peptide 5. The test antiserum is These ports when bound to a microtiter plate in the ELISA described below. There was no immunoreaction with the ripeptide.

The ability of group A polypeptides to inhibit the binding of human anti-EBNA antibodies to solid phase P62 Data regarding this are shown in Table 4 below.

Table 4 Suppression of antibody binding to peptide P62 by 100μg/- of antagonistic peptide , □ Sarashin N Akuji Tayu ni millet [ Belly - 18 - Standing A 6 Person - 1 - [1 Person - Yu T'J 96 80 91 74 31VM 93 81 51 17 9 cV 92 93 93 72 20 , JC9492608874 362869689957B S6o 106 109 93 84 53 *These tests are mentioned in Table 3. I went there.

Almost all tested sera were inhibited by A9, but a higher concentration was required. (Concentrations of R62 or DI required for equivalent inhibition and one serum suppressed by A7) was suppressed. It was not inhibited by short polypeptides A6 and A5.

In other words, the decrease in immunoreactivity compared to the decrease in polypeptide size is due to the following two effects. It appears to be based on the results that: (1) antibodies such as those exhibited by group A polypeptides the effect of lacking a site on the antigen that binds, and (2) the group D polypeptide. A change in the conformation of a polypeptide when its size decreases.

3. Conformation The conformational properties of the polypeptides of the present invention were determined using circular dichroism (CD) spectroscopy. It was tested. Polypeptides P27, R60, R62, R13, R15 and R1 The CD spectrum of 6 was measured. The data, shown in part in FIG. amino acid residue sequence; i.e., Gjl! y R’ G 12 y R” G j! y (in the formula, R1 and R2 are as described above) and -Gay -AZa-' G1y-Gly-Ala-Gj! Polypeptides of the invention comprising both y- has a relatively stable secondary structure or conformation in physiological solutions at 20°C. Which indicates that. The primary conformation of these polypeptides is relatively stable Therefore, human anti-EBNA antibody activity is in response to this special conformation. This is considered to occur.

B, multimer The invention also provides polypeptides in which at least one of the repeating units is a polypeptide as described above. polypeptide containing a number of linked random copolymer polypeptide repeat units Regarding multimers.

The multimer of the present invention can be administered to a mammalian host in an effective amount alone or in combination with a carrier. When done, production of antibodies that bind to EBNA can be induced. Particularly preferred multimers are CMV homologous polypeptides of the invention capable of binding human antibodies elicited by EBNA It's petite.

That is, the multimer of the present invention, like its constituent polypeptides, is immunogenic and has human anti-inflammatory properties. Antigenic to EBNA antibodies. Therefore, these multimers can be diagnosed using the diagnostic method described below. Methods and compositions that can be used to induce the production of anti-EBNA antibodies that are useful in methods and compositions. It can also be used as an antigen in suitable diagnostic methods and compositions.

Multimers with fewer than about 35 amino acid residues in the total multimer are useful as immunogens. It is typically attached to a carrier in its application. Contains about 35 or more amino acid residues in total. Typically, the multimers are sufficiently immunogenic to be used without a carrier.

Polypeptide multimers are produced by combining multiple polypeptide monomers head-to-head using the solid phase method described above. can be prepared by joining together in the tail form, i.e., one complete polypeptide The sequence is synthesized on resin and then over 111 of the same or different polypeptide sequences are synthesized. react the column and then separate the entire multimeric unit from the resin as described above. use. Such head-to-tail polypeptide multimers preferably contain about 2 to about 4 polypeptides. Contains repeat peptide units.

In addition, multimers are polypeptides of random copolymer polypeptides used as monomers. As used herein, the literal term 'polymer' The term refers to multiple random copolymers joined together by other than peptide bonds. - defined as a type of multimer containing polypeptide repeating units.

An exemplary polymer of the present invention has a silica added to both the amino and carboxy termini. Polypeptide monomers of the invention containing a Stin residue (di-Cys polypeptide ) can be synthesized using This di-Cys polypeptide monomer is separated using an oxidation method. Within the molecule, the co-polypeptides are bound together by cysteine disulfide bonds to form an immunogen. Polymers that are sensitive and antigenic can be prepared. The polymer thus prepared is a polymer of the present invention. It contains multiple repeating units with random copolymer polypeptide. These repetitions The units are linked together by the oxidized cysteine (cystine) residues mentioned above.

Use of the present invention for binding polypeptides to carriers or for preparing polymers The presence of one or two terminal Cys residues in the polypeptide The amino acid sequence of the repeat unit should not be construed as changing.

C1 inoculum In another embodiment, the polypeptide of the invention is administered in a pharmaceutically acceptable diluent. EBNA can induce antibodies that are immunoreactive with EBNA when administered in effective amounts. Produce an inoculum or vaccine.

The literal term "inoculum" as used herein refers to the polypeptides of the invention. describes a composition containing as an active ingredient for the production of antibodies against EBNA. used for

When using a polypeptide to induce antibodies, the polypeptide is used alone or in a carrier. It should be understood that it can be used in combination or as a multimer; For reasons of simplicity, these variations are not necessarily described hereafter.

For polypeptides containing fewer than about 35 amino acid residues, the antibody It is preferable to use a carrier for the purpose of inducing production. Polypeptide attached to carrier is used herein as an example to produce antibodies.

The inoculum is for use in diagnostic assays to detect cells expressing EBNA. Can be used to produce antibodies. The antibodies produced by this inoculum were EBN Preparation for inducing passive immunity against 8928 spheres expressing A on its cell surface It can also be used in objects.

The literal term "vaccine" is used herein to refer to the polypeptides of the present invention. as an active ingredient used to induce active immunity in a host animal. Used to describe a type of inoculum. Active immunity involves the production of antibodies, so A vaccine or inoculum may contain the same ingredients but have different uses. Almost In all cases, the components of vaccines and inoculants contain many compounds that are useful in animals. This is different because adjuvants cannot be used in humans.

The inoculum or vaccine contains an effective amount of the polypeptide of the invention in an oxidized polypeptide. Polypeptides of individual polypeptides linked together via peptide-terminal cysteine residues Contained as a multimer such as a mer or as a conjugate attached to a carrier do. However, for ease of explanation, various implementations of the polypeptides of the invention will be described. Embodiments herein include the term "polypeptide" and its various literal forms. Use in bulk.

The effective amount of polypeptide per unit dose may vary depending on the species of animal inoculated, among many others. , depending on the weight of the animal and the method of inoculation as is well known in the art. Inoculum and vaccines have a polypeptide concentration of about 10 μg to about 500 μg per inoculation (dose). Contains. This amount of polypeptide does not include the weight of the carrier when a carrier is used. The weight of a polypeptide. Specific exemplary inocula, described below, include carrier + port. The weight of the lipeptide (conjugate) is shown.

The term “unit dose cap” refers to a physically separate unit suitable for a single dose for animals. each unit contains a predetermined amount of active substance calculated to produce the desired therapeutic effect. Novel unit dose caps of the present invention containing together with a diluent, i.e. a carrier or vehicle. details the unique characteristics of fat active substances and the therapeutic effects to be achieved, and (bl Techniques for the formulation of active substances for therapeutic use in animals as detailed in the specification. are governed by and directly dependent upon the inherent limitations of the invention, and these are the features of the present invention.

Is the inoculum a dry solid polypeptide polymer or polypeptide polymer? These polypeptide-conjugates or polypeptide polymers are Water that is acceptable to the public 2. Dilutions like saline or phosphate buffered saline It is typically prepared by suspending it in an agent.

The inoculum may also contain an adjuvant. Complete Freund's adjuvant (CFA) ), incomplete Freund's adjuvant (IFA) and adjuva such as alum Agents are materials well known in the art and are commercially available from several sources. Wear.

D7 receptor produced against the polypeptide of the present invention (induced by the polypeptide of the present invention) antibodies) and substantially whole antibodies and antibody binding moieties prepared from such antibodies; This contributes to yet another embodiment of the invention. These molecules are collectively known as a book. In the specification, it is called a receptor. The receptor is found in mice, rabbits, horses, etc. It is produced in a mammalian host by immunization with the inoculum described above.

Suitable monoclonal receptors, typically whole antibodies, also include N1eaan etc., Proc, Natl, Sci, U, S, 8, 8 0 H4949-4 953 (1983). Ru. The description of this document is incorporated herein by reference. In short, monoclonal To prepare hybridomas that produce receptors, myeloma or other A self-permanent cell line was obtained from the spleen of an animal hyperimmunized with a polypeptide of the invention. fuse with lymphocytes.

Preferably, the myeloma cell line is from the same species as the lymphocytes. typical In total, 129Gj! x” mice are the preferred animals. Hypoxanthine-aminopterin-thymidine is suitable for mouse myeloma. sensitive (HAT) cell line P3X63-Ag8.653 (ATCC CRL158 0), and Sc210-Ag14 (ATCC CRL1581).

Spleen cells are typically myelinated using polyethylene glycol (PEG) 1500. It is fused with Roman cells. fusion hybrids by their susceptibility to HAF. select. Hybridomas producing the receptor molecules of the present invention can be prepared using the materials and materials described below. Enzyme-linked immunosorbent assay (El, 1) described in Methods section 111D 5A).

Wang Tuclonal receptors should be obtained from hybridoma supernatants as well as the desired Generally, the hybridoma is obtained in a more reduced form from the ascites of an animal that has been introduced. Can be done. Production of monoclonal antibodies using ascites is well known and will be further explained. I don't think it will happen.

The receptor of the present invention binds to the polypeptide that produced the receptor, and The polypeptides of the invention also immunologically mimic the corresponding EBNA antigenic determinants. Join. Thus, polypeptides of the invention can be both immunogens and antigens.

The receptors of the present invention are oligoclonal when compared to natural polyclonal antibodies. This can be explained as This is because they are produced against immunogens that have relatively fewer epitopes than their offspring.

As a result, the receptor of the invention binds to an epitope of the polypeptide, = while , natural antibodies raised against EBNA bind to epitopes throughout the EBNA molecule. Ru.

Antibody-binding portion of the present invention produced in rabbits against the polypeptides shown in Table 1 Exemplary receptor molecules containing the N atl, Acad, Sci, U, S, A,, 76:4350-4 354 (1979) and Bjllings et al., Proc. Natl, A. cad, Sci, +U, S, A,, 80 Near 104-7108 (19 The test was performed using the Imtube Cl sorting method of 83). Further details on materials and and method chapter (III).

All polypeptides in this study were conjugated to protein carriers. When inoculated into a rabbit host with an effective amount of the inoculum described below, the rabbit anti-polypeptide It was found that antibodies were induced.

These receptor molecules are expressed in the EBV-transformed human B lymphocyte cell line Wr-L2. , Rajj and Daudi. I recognized the quality. Some data from these tests are shown in FIG.

In a control test, protein extracts from 8928 bulbs negative for EBV infection (BJAB cells; Scripps Clinic and Ujola, California) (available from Research Foundation) reacts with anti-polypeptide antiserum. Couldn't get a sex band. These data are illustrative of the present invention. We show that receptor molecules immunoreact with EBV infection-specific proteins.

Furthermore, the immunity of rabbit anti-polypeptide antibodies to intact EBNA protein was As shown in FIG. It was also found that it can be blocked by a progenic polypeptide. These results are anti-poly The ideotype (antibody binding site) of the peptide antiserum is specific for the EBNA antigenic determinant. .

A rabbit anti-polypeptide antibody against polypeptide P62 was used in a competitive test to test polypeptide P62. The antigenic relevance of peptides P27, P62, P2O and P89 was tested. This resistance The conjugate and non-conjugate ports were tested in the EL ISA described below. Highly cross-reacted with lipeptides. Anti-polypeptide against solid phase polypeptide P62 Binding of peptide P62 is determined by first incubating the antibody with polypeptide P62. As a result, it was suppressed by 98%. In the same method, for polypeptide P62 Anti-polypeptide binding was 81% with polypeptide P60 and 81% with polypeptide P2. 7 inhibited it by 36%. Polypeptide P89 reacts with anti-polypeptide P62 He didn't suppress his sexuality at all.

Human EB ■ - To determine whether antibodies in immune serum recognize the above antigenic determinants. For this purpose, we conducted a reversal test using serum from patients with EBV-immunized rheumatoid arthritis (Serum 1011). I went there. The results shown in Figure 3 are those obtained when using anti-polypeptide P62. The results were similar. This is shared by polypeptides P27, P62 and P2O. The antigenic determinants shown in this figure mimic the natural immunogenic determinants of EBNA.

E2 diagnostic assay system and method Polypeptides of the present invention, antibodies and antibody binding sites produced against the polypeptides The assays and methods can also be used in diagnostic tests such as immunoassays. the Such diagnostic methods include, for example, enzyme immunoaffirmation, enzyme multiple Doimunoassay method (EMIT), Enzymulink doimunosolvent assay BLISA, radioimmunoassay (RIA), fluorescent immunoassay B. One- or two-step antibody methods, and detectable labeling of receptors or antigens. Or there are other ways of labeling.

Generally, MaggLo + Bnzyme Iwmunoassay + CR Published by CPress ~ Cleveland, Ohio (1981) i and Go1g * an+ M, IF Fluorescent Antibody Methods, To Academic Press, New York, NY (1980) Assay methods useful in carrying out such diagnostic methods and Specific examples of systems are discussed below.

1. EBNA assay Also contemplated by the invention is a method of assaying the presence of EBNA in a biological sample. Ru. In a typical method, a biological sample to be assayed is prepared, and a A record containing antibody binding sites generated against a random copolymer polypeptide. Mix with Scepter molecules. A mixture of receptor molecules is present in a biological sample EBNA is then maintained for a predetermined period of time sufficient to immunoreact with the EBNA. Determine the amount of EBNA molecules present or absent in the assayed biological sample Measure.

useful for detecting EBNA present in one aliquot of a biological sample. A kit-shaped diagnostic system embodying one aspect of the present invention comprises a polypeptide of the present invention. Antibodies, substantially whole antibodies or Fab and F(ab') raised against The receptor molecules of the invention, such as body parts, are contained in one puff cage. This system Also includes an indicator means indicating the presence of an immune response between the receptor and the antigen.

Typical indicating means include radioisotopes such as 125I and 1311; Lucali phosphatase, horse rub in uva oxidase, beta D-galac Enzymes such as tosodase and glucose oxidase, and fluorescein and fluorochrome dyes such as rhodamine. The instruction means is the receiver of the present invention. can be directly connected to a printer. The indicating means may also be a second antibody, an antibody combining site or a second antibody of the present invention. Staphylococcus aureus (S, aur) reacts (binds) with the receptor of eus) can bind to other distinct molecules of protein A. such separate molecules A specific example of an indicating means is "'ll1m5. aureus protein A."

The indicating means allows the immune reaction product to be detected and bound directly to the receptor of the invention. If not, package it separately from the receptor.

Mix with biological samples such as acetone-fixed peripheral blood lymphocyte (PBL) smears In this case, the receptor molecule immunoreacts with EBNA to generate an immunoreactant, and then Indicative means present indicate the production of immune reaction products.

One embodiment of the EBNA diagnostic method is an immunofluorescence assay containing an amplification reagent. be. In such assays, PBL smears are transferred to flat microscopic slides. Fix with acetone. generally produced in accordance with the invention, e.g. in rabbits. Aliquots of antibodies produced from about 10 to about 500 μg were prepared using well-known methods as described above. contact with the slide.

After washing off the unreacted antibody of the present invention, remove the non-specific binding site on the slide. If necessary, blotting is typically performed with a protein such as fresh blood edge albumin (BSA). Click. Then a second reagent (amplifier) such as complement or an anti-immunoglobulin antibody , for example, guinea pig complement can be incubated on the test slide.

After this second incubation, remove unreacted amplification agent by washing. The antibody is then removed to leave only those bound to the first antibody on the assay slide. 3rd test Place the drug (indicator), e.g., an antibody such as goat anti-guinea pig complement, on the test slide. Incubate with The third reagent is a fluorescein compound well known in the art. Socyanate (F ITC), Rhodamine B Isocyanate (RITC), Tetramethylrhodamine isocyanate (TIIITC), 4,4'-diisothi Fluorochromes such as oceanostilbene-2,2'-disulfonic acid (DIDS) Label by binding to a dye.

Wash off all unreacted third reagent after the third incubation above and All FITC-labeled and goat-labeled guinea-pig complements bound to the above complement on the ride. Let it be body. FICT III! detecting the presence of the third reagent using fluorescence spectroscopy; Thus, it can indicate EBV infection.

B lymphocytes known to be infected with EBV were examined for the presence of EBNA as described above. We also used the immunofluorescence assay method described in detail in the Materials and Methods chapter. It was tested. Rabbit antibodies raised against each of the polypeptides shown in Table 1 were able to detect EBNA in the EBV-infected cell line Wl-L2.

A preferred diagnostic system, preferably in kit form, useful for carrying out the above assay method is , in a separate package: (a) a receptor of the invention immunoreactive with EBNA; (antibody'), (bl complement such as guinea pig complement that reacts with the receptor , an anti-immunoglobulin antibody or a second amplification such as S, aureus protein A. reagent, and (C1) capable of binding directly to said amplification means or reacting with said amplification reagent; and an indicating means, which may be a separate molecular moiety, such as an antibody or antibody portion.

The instruction means indirectly induces the immune reaction between the receptor molecule and EBNA through the mediation of an amplifying agent. instruct.

The receptor molecule and separate indicator means of the diagnostic system described above and the amplification reagents described above are It can be prepared as a solution, as a liquid dispersion or as a substantial powder, e.g. in lyophilized form. Can be manufactured. If the indicating means is a separate molecule from the amplifying agent, the indicating means can be puffed separately. If the indicating means is an enzyme, which is preferably caged, the enzyme substrate is also separate from the system. May be provided in individual puff cages. A solid support such as a microscope slide as described above The body, one or more buffers, and acetone are also included in separate packages of the diagnostic assay system. can be included as a di element.

The above packages mentioned in relation to the diagnostic system are the packages normally used in the diagnostic system. It is a page. Such packaging may include glass and plastic (e.g. polyethylene, polypropylene, polystyrene and polycarbonate) bottles, bars plastic and plastic foil laminate packaging materials.

Whole intact biologically active antibodies can be assayed using immunofluorescence assays such as those described above. Contains immunologically active ideotypes that are not necessary in many diagnostic systems, but rather Using only the antigen binding and recognition receptor sites, e.g. the antibody binding site of the antibody molecule. may be used.

Examples of such antibody binding sites are papain and pepsin, respectively, in the art. Fab and F (a b') are well known in the art as z antibody moieties.

2. Anti-EBNA antibody assay ta) General assay Another diagnostic method of the present invention detects anti-EBNA antibodies in an antibody-containing liquid biological sample. An assay such as an ELISA to detect. Therefore, polypeptide P62, C EBV homologous or CMV homologous polypeptides of the invention such as 1, C2, C3 is used as an antigen, preferably by Pharmacia Fine Chemicals Co., Ltd. Product name: Chef Adenx (SEPIIADHX) from Biscataway, Georgia) Cross-linked dextran, agarose, glass pieces, PVC available as polystyrene, cross-linked acrylamide, nitrocellulose, microtiter solid matrices, such as the wells of a plate or the surface of a dipstick or spoon. solid support by being attached to a box or otherwise attached or fixed. Prepare the body.

A liquid biological sample to be assayed for the EBV or CMV homologous polypeptide. Mix with. The thus prepared immunoreaction mixture was used to detect anti-E present in the biological sample. The BNA antibody is then maintained for a sufficient period of time to immunoreact with the polypeptide. A biological sample assayed by measuring the presence of this immune response by means of an indicator. Indicates the presence of anti-EBNA antibodies in the pull.

One exemplary EL ISA using the above method will be discussed later in the Materials and Methods section. Opaque double well plastic spoon polystyrene or polyester as described above. For solids such as wells of 12- or 24-well microtiter plates made of vinyl chloride. The EBV or CMV homologous polypeptide of the present invention adsorbed (immobilized) on a shape matrix. A solid support containing the peptide is used. Non-specific binding sites on solid matrices are After binding of anti-EBNA antibodies such as bovine serum albumin (BSA) or immune It is typically blocked with a protein that does not contain the reactive sequence. unconjugated polypeptide The compound and BSA are removed from the matrix by washing.

A biological sample such as human saliva, serum, blood or plasma can be mixed with the solid support described above. Together, an immunoreaction mixture containing a solid phase and a liquid phase is prepared. This solid/liquid phase mixture The anti-EBNA in the biological sample immunoreacts with the polypeptide antigen and binds to the solid phase. for a sufficient period of time (e.g., 2-60 minutes) to generate an immunoreactant. solid phase and the liquid phase generally separate thereafter.

A second label indicating means-containing antibody that reacts with the first anti-EBNA antibody, and an antibody binding portion. Even if a solution of protein A is then mixed with the above solid phase, Another or a second solid/liquid phase immunoreaction mixture is prepared. As an example If the first antibody is from a human biological sample, the second antibody is horseradish (Western This is a goat anti-human Ig antibody labeled with peroxidase (HRPO). further Useful enzyme labels include alkaline phosphotase, beta-galactosidase and glucose oxidase. prepared from a solid phase and a second labeled antibody solution. The mixture produces a second reaction between the first antibody and the indicator means, such as an immunoreaction between the two antibodies. for a predetermined period of time (e.g., 2-30 minutes) sufficient to generate a solid-phase bound immunoreactant. hold The solid and liquid phases are then separated.

The second antibody described above may also be a mouse anti-1gG, anti-1gM or anti-IgA monoclonal antibody. Immunoglobulin such as antibody (e.g. IgGSIgM, IgE, 1.A or or IgD) is also specific and immunological for only one of the group or its binding site-containing moiety. Epidemic reaction.

Such antibodies are present in biological samples, as shown in Tables 6-10 below. It has the ability to identify immunoglobulin groups of anti-EBNA antibodies. Furthermore, the second antibody Alternatively, the antibody combining site may be of two types on immunoglobulin short chains (e.g., kappa or is specific and immunoreactive only for lambda)01. These antibodies are obtained from biological samples. has the ability to identify the isotype of immunoglobulin molecules present in a sample.

Enzyme labeling substrates such as hydrogen peroxide for peroxidase and alkaline phosphorus of 0-phenylenediamide or p-nitrophenyl phosphate for hotase A solution containing a color-forming dye such as is then mixed with a solid phase and this mixture is further maintained for a predetermined period of time. Then, a predetermined wavelength (e.g., 490 or 4 05 nanometers) after a predetermined period of time (for example, 60 minutes), Anti-EBNA antibodies are present in the biological sample compared to the optical density of the control Decide whether or not.

In another embodiment of the above assay, two solid supports are used.

Each solid support is an EBV homologous random copolymer immobilized on a solid matrix. Contains polypeptides.

A first aliquot of a patient liquid biological sample, such as blood, plasma or serum, is A solid/liquid phase mixture is prepared by mixing with a solid support. mixture present in the sample The anti-EBNA antibody immunoreacts with the polypeptide to form a solid containing anti-EBNA antibody. maintained for a predetermined period of time (e.g., about 2-30 minutes) sufficient to generate a phase-bound immunoreactant. hold A similar process was performed for the 27th recoat of the patient liquid biological sample and the 2nd solid support. Of course, each first and second aliquot is carried out on its respective solid phase support. may be mixed with the body in any order or simultaneously.

After separating the solid phase and liquid phase obtained from the above mixing and maintenance steps, the first solid support is Labeling of solid-phase-bound anti-EBNA antibodies present in the carrier and its immunoreactants in an aqueous medium Mix with anti-human IgG antibody. This mixture was added to a solid-phase bonded host containing anti-1gG antibody. ) Maintain for sufficient time to immunoreact with IgG antibodies. present with a second solid support Solid phase bound antibodies are similarly mixed and maintained with standard anti-Human IgM antibodies.

Thereafter, the relative amounts of labeled anti-1gG and anti-rgM antibodies are measured.

As shown later in Tables 6 to 10, a relatively larger amount of labeled anti-1g than anti-1gG The presence of M indicates that the patient is in an acute state of EBV-1M infection, whereas the presence of anti-Ig The presence of a relatively higher amount of labeled anti-18G than M indicates that the patient is in the convalescent stage of the disease. Approximately equal doses of anti-1gM and anti-18G will help patients transition from an acute to a recovery state. to show that

Another embodiment of the invention is a polystyrene 24-well microtitre A solid matrix such as a lip or double well “spoon 5” and a conjugated to Trix) or otherwise immobilized to form a solid matrix. A solid support containing the EBV or CMV homologous polypeptide of the present invention The system preferably includes a diagnostic system in kit form containing a peroxidase-labeled goat antibody. Separately packaged anti-human Ig with binding directing means such as human Ig antibodies Antibodies may also be included, as well as substrates for the binding indicating means such as hydrogen peroxide and o - Color-forming dye precursors such as phenylene diamines can also be added to further separate May be included in a puff cage. As mentioned above, these anti-human Ig antibodies Specific for human IgM, IgG, IgA or IgE only or all human antibodies It may be an antibody that immunoreacts with the patient. Stabilized hydrogen peroxide may also be included in the kit. Alternatively, it may be provided by the end user. Useful in assays using this system Buffer salts may also be included in dry or liquid form in one or more separate puff cages. It can be done. Human anti-EBNA antibodies and human antibodies without anti-EBNA antibodies (0! Separate puff cages containing human antibodies) were also used as positive and negative controls, respectively. can be included as An amount of each of the above components sufficient to perform at least one assay. for the presence of anti-EBNA in biological samples such as serum. Assays can be performed with this diagnostic system using the methods described above.

fb) EB V-IM Assays Above and Further Later in the Materials and Methods Section (III) details [! One example EL I SA similar to LISA Anti-EBNAI confirmed using anti-complement immunofluorescence (ACIF) as described above using Presence of anti-polypeptide immunoglobulin in the serum of 91 people with sex Ii heptalotib Screened for presence. If each serum was assayed at a 1=20 dilution, 9 All one person has polypeptides P27, P62, P2O and F16, F14 and positive for F15 (i.e., bound) at a high dilution of 1:320. 83 out of 91 immunoreactions with polypeptide P62- in EL I SA did. That is, the anti-FBNA antibody titer confirmed by ACIF and the anti-peptide activity of each serum. There appears to be an excellent correlation between

Furthermore, the above results demonstrate that the present EL method is simple and easy to use with the ACIF method. It shows excellent correlation with the ISA method.

Furthermore, the above results demonstrate that the polypeptide of the present invention can be used in diagnostic assays for anti-EBNA antibodies. This shows the usefulness of the peptide.

Table 5 below shows cases of EBV-induced infectious mononucleosis (IM; i.e. EBV-IM). Both cases show reactivity of sera obtained from two subjects before and after (SB and MV). In this case, antibodies that bind to polypeptides P27, P62, and P2O are produced prior to infection. It was not present during the period, but appeared after infection. In contrast, binding to polypeptide P89 No combined antibodies were produced by either of these two sera.

In a further study, a previously reported panel of 27 EBV non-immune donors (Catalano et al., J, Cl1n, Invest, +65:123 +11245 (1980)) was added to the polypeptide in the above EL I SA. It was screened for binding to P62 and P2O. blood used Sei's EBV immune status is determined by the presence of Epstein-Barr virus cutaneous antigen (VCA). or determined by absence. Those who do not have serum antibodies against VCA (VCA- ’) are not infected with EBV, vCA positive (VCA’) are infected with EBV and are typical. They generally have low levels of anti-EBNA antibodies. As you can understand from the following table 5 None of the sera showed significant reactivity to either polypeptide.

Table 5 ■Antibody VCA against EBNA peptide in human serum “Healthy 1” 155 9 58 154 23VCA “Healthy 23 516 819 145 16SB ago Mononucleosis 11 13 51 9 Post-SB mononucleosis 33 514 115 23 MV pre-mononucleosis 15 77 72 29MV post-mononucleosis LOT 631 162 25VC^-Healthy (n=27)' 67 ± 23’ ND” 45 ± 18 NDl, all sera were tested at a 1:20 dilution. I tried it.

2. Optical density at 405 nanometer light wavelength after 30 minutes of substrate incubation. It was measured.

3. VCA serum from a person who does not currently show clinical signs of EBV-related disease 4. Current or past disease as indicated by the absence of antibodies to VCA. Sera from 27 people with no clinical signs of EBV-related disease 5. Average optical density ±1 standard deviation 6. Not measured To test the correlation between the above ELrSA and ACIF diagnostic method during ongoing infection, 8 patients Preserved sera from university-aged students were collected sequentially after the onset of infectious mononucleosis (IM), and It was tested using the EL I SA method described above using P62. All students have AC 1% method of IF, Hencle et al., J. Infect, Dis, 130:23 1-239 (1974) showed anti-EBNA titer from 1 month.

In half of the students, anti-P62 antibodies were present as shown in patient 15 in Figure 4. The anti-EBNA titer increased in proportion to the anti-EBNA titer. In the other half, polypeptide P62 We detected antibodies to ACIF in the first month after onset of symptoms, but these antibodies It was detected later using the method. These results apply to patients 14 and 2 in Figure 5. is shown. Therefore, the anti-P62 antibody is a standard anti-complement immunofluorescent antibody. It was possible to detect it using the ELISA of the present invention before it could be detected by optical assay.

Primarily responsive to an individual's immune response at a given time during the course of infectious mononucleosis To distinguish between certain immunoglobulin groups, human 1gG or IBM-specific The second (indicator) antibody was EL　r　S as described in the Methods and Materials section (If). It was used in A. Table 6 below shows that for polypeptide P62 in ELISA The results of this study with sera from two patients from different time points during EBV infection measured by Show results.

Table 6 Anti-peptide activity detected by polypeptide P62-binding ELISA after mononucleosis infection Appearance Patient MV Patient 15 Time after infection 1gM' IgG" IgM' IgG" Pre-infection 103' 74 ND' ND1 week 245 80 152 1131 months 87 37 2 25 118 3 months 136 60 208 11812 months ND ND 249 375 21 months 145 600 185 9941, the patient's 1gM value was H) IgM The specificity was determined using a second antibody.

2. Patient RGC values were measured using a second antibody specific for human IgG.

3. Optical density was measured at a light wavelength of 405 nanometers.

4. Not measured.

By using this ELISA, the appearance of IgM antibody titers is small, but reproducible and found in all sequentially tested sera. ELTSA Af The immune response measured by Sei [gM appears before IgG during EBV infection] It's normal.

The appearance of IgM antibodies before IgG antibodies is particularly well demonstrated in patient 15 in Table 6. ing. The above results demonstrate that infection by EBV can be caused by at least one polypeptide of the present invention. This again shows that this results in the production of antibodies that react with the compound.

In a larger anti-polypeptide study, the aforementioned 19-person VCA” serum panel The polypeptides P27, P62, P2O, F12, F1a, F14, F15 and and F16. No positive VCA-serum found. won.

Sera from clinically normal individuals positive for VCA antibodies were tested at two dilutions.

A typical result is that all VCA' individuals are positive, as shown in Table 7 below. That is, they had antibodies that bound to each of the polypeptides.

Sera from numerous patients with rheumatoid arthritis (RA) were also screened using the above ELISA. did. These results are also summarized in Table 7.

Table 7 Average anti-peptide value in human serum measured by EL I SA Average antibody activity against polypeptide 1 Patient group Serum count P27 P62 P2OF12 F1a F16 healthy-2 1/20” 19 22 17 77 34 41 47 Healthy +4 1/20’ 26 521 854 394 12B? 0. 733 healthy +4 1/32 0348 90 223 70 37 37 166A″’ 1/20’ 28 597 999 564 118 113 843RA” 1/320”48 126 348 122 50 50 2311, activity was determined by serum infusion for 30 minutes. It was measured as optical density at a light wavelength of 405 nanometers after exposure.

2. VCA negative individuals 3. Dilution rate at which serum was tested 4.vcAlli individual 5. Healthy VCA positive individuals diagnosed with rheumatoid arthritis (control Differences between patients with RA and RA patients are best understood at a serum dilution of 1:320. RA patient The antibody titer was determined using the Wilcox Rank Sum method. This dilution was significantly higher for each polypeptide tested when analyzed using significance value of 99% or higher).

Schaublein's disease, systemic lupus erythema (SLE) and progressive systemic sclerosis (P Patients with SS) were also screened at both high and low serum dilutions. these The only difference between the patient group and healthy subjects is the polypeptides P27, PO2, P2O, Fl6, There were relatively high mean titers in PSS patients for Fl4 and Fl,5. this Their results suggest that EBV may be present in previous EBV-related diseases or in these autoimmune diseases. It is probably based on These data are used in ELIS as a diagnostic method. This does not impair the usefulness of A.

The relative amounts of IgM and IgG antibodies thus determine the relative stage of an individual's disease. It can be used to (1) Increase and then decrease, and IgG anti-EBNA antibody production The above-mentioned reaction phase of IgM anti-EBNA antibody production that overlaps and continues until the time of accumulation of IgG antibodies. (2) This reaction phase response continues (given the fact that Ig The normal appearance value of M changes to IgG in the recovery stage.

Thus, the data in Table 6 shows that during the early acute stage of the disease, anti-EBNA IgG antibodies This also indicates the presence of large amounts of anti-EBNA IgM antibodies. The IgG level is begins to rise during post-recovery, during which the 1 gM value decreases, and both values intersect, i.e. I The ratio of gG to 1 gM is [at some point during the acute phase and the recovery phase].

These findings are generally consistent with conventional knowledge of primary immunity and disease states. [For example, Hood et al., Immunology The Benjamin/ CuCumm1n Publishing, Menlo Park, California ( 1978) pp. 39-46; Mims and White, ViralPath genesis and Ia+munology, Blackwell 5 scientific Public-atiOnS, Boston (1984) pp. 105-106; and Immunology, 2nd edition, edited by Bach, Jo. hn Wiley & 5ons, New York (+982), 337-33 Please refer to page 9]. These findings indicate that IgG antibodies are detected early in infection, e.g., day 2. IgM antibodies increase over a period of approximately 3 months, reach a peak, and then decline. The state of anti-VCA rgM antibodies and IgG antibodies that disappear after several injuries during the acute phase. (Cecil, Textbook of Medicine.

Edited by Beeson et al., It4. B, 5unders, Pennsylvania (1 979) pp. 264-268].

These findings are also echoed in some respects by Henle et al., J. Infect, Dis.

130:231-239 (1974). These researchers Reported the appearance of low titer anti-EBNA antibodies 2 to 4 weeks after onset of symptoms in the serum of patients with 2.3. In their study, low but relatively high titers were observed from the second month after onset to 12 months after onset. in one or more sera as the time from onset increases by the end of It is said that it is appearing. The methods used by these authors are relatively insensitive and likely Only very few IgG antibodies would have been measured.

Thus, a detectable appearance of antibodies during the recovery phase could be expected. More recently, ^d niman etc., Chapter 33 in Concepts in Vir alPathogenesis I[, Notkjns and 01dstone &I, Springer-Verlag, New York (1986), 32 Page 6 states, without identifying the antibody group, that anti-EBNA antibodies are usually detected 1-2 days after infection. It is reported that they begin to appear after several months.

The results shown in Table 6 show that appropriate amounts of IgG and IgM anti-EBNA antibodies were Comparisons were made using the lipeptides as antigens for these antibodies and the stages of EBV-IM, i.e. , further demonstrating its effectiveness in making acute and recovery diagnoses. some of these results Sm1th et al., J. Infect, Dis., 154:885-889. (November 1986), the description of which is incorporated herein by reference. Ru. Some illustrative data from the article is shown in Table 8.

Table 8 Serological data from patients with acute infectious mononucleosis 1.519 1.079 0. +99 5.4222, serum from a positive donor 3. Serum from a positive donor The results in Table 8 report the rise and fall of IBM anti-polypeptide antibodies over time. , the decrease in IgM was observed in both those who were positive and negative at the beginning of the assay period. Patient's 1. This corresponds to an increase in G anti-polypeptide antibodies. EBV viral envelope All antibodies (IgM and/or IgG) against the antigen (VAC) One serum sample was tested in a total of 13 patient studies. .

Sera from one of the philophilic-negative patients and two of the 3 philophilic patients were collected shortly after disease onset. had relatively high JgG anti-EBNA polypeptide antibodies at time. But long et al., the ratio of IgG to 1 gM was initially greater than 1, but in each patient increased; each serum sample had a double rise in IgG anti-pedipeptide antibody levels showed a general decrease in gM anti-polypeptide antibody levels. That is, the tested patient serum In all cases, IgG anti-peptide antibodies versus 1 gM anti-polypeptide antibodies present. The increase ratio predicted the convalescent phase of the disease when the maximal IgG response was observed.

Blood samples should be collected from patients clinically diagnosed as having IM or clinical data entered. If this is not possible, the presence of anthropophilic antibodies in the serum has been determined1. Obtained from 9 people. These samples were prepared using polypeptide P62 as a solid-phase bound antigen. The assay was performed using the ELrSA assay of the present invention. IgG in these sera, Measure IgM and IgA values, and calculate the ratio of anti-EBNA IgG and IgM values. compared. IgA4M showed no appreciable correlation. This IgG and IgM The results of the assay are shown in Table 9 below.

Table 9 Anti-EBNA antibody level in human-positive serum 1 0, 0+8 0.609 0.0292” 0.055 1.074 0 ．． 0513 0, 133 0.412 0.3224 0, 035 0.545 0.0645 0, 087 0.491 0.17762 0.319 1. 046 0.3057 0, 016 0.388 0.0418 0, 315 1.245 0.2539 0, 1+8 1.255 0.09410 0.3 00 0.753 0.039 + 1’ 0.019 0.918 0.02 0+ 2 0.002 0.415 0.00513 0.2g2 0.928 0.300 + 4 0.004 0.572 0.00715 0.007 0.779 0.009 + 6 0.009 0.396 0.02317 0 ．． 005 0.446 0.01118 0.002 0.404 0.005 19 0.002 0.730 0.0031, 0D490=Materials and Methods Optical density at 490 nm measured as described in Chapter I [IK] 2. Serum has a relatively high IgA value, ie, OD490 of 0.300 or higher.

The data in Table 9 again show that serum from patients with active acute disease has low rgGO values. Relatively high levels of rgM EBNA antibodies bound to polypeptide antigens when compared to This indicates that it contained

These data also compare with sera from patients with VCA as shown in Table 5. High levels of anti-EBNA antibodies are shown. Presence of negligible amounts of IgG anti-polypeptide antibodies Hen1e et al., J. Infect, Dis, +130:231-239 (1974).

Approximately 10% of the sera from acute EBV-induced rM have allergenic antibodies as determined by conventional serology. Does not include the body. Since heterologous antibodies and anti-EBNA antibodies should be unrelated, Some additional sera from negative [M patients were analyzed in the assay used in Table 9]. did. The results are shown in Table 10 below. These patients are clinically or normally Acute IM was confirmed by BV serology. Each serum is positive for VCA-1 g M and EBV early antigen dissemination (EA-D) titers, these have negative EBNA titers ( Acute IM features despite the presence of heterologous antibodies in relation to is usually regarded as

Table 10 Anti-polypeptide EiNA levels in positive-negative serum from patients with acute illness OD4901-Lou Sample number" IgG IgM IgG/rgM1 0.706 1.449 0.492 0.836 1.232 0.6B3 0.182 0.713 0.2554 0.013 0.694 0.0185 0.351 0.7 36 0.4766 0.033 -0.841 0.0397 0, 029 0.758 0.0388 0.011 0.456 0.0249 0.01 4 0.538 0.02610 0.163, 0.757 0.2151 , see note 1 of Table 8. 2. Each serum sample was assayed positive for VCA-1gM and EA-D. The test result was determined to be negative for EBNA antibody by ACIF.

In all cases mentioned above, anti-EBNA polypeptide EL [Ig in SA The ratio of G to 1 gM was less than 1.0 and also less than 0.7. these days The anti-EBNA polypeptide P62 ELISA is an IgG/IgM anti-polypeptide assay. When the peptide ratio identification method was applied to the above ELISA data, the IM of positive-positive individuals It is clear that it is possible to detect

Presence of heterologous antibodies and IgM anti-polypeptide antibodies obtained by the assay of the present invention Further explanation of the irrelevance of early detection of anti-polypeptides is provided by the data in Table 1.1 below. Shown by ta. These data also show that using the assay of the present invention compared to ACIF. The data in Table 5 regarding early detection of these antibodies is supplemented.

Therefore, eight pairs of countersera were tested using conventional commercial assays for human-like antibodies and anti-polypeptide assays. Assayed by P62 ELISA. In each case, the first serological test The results were negative using the former method and positive using the latter method. At the time of the first blood draw, each individual He showed symptoms of IM. Therefore, in these patients, IgM anti-polypeptide P62 antibodies are present in serum prior to heterologous antibodies when confirmed by routine serology. Appeared. In the first two cases in the table, the number of days between blood collection days is 14 and 8 days, respectively. Met.

Table 11 Anti-polypeptide P62 antibodies are detected before heterologous antibodies in acute IM 1 0.122 0.489 0.25 -0.399 0.940 0.42 +2 0.064 0.512 0.125 -0.749 1.10 0. 68+ 3 0.0 +, 3 0.364 0.036 -0.04 0.592 0.0 68 +4 0.009 0.597 0.015 -0.03B 0.696 0.055 +5' 0.007 0.472 0.015 -0.079 0.624 0.13 +6 0.014 0.363 0.04 -0.08 6 0.937 0.09 +7 0.009 0.447 0.02 -0. 021 0.584 0.04 +8 0.023 0.466 0.05 - 0.951 0.50 1.9 + 1. See note 1 in Table 8. 2. The first serum blood draw was obtained at the onset of symptoms of acute IM. The second blood draw is from the same patient. obtained from a person shortly thereafter. Acute IM is the ultimate development of heterologous antibodies in each case. This was confirmed by the presence of

3. The presence of heterologous antibodies was measured using a commercially available kit.

The aforementioned ratio assay has a quantitative aspect that measures the presence of anti-P62 antibodies. However, the relative amounts of anti-P621gM and IgG antibodies present in human biological samples are It is mainly used to quantitatively confirm that the This quantitative assay is The process usually takes 2.5 hours after preparing the solid support.

A more quantitative ratio approach that requires only about 6 minutes after preparation of the solid support. He also developed a system. This assay uses similar methodology and chemistry to the assay described above. However, it relies on a visual comparison of the color intensity produced by the aforementioned indicating means and the substrate. Anti-peptide P621gG and IgM antibodies present in samples assayed by relative amounts and thereby whether the disease state is acute, convalescent or acute Make sure you are on the road to recovery.

In a preferred embodiment, the solid support is a white opaque polystyrene “spoon”. The solid phase “spoon” matrix contains polypeptide P62. The wells are configured to include two adjacent wells in which the cells are immobilized. Each well is The dam is separated by a dam that prevents liquid from entering from one well to the other. It works to prevent

Assays for anti-IgM and anti-1gG antibodies were performed as generally described above. can be performed separately, but preferably in parallel (analyzing each antibody type in each well). ) at substantially the same time. Additionally, optical density values or other label signals can be mechanically measured. Rather than determining the color intensity of each well, the user simply visually compares the color intensity of each well.

This assay does not require about 1/1 the time of the previously described assay, but Shows extremely high specificity and sensitivity. Additionally, physicians and patients can share assay results with each other. Suitable for use in a doctor's workplace so that a person can view the device while they are present .

Further details of this assay are described in Materials and Methods.

(c)’EBV-IM and CMVIM assay Epstein-Barr nuclear antigen is a unique sequence of 200 amino acids containing only the amino acids glycine and alanine Contains.

Peptides prepared from these sequences were prepared from blood samples from VCA-positive individuals, as described above. It is immunoreactive with serum. Furthermore, a peptide from this sequence, P62, is most immunoreactive with sera from patients with BV infection (Rhodes et al., H erpesvirus+R, Rapp, Alan R, Ltss, New York, 487-496 (1984); Rhodes et al., J. o1ogy, 134:211-216 (1985) iSs i th et al., J, I nfect, Dis, 154:885 889 (1986); Y et al., J. Cl1nLab Analysis 1:153 162 (198 7)), patients with acute EBV-rM have several 171EBV-positive cell lines [Ge tosky et al., J. C11n Lab Analysis+ 1: 153 : 162 N 987); Rhodes et al., J Exp Med, 165 IgM antibody against EBNA protein in Contains when detected by blotting method.

The results explained later show that not only EBV-IM but also cytomegalovirus (CMV) ) Sequential sera from induced mononucleosis-like disease (CMV-IM) were detected during the acute phase of the disease. Shown to have [gM antibodies specific for the NAP62 epitope. Acute C Sequential serum samples collected from eight patients with MV infection, all during the acute phase. This same response shows a sharp rise in IgM anti-P62 antibodies in patients with past EBV sensitivity. seen in patients with CMV infection as well as in one person infected with CMV before EBV. Sudden Immunoblotting of sera from both sexually transmitted EBV and acute CMV infected IM patients. The reason is that the same group of IgM antibodies are induced by both viruses and that the antibodies have not yet been detected. It was shown that it reacts with many normal cell proteins present in infected cells. That is, The presence of these IgM antibodies is diagnostic of acute infection with either EBV or CMV. It is.

(i) Sequential sera from patients with EBV-IM were detected at P62 and E during the acute stage of the disease. Ig recognizing peptide EBNAP62 with IgM antibody against BNA-1 M antibodies are detected in ELISA assays in the acute phase of EBV-IM (Rhodes et al., Herpesvirus, R, Rapp [, Alan R. Li5s, New York, 487-496 (1984); Rhodes et al., J, 1mmuno1ogy, 134:211-216 (1885) H5 mith et al., J. Infect, Dis, 154: 885-889. (1986); and Geltosky et al., J., Cl1n, Lab. analysis, l: 153-162 (1987)), these observation beams , B, sequential sera from patients with EBV-IM to EBNAP62EL This was confirmed when immunoreactivity was analyzed by ISA (Figure 6).

These data demonstrate that VCA IgM and The acute phase of the disease (1985) corresponds to an early rise in IgG and IgG antibodies (1:2). ■It shows an early rise in gM antibodies during the month of March to March. Serum from this patient is for P62, which peaked in the March-May 1985 samples after the acute phase of the disease. He had a slow rise in IgG antibodies.

The TgG/IgM ratio was determined from samples taken in May 1985, when the patient was in the late recovery phase of the disease. It exceeded 0.7. At this point, the anti-EBNA titer is included, and the VCA-1g M titer is It was slightly detectable by conventional EBV serology.

The dramatic rise in IgM-specific antibodies during the acute phase of the disease has been demonstrated by immunoblotting. (Figure 7), the same serum sample used in the above ELISA was used in a blotting test to detect antigens present in EBV-transformed B cell lines. I put it out.

As can be seen from Figure 7, the IgM antibodies present in the patient's serum are 1% in the extract. Recognize the eight major bands of a series and the smaller bands. Most of these proteins IgM antibodies to which were elevated in the first two serum samples and in the third It peaks in the sample and then declines. This was observed using peptide ELISA. This is the same temporal arrangement.

The proteins recognized by IgM antibodies include 77KD EBNA-1 and EBNA-1. Epitopes related to the glycine-alanine region of (Rhodes et al., J. Ex. p, Med, 165: 1 containing 1026 1040 (1987>) There are a series of normal cell proteins (92, 82, 80, 69, 62 and 58KD) , 120 and 29KD proteins have no sequence in common with EBNA-1, but (Rhodes et al., J. Exp. Med., l 65: 1026-10 40 (1987)), and appears to peak early. That is, synthetic peptide P6 fgM antibody against 2 and IgM antibody detected by Western blotting There is a good correlation between the majority of

The IgG antibody against EBNA-1 (77KD) in patient B. (Figure 7), the observation is consistent with the EBNAP62 ELISA data. Ta. On the left side of the plot in Figure 7, VCA positive (VCA”), EBNA Strength against 77KD EBNA bunt from -1 positive ([!BNA-1゛) person A strong IgG response is shown as a control. IgG anti-EBNA-1 antibodies are a disease 51 days after onset (fourth time point), i.e., IgG antibodies against the peptide begin to be produced. was detected first in the plot at the same time.

(ii) Sequential blood from patients who had CMV-IM and subsequently developed EBV-IM. Qing Sequential serum samples from patient S were also EBNAP62ELISA and clinical/serological data of 0 subjects analyzed by Western blotting. The data have been published and classical serology indicates that this patient had an acute CMV infection and Three months later, he was shown to have acute EBV disease.

Anti-peptide ELISA test using sequentially collected serum from patients R, S. The results are shown in Figure 8. just as patient B had during his acute EBV infection. , a clear response to P62 peptide detected by EL I SA. I had it.

IgM anti-pedipeptide signal 1 was high and high in three serum samples obtained during the disease. It was high in the initial samples of 2 diseases. Whether antibody levels decrease between the two diseases It is unknown whether IgG antibodies against polypeptide P62 will last until one year after the second disease. It was not detected until several samples were obtained.

We would also like to analyze samples from this patient by immunoblotting (Figure 9).

[g M antibodies were found during both infections. It should be noted that IgM antibodies are present in both diseases. recognition of the same antigen. The IgM antibody value measured in each plot is and the findings were at its highest at the beginning of the second infection and declined thereafter. The findings were similar to those in the Tide P62 ELISA. IgG against EBNA-1 is The normal VCA’, EBNA-1” command in the right lane should appear only at the last moment. (the next weakest band after the control).

Both patients had IgM antibodies to many of the bands seen in the Western plot. showed an early IgM anti-peptide P62 response that correlated with IgG anti-peptide P6 2 The response is much slower and the appearance of IgG antibodies that are specific for EBNA- and white matter. It was correlated with This has been reported previously (Rhodes et al., Help esvirus+ R, Rapp&! , ^lan R, Li5s, New York Ku, 487-496 (1984); Rhodes et al., J, Jma+un ology+134:211-216 (1985) H3sith et al., J , Infect.

Dis, 154:885-889 (1986); Geltsky et al. J, Cl1n, LabAnalysis, 1: 153 162; and and Rhodes et al., J. Exp. Med, 165:1026-1040 (19 87)).

The surprising finding is CMV! ! ! that the stain contains IgM antibodies with these same properties. It is. That is, the IgM antibodies that appear during CMV infection are the same as those produced during EBV infection. It reacts with a series of antigens that are apparently the same size.

(iii) Serum CMV-IM from 10 patients with acute CMV-IM Sera from 10 patients with were tested in ELISA and by immunoplot. against EBNAP 62 and immunoplot antigens in the serum of patients O,S. Whether the IgM antibodies detected are specific for this patient or whether this response is We determined whether it could be detected in serum from other patients with MV-IM. . All of these patients had positive IgG VCA titers and positive EBNA by rFA. had previous EBV infection as indicated by -1 titer.

Representative anti-peptide P62 data from two of these patients are presented in sections 10A and 10. All 10 patients received IgM anti-peptide therapy during the acute phase of CMV disease, as shown in Figure 8. showed a clear increase in deP62. IgM antibodies last for 1-2 months and then usually Returned to value.

In contrast, the IgG anti-peptide P62 antibody starts with a positive value and each patient has an EBV ’, VCA”, and EBNA-1”), which hardly changed during the course of the disease. Didn't show it. In fact, of the seven patients with sequential serum samples, four showing a slight increase in IgG anti-peptide P62 antibody levels (e.g., Figure 10B); One person showed a decrease (Figure 10A), and two showed no change.

The same serum was also analyzed by immunoblotting.

All of the patients are in the acute phase of the disease similar to that seen in Figures 7 and 9. showed a transient increase in IgM antibodies against many antigens. IgG anti-EBN Almost no change, if any, could be detected in the A-1 antibody value.

That is, all test patients with acute CMV infection received IgM anti-peptide P62 antibodies. showed an obvious transient increase and small variable changes in IgG antipeptide bond. .

The ratio of IgG to 18M explains the anti-peptide P62 data from EBV-IM patients. This is a convenient way to As mentioned above, this ratio is less than 1 during the acute stage of the disease. is small and increases to more than 1 during the recovery period, where this ratio is (Rhodes et al., J. Exp. Med, 165: 10 26-1040 (1987)), the advantage of this ratio analysis method is that the background and cutoff (Geltosky et al., J., C. 11n LabAnalysis, 1: 153 162 (19B?)).

When analyzed by the ratio method, this study shows that the For example, patients with (Figure 10A) showed a major response. Here, most of the premorbid samples A ratio of This ratio drops below 1 (to 0,661) during the acute stage and then It rose again during the recovery period. 8 of 10 patients with acute CMV-■M infection All of the individuals tested positive in the EBNAP62 ELISA by ratio analysis.

Patients S, G, and G, S show another type of response to P62. did. Both show an increase in IgM during acute illness, but the highest levels remain lower than IgG levels. It is. These sera are negative in the ratio analysis; in the case of two people, the false negative result is large. was based on the initial IgG value. Previous CMV infection was associated with EBV-IM. Although relatively rare in individuals with loops, some false negative results 71g This can occur when using the M ratio method.

(iv) Direct comparison of antigens recognized by EBV IM and CMV 1M serum A very similar pattern of antigens was detected by 1gM antigen during acute EBV and CMV infections. (See Figures 7 and 9).

To directly compare these antigens, serum from patients with either disease was Immunoblotting was performed in parallel with the same cell extracts. The cells used for the extract were 562 cell line.

Importantly, note that these cells do not contain EBV or CMV sequences. Is Rukoto. That is, any antigens detected are normal cell proteins.

Data from this study regarding binding by IgM antibodies are shown in Figure 11A. lane 1 ．． 2. 9 and 10 were tested with serum from EBV-■M patients, and the remaining lanes was tested on serum from patients with acute CMV-IM infection.

As can be seen from the drawings, 92.82-77.69.62 and 58KD Major antigenic bands have the same reactivity as serum from patients infected with either virus . Some small intensity bands were also seen in both patient groups. CMV patient There is a band around 50KD as found only in This is an exception. All other bands were found in serum of 1 or 2.3 or common in all serums. That is, any virus infection also induced IgM autoantibodies against the same group of proteins.

The same set of sera was blotted with extracts of CMV-infected fibroblasts. Ig G antibodies were detected in the test shown in Figure 11B.

In Figure 11B, all CMV patients have 50KD major antigen, 64KD I for another antigen, and for other antigens present only in 2.3 CMV patients. It can be seen that it had gG antigen. In EBV-infected patients, However, almost no response was observed.Ongoing research was seen in this trial. It shows that all of the bands are based on the viral code CMV protein.

In conclusion, acute infection with CMV or EBV induces a common group of IgM antibodies. Ru. In contrast, IgG antibodies are specific to proteins from the virus that causes the disease. and IgG antibodies have no reactivity with host components (Rhodes et al., J. Bxp, Med.

165:1026-1040 (1987)).

(v) Inhibition of antibody binding by peptide P62 Peptide P62 is induced by EBV. It has been previously shown that it suppresses antibody binding to many TgM antibodies that have been raised. (Rhodes et al., J. Exp. Med, 165: 1026 -1040 (1987)). This means that two EBV'''' sera are combined with EBV''''B It is shown again in Figure 12 blotting on extracts of cells.

In Figure 12, lane 1 contains serum from patient B, 15 days after onset of illness. lane 5 contained another acute EBV-[M serum. lane 2 and 6 are each serum further containing 400 gg/- of peptide P62. child These tests were performed at a higher hemodilution than shown in Figure 7, and the most reactive bands were so that it can be easily seen.

As can be seen from Figure 12, polypeptides are associated with several protein bands. The earliest visible band is the 82-77KD band. It was de. Ongoing research indicates that this area may involve EBNA-1 and one or more cellular proteins. This indicates that it contains white matter.

Lane 3 contains serum from patient R.S., 12 days after onset of CMV disease; 4 was the same serum + peptide P62 as mentioned above, also 82-77K 1 gM reactivity with the D band and some in the 58-62 KD range is the EBV peptide. suppressed by.

A test for inhibition of IgG antibodies seen in a blotting test was also performed. Leh Lane 7 contains serum from patient R.S., 514 days post-onset, and lane 9 contains serum from patient R.S. Lane 11 contains serum from patient R.S., 417 days after onset of symptoms; lane 11 contains serum from patient E. It was from a healthy adult with BV infection. Lanes 8.10 and 12 are 50 μg/d. Each serum contained P62.

The only protein detected was the EBNA-1 band at 77KD. This phlegm White matter connectivity was suppressed by the peptide in all cases.

These studies indicate that CMV infection is caused by infections from the EBV EBN A-181 region. It is concluded that the IgM autoantibodies that recognize peptides are produced. These Ig The M antibody has the same mobility as the epitope produced during EBV anger infection. recognizes a series of cell autoantigens that share specificity.

(d) Assay for nasopharyngeal carcinoma (NPC) As mentioned above, EBV It is related as the cause of (NPC). Nasopharyngeal cancer: 100/100 It is a pleomorphic cancer in China with a high index of about ,000 people per year.

Healthy VCA” subjects with IM using EBV-infected Wi-L2 cell extracts Western plot analysis with sera from patients with NPC and six patients with NPC , IgG immunoreaction with EBNA protein, EBNA protein with serum from IM patients. rgM and IgG responses by proteins and EBNA proteins from NPC patients IgG, IgM and IgA responses to the tissue were shown.

Recent studies have shown that serum levels of IgG and rgA antigens for various EBV antigens are have been shown to be significantly elevated in NPC patients (Henle et al., In t, J, Cancer s 17: 1-7 (1976); Desg Ranges et al., (It, J, Cancer's), 9'627-63 3 (1977) i and Pearson et al. Cancers 51160-26 8 (1983)), and more specifically, Henle et al. ) high 1gA against these antigens, in some cases corresponding to 1g0 titers against the antigens; Desgranges et al., who reported serum titers, used saliva to perform VCA and EA (early We reported the discovery of neutralizing 1gA and IgG antibodies against 1gA and IgG antibodies. p@ar son et al. of the diagnostic evaluation of IgA anti-VCA assay in combination with IgG anti-EA assay. reported efficacy. These studies showed that the IgA anti-EBNA in the samples they tested The presence of antibodies was not reported.

A large number of sera from patients known to have NPC were transferred to a solid matrix. A solid E of polypeptide P62 was immobilized to prepare a solid support as described above. Assayed using LISA. These sera were tested using standard serological assays. I also assayed it. The results are shown in Table 12 below.

Table 12 ELISA titer of serum from NPC patients Anti-EBV titer 1, anti-P62 titer 2 Sump 7 and go to VC 8 A I A-IA I M I G1 320 120 6 0 0, F+17(+) 0.642 1.6862 960 40 60 0 ．． 376 (-) 0.541 1.5703 640 120 15 0.73 7 (+) 0.247 1.143432ON 30 0.289 (-) 0 ．． 173 0.9585 1920 320 240 0.871 (+) 0. 303 2.0006 960 80 80 0.73B(+) 0.334 0.9157 640 320 10 0.675 (+) 0.216 0.9 938 960 640 320 0.667 (+) 0.47B 1.160 9 480 NT 15 0.701 (+) 0.417 1.32410 6 40 60 20 0.024 (-) 0.159 0.20711 1280 240 80 0.466 (+) 0.142 1.13912 240 4 0 80 0.65N+) 0.725 1.36013 1280 120 160 0.421 (+) 0.190 1.566NT = Not tested 1. Anti-EBV polypeptide P62 ELISA was performed using the serum described above in the Materials and Methods section. I did it as described in. Plus sign (+) indicates optics deemed positive by NPC Concentration values, the minus sign indicates the optical value considered negative for NPC.

As can be seen from the above data, hematological assays for VCA, EA and IgA. Sei correlated well with relatively increased IgA anti-peptide P62 titers. IgA The anti-peptide P62 titer is determined by the presence of IgM or IgG antibodies in the serum. Not restrained. Furthermore, the JgM to 1gG ratio is determined by whether the patient has acute IM or not. Can understand what is meant by something. Furthermore, anti-EBNA titer was measured with ACIF. At that time, it turned out that it was not related to the disease.

In a test of serum from healthy individuals without NPC, 0.0 aq of 0.3. Set the value to this It was found as a control value for IgA assay. This special assay format 0,0a significantly exceeds 0.3. Value as NPC positive indicator I took it as That is, in the table above, 10 out of 13 bloods are positive for NPC. Therefore, it is indicated by a plus sign (+).

The general assay method for detecting anti-EBNA antibodies described above also detects the presence of human NPC. It can be understood that it is effective for detection.

Using antibody-containing biological samples such as serum, plasma, saliva, sputum, and throat washes. Living The body sample is mixed with the solid support and aqueous medium as described above. Here, Ig The amount of A antibody was assayed and a relatively increased amount compared to the control indicated the presence of NPC. vinegar.

(e) Immunological cross-reactivity of EBV and CMV-related peptides in herpesviruses Human cytomegalovirus (CMV) is associated with birth defects and opportunistic infections in immunosuppressed individuals. It has been implicated as an important pathogen that can cause cancer. CMV is also caused by EBV It causes "mononucleosis-like" lesions similar to infectious mononucleosis (EBV-IM).

of patients with acute phase EBM-IM using a polypeptide P62-based ELISA. During clinical studies to detect the presence of anti-EBNA antibodies, approximately 60-70 of CMV-IM It was noted that % of allergy-negative cases were found to be positive in P62 lineage EL [SA]. .

The presence of antibodies in CMV-IM patients that can immunoreact with P62 may be due to existing CMV infection. CM that immunologically cross-reacts with EBNA or as a result of reactivation of latent EBV infection It was thought that this was either a result of the production of V protein.

P62 cross-reactive antibodies induced by CMV-encoded antigens in serum from CMV-infected individuals In order to test the possibility of containing Synthesized chido.

Table 13 Go1)-L4 Ne Amino Tori 1 1. The amino acid residue sequences of peptides C1, C2 and C3 are each 5tapr According to aus et al., J. Virol, 57: 591 602 (1986). It is expressed by the coding end region of a 2-2 kilobase CMV RNA reported by corresponds to the part of the polypeptide for which it is expected.

Serum samples obtained during IM from selected patients were treated with polypeptides P62, C1 and IgG and IgM immunoreactivity to C2 in Methods and Materials. Chapter 1 [[Similar to that described in D] Assay was performed using LIS^. Figure 14 is Patients with EBV infection leading to the onset of acute IM and then during the convalescent phase of the disease ( The results of this test using serum samples obtained from DB) are shown. These results are EB The aforementioned pattern of anti-P62rgM antibody detection and anti- -R6:1MgG (and concomitant decrease in anti-P62IgM) during the recovery phase of antibodies; This shows an increase in the number of people.

Figure 14 also shows that the CMV-related peptides CI and C2 were tested on the solid phase of ELISA. When used as antigen (target), the same sequential samples obtained from the patient DB Also shown is the presence of anti-CI and anti-C2 IgM antibodies in the acute phase of the virus.

However, IgM anti-C1 and anti-C2 antibody responses decline with the onset of the convalescent phase. However, there is no concomitant increase in anti-CI or anti-C21gG antibodies detected. Sudden Primary CMV infection resulting in acute EBV-IM followed by secondary CMV infection resulting in acute EBV-IM. Serial serum samples from a second patient (DS) with subsequent EBV infection were collected as described above. and assayed in R62, C1 and C2 ELISA. Shown in Figure 15. As shown, each ELISA tested 1 gM of each anti-inflammatory drug during the acute phase of CMV and EBV infection. Peptide antibodies were detected.

The immunological activity of CMV-related peptides induced by CI, C2 and C3 The antibodies were further tested by testing their ability to immunoreact with EBNA. C Each rabbit anti-peptide antiserum to I, C2 and C3 was tested in Methods and Materials. Express EBNA using immunoblotting similar to that described in Section 111G. The cells were immunoreacted with extracts of the cells. The results of the immunoblotting test are anti-CL. It is shown that anti-C2 and anti-C3 rabbit antibodies immunoreacted with EBNA.

Furthermore, anti-CI and anti-C2 rabbit antibodies were added to immunoblotting acid. When maintained with various peptides, the immune reaction with EBNA protein was blocked. It was done.

Therefore, the amino acid residue sequences of polypeptides CI, C2, and C3 are found in the CMV genome. These peptides correspond to the sequences encoded by the polypeptides of the invention. It should be noted that

This is because these peptides have the formula; %formula% (In the formula, R', !:R'' are individually the same or different, but Ala, From ＾rg, G11y, Leu, Pro, Ser and Thr. (R1 and R2 are not both Gβy) This is because it contains residues. Furthermore, the above results show that CI, C2 and C3 are EB It is possible to immunoreact with IgM antibodies induced by NA and to these peptides. This shows that more induced antibodies immunoreact with EBNA.

Both EBV and CMV produce non-malignant lymphoproliferative diseases, the acute phase of which It is characterized by the production of IgM antibodies that immunoreact with the polypeptides of the invention. Anti-F, B NA antibodies are not typically found to be associated with malignant lymphoproliferation. Therefore, the present invention provides a method for treating virus-induced non-malignant and malignant lymphoproliferative diseases. The present invention provides a diagnostic system and method for distinguishing.

3. Preparations for passive immunization Patients with latently infected B lymphocytes expressing EBNA on their cell surface is a preferred compound produced by a synthetic polypeptide of the invention that is immunoreactive with EBNA. or the receptor of the invention as a whole antibody. These recipes The receptor has an effective amount of receptor dispersed in a pharmaceutically acceptable diluent. Administer in unit doses.

Effective amounts of such antibodies vary depending on the reactivity and type of antibody. Typically A range of about 0.5 to about 25.0 μg/kg patient weight is considered effective. Antibodies are given intravenously , intramuscularly or intraperitoneally, with several doses given 3 to 20 days apart. antibody can also be administered in conjunction with surgery or chemotherapy.

The antibody is obtained from the polypeptide of the present invention from the serum or serum of an animal species different from that of the patient to be treated. Antibodies to the virus can be obtained by producing them using the inoculum described above. Wear. Antibodies can also be isolated from hybridoma cell lines using methods well known in the art. It can also be obtained from monoclonal sources such as ascites fluid by preparation. all Antibodies are preferred binding sites because they can activate the complement system when forming immune complexes. Yes.

■Methods and materials A. Polypeptide synthesis The polypeptides of the present invention are described by Merr IFIELD et al., J. Am, C. lew, Soc, +85:2149-2154 (1963) and Houg h ten et al., Int.

J, Pept, Prop, Res, 16: 311-320 (198 It was chemically synthesized by the solid phase method described in 0). The solid phase method of polypeptide synthesis is Commercially available from Vesokman Instruments I., Berkeley, California, USA. performed using an available Beckman Model 990B polypeptide synthesizer. Ta.

For polypeptides with fewer than 35 residues used in the inoculum, the system Adding a phosphorus residue to the amino or carboxy terminus to form a protein carrier described later. This encouraged the coupling of

The composition of all polypeptides was confirmed by amino acid analysis.

In preparing the synthetic polypeptide of the present invention by the above-mentioned solid phase method, amino acid residues are It is attached to the resin (solid phase) via an ester bond of the carboxy terminal residue. polype The peptide can be attached to the carrier by a Cys residue or can be attached to a carrier by a terminal Cys residue. When combined, the carboxy-terminal residue whose Cys residue is esterified with the resin It is convenient to use it as

The α-amino group of each added amino acid adds the amino acid to the growing polypeptide chain. It is typically protected with a tertiary-butoxycarbonyl (1-BOC) group prior to oxidation.

The t-BOC group is then removed before adding the amino acid to the growing polypeptide chain. do.

Reactive amino acid side chains were also protected during polypeptide synthesis. Typical side chain protecting groups are: 0-(p-bromoben diroxycarbonyl); threonine, serine, aspartic acid and glutamine 0-benzyl for acid; S-methoxybenzyl for cysteine; di-benzyl for histidine. Nitrophenyl; 2-chlorobenzoxycarbonyl for lysine and arginine Toshiru for use.

The protected amino acids were recrystallized from a suitable solvent and analyzed by thin layer chromatography. Get a single spot. Cambling is a 10-fold molar excess of protected amino acids and dicyclo hexylcarbodiimide on a few milliquivalents of the starting N-terminal amino acid. Therefore, this is typically done. A 2 molar excess of both reactants may also be used. in asparagine Add an equimolar amount of N-hydroxy-benzotriazole to the protected amino acid and Methyl alumant is used as a solvent. All coupling reactions are G15i n, eight na1. Glee, ^cta, 58: 248-249 (1972 ) was more than 99% complete according to the picric acid test.

After preparation of the desired polypeptide, a portion (approximately 1 g) of the resulting protected polypeptide was Treated with N1 anisole, anhydrous hydrogen fluoride about 20 wl1. the dry ice temperature The 0 reaction mixture condensed into the reaction vessel was stirred and kept at about 4°C for 1 hour! Separate one unit The polypeptide was removed from the resin. Hydrogen fluoride at a temperature of 4°C with a flow of N. After evaporation, the residue was extracted three times with anhydrous diethyl ether The filtrate was removed and the residue was dried in vacuo.

The resulting vacuum-dried material was extracted with a 5% acetic acid solution (3 times at 50°C) to obtain free polypeptides. Separate the putide from the resin. The extract-containing solution was lyophilized to form monomeric unoxidized polyester. The peptide was obtained.

The generated synthetic polypeptide was analyzed using an enzyme-linked immunosorbent assay (E can be used as a reagent in LISA) to detect anti-EBNA antigens. synthetic polype Polypeptides are also commonly prepared by combining this polypeptide with a carrier to prepare a conjugate. and then administering an effective amount of the conjugate as described below in a physiologically tolerable manner. It can be used to prepare adhesives by dispersing it in a diluent.

In addition, the synthetic multimer of the present invention combines the carboxy-terminal residue of one polypeptide with the second polypeptide. together end-to-end (head-to-tail) by an amino bond between the amino-terminal residues of the repeptide It should also be noted that they can be prepared by in-phase synthesis of multiple polypeptides of the invention linked together. I want to be understood. Such synthetic multimers are preferably single long polypeptide multimers. I-(3-dimethylalcohol) is preferably synthesized after each synthesis. (minopropyl)-3-ethyl-carboxylic acid such as Also prepared as individual polypeptides linked together using diimide reagents in water can. Total number of amino acids contained in a multimer prepared as a single polypeptide chain is preferably about 50 or less, so that up to about 8 polypeptides of the invention can be contained in a single-head, single-tail multimeric chain synthesized as a single polypeptide. synthesis The analog-tail multimer is more preferably a synthetic chain of the present invention having 2 to about 4 blocks linked together. random copolymer polypeptide, and up to about 40 amino acid residues in total. contains.

B. Preparation of polymer Polypeptides of the invention include multiple polypeptide repeat units linked together Antigenic and/or immunogenic polymers (synthetic multimers) can be prepared. Like that Polymers typically have the advantage of increased immunogenicity and antigenicity. In addition, high A carrier is typically not required when using molecular immunogens. different polypeptides When the monomers are used in the preparation of polymers, for some EBNA antigenic determinants The ability to immunoreact with antibodies can be obtained. A further advantage is that such polymers Antibodies immunoreactive with some antigenic determinants of HBNA when used in an inoculum of It is the ability to induce

The polymers of the present invention are as described above and have both amino and carboxy termini. However, by synthesizing polypeptides containing cysteine residues, we create “di-Cys-terminal” polypeptides. Prepare putide. For example, each polypeptide in Table 1 and the polypeptide in Table 2 D1 and D2 with additional Cys residues at the amino and carboxy termini, respectively. A di-Cys-terminated polypeptide can be prepared in its reduced form by synthesis containing After 0 synthesis, in a typical laboratory preparation, 10 μ of the di-Cys polypeptide ( 250 milliliters (R1) of 0.1 Moles (M) of ammonia dicarbonate dissolved in the buffer are then dissolved. The di-Cys-terminated polypeptide was added to the resulting solution by gently incubating it in air for about 18 hours. merca, which can be detected by stirring or by the Ellman test. Air oxidation until no ptane is present* (Ellman, Arch.

See Biochew, Biophys, 82: 70-77 (1959). . ] The polymer thus prepared (synthetic multimer) contains oxidized cysteine (cysteine). multiple synthetic random copolymer polypeptide repeats linked together by Contains units. Such polymers may have a head-to-tail shape as well as a head-to-head or tail shape. - Contains the 9th position of the polypeptide repeat linked together in a tail shape. That is, two poly The amino terminus of a peptide repeat unit is simple, as are the two carboxy termini. may be linked together via a single cystine residue, since both polypeptide termini This is because the bonding groups are the same.

Coupling to C1 carrier Keyhole limbe sotohemocyanin (KLH) using synthetic polypeptide as a carrier ) was described in Liu et al., Biochem, 80:690 (1979). coupled by a method. In short, 0.4 milligrams (■) of carrier. m-maleimidobenzoyl-N-hydroxysuccinimide ester of 51■ Activation and then conjugation of the polypeptide with amine or carboxy-terminal cysteine. conjugates containing from about 10 to about 35% by weight polypeptide. A gate was prepared.

Adding one or more additional amino acid residues to the amino or carboxy terminus of the synthetic polypeptide In addition to the ends, binding of the polypeptide to the carrier can be facilitated. I mentioned earlier cysteine added to the amino or carboxy terminus of a synthetic polypeptide. The residue was found to be particularly useful in preparing polymers with disulfide groups. has been done. However, it is well known in the art for conjugate preparation. Other methods of can also be used. Further exemplary coupling methods include Mitchell (Mi chael) addition reaction products, aldehydes such as glutaraldehyde (Kl 1pstein et al., J. Infect.

Dis, 147:318-326 (1983), or multiple points. As previously described for linking lipeptides together to prepare synthetic multimers. For example, using a water-soluble carbodiimide to form an amino bond to the carrier. There is the use of rubodiimide technology.

Useful carriers are well known in the art and are generally the protein itself.

Examples of such carriers are keyhole hemocyanin (KLH), edostin, tyrog. Robulin, bovine serum albumin (BSA) or human serum albumin (H3A) albumins such as, red blood cells such as sheep red blood cells (SRBC), tetanus ibis soid, cholera toxoid, and poly(D-lysine: D-glutamic acid) Polyamino acids such as

Also, as is well known in the art, synthetic polypeptides can have intermediate linking groups on their carriers. It is often advantageous to combine by

As mentioned above, glutaraldehyde is such a linking group.

However, when cysteine is used, the intermediate linking group is m-maleimidobenzoyl N-hydroxysuccinimide (MB S) is preferred.

Additionally, MBS is an ester amide as disclosed by Lui et al. (supra). It can be initially added to the carrier by an exchange reaction. Then, thiol acetic acid (CHz C03H ) by addition of a blocked mercapto group to a maleimidonic double bond. can be done. After splitting the acyl blocking group, the disulfide bond is deblocked. Formation between the mercaptan group and the mercaptan of the additional cysteine residue of the synthetic polypeptide make it happen

The choice of carrier depends more on the final use of the immunogen than on the determinant moieties of the immunogen and is Based on criteria not specifically included in the invention.

For example, if an inoculum is to be used in an animal, it may be unsuitable for that particular animal. A carrier that does not cause any reaction should be selected.

D. ELISA Anti-peptide antibody binding and inhibition tests can be performed using EnzymeLink as described below. It was carried out by Doimuno Solvent Afsay.

In short, a microtiter well (Costa, Cambridge, MA) 3590) containing the polypeptide, typically at a concentration of 10 μg/'- 100 microliters (μl) of BBS (10 millimolar (mM) boron) Sodium (p) 18.3), by adding 150mM NaCj coated with individual polypeptides as antigens. of the well and antigen-containing solution. Contact is maintained at 20° C. for a predetermined period of time, typically 15 minutes, to remove the antigen-coated solid phase. Prepared. The in-phase and liquid phases were separated and the wells were washed three times with BBS.

Non-specific binding sites were isolated with 200 microliters of 1% bovine serum albumin (BS). Prepare another solid/liquid phase mixture by mixing A) into each well. The compound was blocked by keeping it at 20°C for 30 minutes. Separate each phase and filter Excess unbound BSA was removed by washing three times with BBS.

Rabbit and human serum (biological sample aliquots) were tested for anti-polypeptide activity. Add 1:20 diluted serum in 100 μl OBBS per well to solid/liquid phase composition. It was assayed by preparing Contact between diluted serum and antigen-coated solid phase The cells were kept at 20° C. for a predetermined period of time, such as 1 hour, to allow immunoreactants to form. solid phase and The liquid phase is separated and then the in-phase, i.e., antigen-coated immunoreactant-containing wells are separated. Washed three times with BBS.

Antibodies in human serum that have immunoreacted with the adsorbed polypeptide are alkaline phosphotase complexes. Jugate goat anti-human 1g antibody (Tago, Burlington, Calif.) Detected using an indicating means including: Rabbit serum immunoreacted with adsorbed polypeptide The antibody is an alkaline phosphorus acid-gated goat anti-heron Ig antibody [Mizuri Kirkegaard & Perry Laboret, Gaithersburg, - The detection was carried out using an indicating means including the following. In either case, 1 00. c+1! Add the indicated antibody diluted 300 in BBS to each well for further fixation. / A liquid phase composition was prepared. This solid-liquid phase composition was heated for 1 hour, i.e., in the solid phase. was maintained at 20°C during the formation of a reaction product between the human antibody bound to the indicator means and the indicator means. . Each phase was separated and the solid phase was washed three times with BBS.

An alkaline phosphotase conjugated antibody bound to a polypeptide-specific antibody is Separate enzymatic hydrolysis of p-nitrophenyl phosphate to p-nitrophenol It was detected by photophotometric measurement. In short, 100 μl of p-nitrophenyl Phosphe-I (2mM MgCj!, (pH 9,8), 50mM sodium carbonate +wg/M in Limeium was added to each well. The enzymatic reaction was carried out for 1 hour, then 4 hours. The optical density at 0.05r+m was measured at the 70-Laboratory in Ingleund, California. TITERTEK vector photometer available from Measured in a turret.

Additions used in this study to measure the IgG to 1gM ratio to assess EBV disease status The assay was performed by Smlth et al., J. Infect, Dts.

+54:885-889 (1986) and Gettosky et al., J.C. 11n Lab Analysis, 1: 1.53-162 (1987) It was done as described. In short, as a solid phase matrix - this micro Fill the titer well with 20 micrograms (μg) of 50 μρ polypeptide P62. /-Ballate buffered saline CBBS; 0.5M sodium borate, 0.15M NaCe, and 0. OOl, MgClt, pH 8,33 for 12 hours at 4°C. A solid support containing peptides coated with and immobilized on a solid matrix. body was prepared.

The solution is then removed and the plate placed in 300μi phosphate buffered saline (PBS). Block with 10% normal goat serum at 37°C and evacuate each well for 60 min at 37°C. It was dried with.

At this point, 200 tt i' of 0% goat serum in PBS was added to the wells. . 10μll? JN patient sample serum was mixed into each well containing goat serum and solidified/ A liquid phase mixture was prepared. This mixture was maintained at room temperature for 1 hour to remove any antibodies that may be present. A solid phase-bound immunoreactant containing these antibodies is prepared by binding to a solid support. Ta.

0.05% Tween□N-20 (polyoxyethylene (20) Separate the solid phase and liquid phase by washing 5 times with sorbitan monolaurate]. After that, horseler dish peroxidizer for human IgG or IgM was added. Ze (HRP O) conjugated mouse monoclonal antibody Add 200 μl of a solution of Tick Co., Raritan, NJ) to each well. A second solid/liquid phase mixture was then prepared. This second solid/liquid phase mixture is further The enzyme-conjugated antibody was maintained in the first solid/liquid phase mixture for another 1 hour to form a solid phase bond. was bound to the conjugated antibody.

Separate the same phase and liquid phase, and remove the solid phase 5 times using the above Tween-2f3/PBS solution. After washing, o-'noenylene diamine (○PD; 2■ dissolved in water of 3- Citrate buffered tablets of Binotoman-More, Washington, New Chassis. The color was developed with a mixture of (Crossing) and hydrogen peroxide. The resulting solution was heated for 30 minutes. It was maintained at room temperature for a while. 501 reactions! 7! Stop with a mixture of 2NH (l), Absorption was measured at 490 nanometers (nm).

The results of EL ISA using polypeptide P62 as an antigen are the above absorption values. Standardized by multiplying the positive control ratio by LJ2 Positive Control Ratio - Reference Absorption Value/Average Absorption of Positive Control Positive Control A reference absorption value of 1.0 was chosen. Observe the reference absorption value of the positive control. divided by the average absorption value. A positive code for each absorbance value for the control and each test sample. Standardized ELISA values were obtained by multiplying by control ratio. Cutoff value (average OD +SD) has been previously disclosed (Geltosky et al., J, C1tnl , ab Analysis.

1: 153-162 (1987)). Under these standardized conditions, 0. An ELISA value ratio of rgG to 1 gM greater than 7 was determined for the healthy asymptomatic group6. Absorption values lower than 0.7 were considered to indicate acute infection by EBV (Smi th et al., J. Infect, Dis., 154:885-889 (19 86)).

Yet another similar but much faster assay was also developed and used herein. I'm there. In this case, the solid phase matrix used was a so-called "spoon". .

The particular spoon used extended approximately half the length of this spoon and having a generally flat upper portion suitable for holding with the fingers (in normal use) Opaque white polystyrene device. The lower part (lower half) of approximately 273 ■ It is narrower than the upper part and has a cylindrical shape.

The bottom part, approximately 173 subuns, is approximately the width of the upper part and has two dish-shaped indentations. It includes a second flat portion forming a well, the recess having a width equal to the width of the flat portion. The diameter of the rim is somewhat larger. The two dish-shaped depressions are the flat surfaces on which the debris forms. They are separated by a dam that runs approximately perpendicular to the plane. Direction downwards into these indentations. The top surface of the dam facing is 1M2 for 1gM and “G” for IgG. The vertical wall of the dam adjacent to each indentation has a marking such as It is formed at a point facing the These points and the corresponding markings on the top of the dam are Connect to indicate what [gM or IgG] is being assayed in each well.

The flat parts mentioned above have substantially the same width, and this width and the length of the spoon are balanced. Tests like 13X100 or 16X160 mm (saw) Easily fits into and extends from tubes, beakers or other liquid-containing devices It has become so.

Typical dimensions of such spoons are approximately 10”], 2m+w wide, approx. 4-mm thick and a total length of about 130-160 I, and about 200-250 μl of liquid. It has an indentation that can hold its body.

The preferred spoon is the flat part of the top, with the tip used at the top 1/3. The wider section is larger than the diameter of the stop tube, e.g. so that the spoon does not touch the bottom of the tube with the bottom of the spoon. It can be hung in a test tube for better retention.

Furthermore, the spoon has a protruding leg the length of which is the thickness of the spoon on the opposite side of the indentation. provides a substantially stable horizontal base that can be rested on a laboratory bench.

Place one series of indentations of such a spoon in BBS of pH 4M 8.3-8.5. Polypeptides were prepared by mixing solutions containing 00 μg/− of polypeptides. Coated with P62. Keep the solution in each indentation for about 18-24 hours Immobilize the repeptide in the matrix provided by the spoon indentation, and Therefore, an in-phase support was prepared and the solid phase and liquid phase were separated.

The non-specific binding sites on the solid support thus prepared were isolated in PBS at pH (17.4). Mix with a solution of 10% BSA or other non-human protein for 90 minutes at 37°C. Profiled by. If the spoon is not used immediately, add 20% grise Phosphorus (v/v) is included in the blocking solution to aid in preservation. after that, The solid and liquid phases were separated and the spoon was dried at a temperature of 37° C. for 1 hour.

When used, 100 μl of a liquid biological sample such as blood, plasma, serum or saliva was added to each indentation of the spoon. Each aliquot of sample from one individual was I used it in each indentation of the spoon.

The resulting solid/liquid mixture was maintained for 2 minutes to prepare each solid phase-bound immunoreactant.

Separate the same phase and liquid phase, each containing 5w1L of PBS containing 0.05% Tween-20. Washed in two 13X100+m test tubes. Tap water cleaning solution Effectively used in the process.

Thereafter, 100 μl of HRPO-conjugated anti-human IgG antibody was added to the Mix with the solid phase support in the wells marked with and also add 100 μl of HRPO conjugate. Gated anti-human anti-human Ig whole antibody mixed with the solid phase support in the well marked “M”. Two second solid/liquid phase mixtures were prepared. The resulting mixture was kept at room temperature for 2 minutes. Ta.

′　The solid phase and liquid phase were separated. The solid phase was then washed with the PBS/Tween-20 solution described above. Washed twice.

After this washing step, the aforementioned OPD or ABTS and Add 100 μl of a chromogenic solution, such as a solution containing 3% hydrogen peroxide, to each solid support. mixed with. For this step, place each spoon horizontally on the spoon leg on the laboratory bench. I placed it on. Keep the chromogenic surface/liquid mixture so prepared at room temperature for an additional 2 minutes. The color reaction was carried out using 50μI of 1% sodium dodecyl sulfate solution or the 2N solution described above. It was stopped by mixing HC1.

The above assay takes just over 6 minutes once the solid support is prepared. . The resulting assay is completed by visual inspection of the color intensity within each indentation. That is, , if the colors are strong in the IgG assay, the patient is in recovery and one color is 1 gM up. If the color is strong, the patient is in the acute phase of IM: the colors are approximately the same intensity. In some cases, acute phase IM is transitioning to recovery mode.

Results using the approximately 6 minute assay described above take approximately 2.5 hours to complete. The results were comparable to those obtained with the microtiter plate calculated ratio assay described above. (al sudden sexually transmitted EBV-IM infection, (b) experienced the virus and was serologically VCA+; from healthy laboratory subjects and patients with telIM-like disease to clinical laboratories. Thirty serum samples from each panel with samples provided were compared.

28 of 30 sera from patients clinically identified as being in the acute phase were It was determined to be acute in the assay. For 1 gM value below the specified lower limit Two sera that did not show results in the long-term assay also showed results in the short-term assay. It was determined that this was acute phase serum.

General agreement is the result determined for one serum in a short visual assay. Further examples of results were shown by sera from other groups, but the results remained was not redetermined in the quantitative assay. Additionally, there are a few examples with different results. Were present.

In subsequent clinical trials, the approximately 6 minute assay described above reduced the acute phase by approximately 94% to approximately 95%. It was found to have specificity and sensitivity for EBV-IM.

E, cell culture The ability of the receptor molecules of the invention to immunoreact with EBNA produced in cells is described above. WIL, 2, R3ji, Daudi and BJAB cell lines as It was tested using Wl-L2 cells (ATCC CRL8155W1-L2-NS , American Type Culture Collection, Hecesta, Maryland) is genetic EBV genome-positive non-producer B-lima from a human patient with anemia of sexual cells. It is lymphoblast type.

Raji cells (ATCCCCCl86, American Type Culture Collection , Bethesta, Maryland) is EBB genome positive from Burkitt's lymphoma. It is an EBNA-producing lymphoblastoid cell line. Epstein, J. Nat. Cancer In5t, 34: 231 (1965).

Daudi cells (ATCCCCCl213, American Type Culture Collection) 6BJAB cells (Ran, Bethesda, MD) are also an EBNA-producing cell line. The Scrinisok and Research Foundation of Ujola, California This is a non-EBNA-producing lymphoid cell line available in the market.

Each of the above cells was incubated in RPMI 1.6 with 2mM l-glutamine and 10% calf serum. 40 medium (Moore, +, Am, Med, ^5soe,, 199 :519-524 (1967) i and Morton, In Vitro6 : 89-100 (1970)).

CMV strain AD-169 cells were obtained from Dr. P. R. of the University of California, Sun Diego. It was provided by ichwas. The virus is Human Geneticsochumieta Human fibroblasts obtained from a host cell system (Camden, New Jersey) The cells were grown in the cell line GM-2504. Each cell was placed in DME containing 10% calf serum. The cells were grown in M medium and infected just before confluence at a multiplicity of 3. After 9 days, the cells were incubated with PBS. The extract was collected by washing twice, and the extract was combined with 10 μg/wd of phenylmethyl 101d DEM containing sulfonyl fluoride [2% SDS, 2% 2-mer captoethanol, 0.0004% bromophenol blue, 40 mmol (m M) TriS-HCZ, pH 6.8 and 15% glycerin] for sticky feeling Prepared by adding directly to 150-tissue culture flasks containing stained cells. .

Scrape the flask with a cell souffle bar, remove the solution, and incubate for 2 minutes at -20°C. saved.

F, whole cell extract EBNA-producing and non-producing (control) cells were prepared to prepare the cells of the present invention. We investigated whether the receptor molecule is useful for diagnosing EBNA expression. Cultures as described above Cells from phosphoric acid containing 0.2 mM phenylmethylsulfonyl fluoride Salt buffered saline (PBS; 150mM NaCl, 10mM sodium phosphate, p H7,4) and 10mM N aC1, 10mM Tris-HC1, pH 7,4,1,5mM MgC1, , 0.2 mM phenylmethylsulfonyl fluoride); by sonication into 3-5 volumes of RBS prepared to ~0.35 molar (M) NaCl. It was dissolved. After 30 minutes on ice, sonicate at 10,0 OOX g for 15 minutes. Cell debris was removed by centrifugation.

G, Immunoblotting procedure The cell extract obtained above was treated with anti-EBNA antibody or the receptor of the present invention for EBNA. Human serum known to contain the target molecule was used in the assay. extract was concentrated by precipitation with 2 volumes of ethanol for about 18 hours at 20°C and then sample buffer (SB; 10% glycerin, 2% 2-mercaptoethanol) 1% sodium dodecyl sulfate (SDS), 0.002% bromophenol Blue, 40m MTris-HCI! (pl+7.4)] or for 5DS-polyacrylamide gel electrophoresis (SDS-PAGE) Diluted 1:6 in sample buffer. 7.5% polyacrylamide gel Casting, Laemmli, Nature+277:68 (1-685 (197 0), supply 50 to 200 μg of total protein to each lane and perform the test. I tried it.

After electrophoresis, the protein bands from the SDS polyacrylamide gel were transferred to a nitrocell. Solid support in the form of loin sheets (Schletcherand 5chue11 (Detroit, Michigan); Towbin et al., proc.

Natl, ^cad, sci,, Il, S, A,, 76:4350- 5354 (1979). This transcription is bio -Radotrans-plot device (Bio-Rad, Suchmond, California) ) with 12.5mM Tris hydroxide, 90mM glycine and 20% After 6 transfers carried out in methanol at 70 volts for 2-3 hours, the nitrocellulose filament 2% BSAS (w/v) or PBS in PBS for 1 hour. Non-specific binding was reduced by saturation in 2% powdered milk (w/v) in BS. So This plot was then added to 0.1- EBNA positive human serum or 2- PBS or were immunoreacted with rabbit anti-polypeptide antibody in 2% milk for 1 hour at 37°C. .

The anti-peptide antibody bound to the EBNA protein allows each protein to react with the indicating means. It was detected by In this case, a 20m ItS1 marker (200,000 counts/min/ad (cpsi/d), 10-counts 7 min/■ (cp+s/ ■)] S. aureus protein 8 was contacted with the immunoreaction product for 30 minutes at 37°C. I set it. Each plot was washed with PBS and placed on Kodak XAR X-ray film overnight at -70°C. exposed it.

In another procedure, cell protein extracts were added to a 7.5% aqueous solution in a 13 cm wide slot. It was placed on a lylamide gel and subjected to electrophoresis. Electrophoresis gel contents as described above. and transferred to nitrocellulose (Billings et al., Proc. Natl. , Acad, Sci, U.S.A., 80: 7104-7108 (1 983); Rumpold et al., J. Tmmunol, 1 3 8: 5 9 3 -599 (1987) ￥ Rhodes et al., J, Exp, M ed., 165: 1026-1040 (1987)). serum powder mill PM buffer (0.05M borate, 0.15M NaCl, p) 1 8.3) dilute the above strip 1:20 or 1:50 in 3% milk powder). The protein was brought into contact with the sample at room temperature for 1 hour to allow an immunoreaction with antibodies in the serum containing the above protein. Ta. Wash each strip and collect the immunoreaction using affinity-purified rabbit anti-human IgM. (Jaclyn Laboratories, Upondale, Pennsylvania) or the same supplier. 0.6 μg/d of affinity-purified heron anti-hiatin) IGC antibody from a source (in PM). ) was measured by incubation with a solution of

After washing, each strip was subjected to IK! as previously disclosed. +1 goat anti rabbit Detection solution for IgG antibodies (Ru+mpold et al., J, Tmmunol.

128: 593-599 (1987); R)+odes et al., l1erpes Virus.

Edited by R. Rapp, A1. an R, (, iss, Co., Ltd. 1487-49 6 (1984); Rhodes et al., J. lvsunology, 1.3 4: 2 11-216 (1985): Swh, h, etc., J, In fect, Dis, 1 54: 885-889 (1986); Gelt osky et al., J, Cl1n, Lab An-a [ysts, 1:153-1 62 (1987)? and Rhodes et al., J. Exp. Med, 16 5:1026-1040 (1987)], and the hand appears to have been moved forward. Detected by autoradiography.

Synthetic peptide inhibition was achieved by diluting serum 1=50 in PM and adding 400 μl of free peptide. g/d plus TgG at 20-50 μg/wd to suppress IgM antibodies. Antibodies were suppressed. This solution was kept at 4°C overnight (ink 1.bate) and then It was used in the 7th Pro.

H9 immunity The receptor molecules of the invention can be used to induce animals to contain the aforementioned polypeptides and/or multimers. contains all antibodies produced in an animal by immunization with an inoculum containing polypep Both tide and multimers are used alone or in combination with keyhole limpet hemocyanin (KL). It can be conjugated to a carrier protein such as H) and used in an inoculum. death However, polypeptides are preferably used as conjugates, and multimers are Preferably used alone.

Conjugate the rabbit in complete Freund's adjuvant (CFA) at 1.0 μl. 1 month later in incomplete Freund's adjuvant. It was boosted with 0 ■ conjugate (IFA, ). Each immunization is done once in the hindquarters. It consisted of a subcutaneous injection.

Rabbits were bled 1-2 months after boosting.

The serum containing immunologically active antibodies is then subjected to methods well known in the art. It was prepared from blood samples. These antibodies contain one or more polypeptides of the invention and immunoreacted with EBNA antigenic determinants. Thus, these antibodies up-regulate EBNA. It can be used in systems where

Individual inocula were prepared in CFA or IFA as follows: P Dissolved in 1) H7,2 in BS (about 0.5-). Then add an equal volume of CFA or The IFA was mixed with this conjugate solution to form a conjugate with a water:oil ratio of I:1. An inoculum containing gate, water and adjuvant was prepared. Then homogenize the mixture. The inoculum was obtained by clarification. The volume of inoculum prepared by Yes, some conjugate, PBS and adjuvant were lost during emulsification. . Substantially all of the emulsion recovered is placed in a syringe and then It was introduced into rabbits as follows. The amount of inoculum inoculated into the rabbit was determined before the emulsification process. It is thought that this amount was approximately 90% of the existing amount.

The inoculum storage solution described above is exemplary of the inoculum of the present invention, as described herein. However, this solution produces receptor molecules that immunoreact with EBN/l. Can be used.

Immunofluorescence procedure Another specific method for detecting EBNA in biological samples is to use the receptors of the present invention. -Detect the products of the receptor-EBNA immunoreaction using molecules and fluorescent color-indicating means. It's about putting it out.

In this study, 2X10'W I-L2 cells grown as described above were used. A cell centrifuge (CYTO5PIN, Cesia, Runcorn, Astomor, UK) The mixture was spread onto a flat microscope slide using a fluorophore (transducer). 20℃ for 5 minutes After air-drying, cells were fixed in acetone for 2 minutes and then air-dried at 20°C for 2 minutes. I set it. Slides were stored at -20°C until use.

Immobilized Wl-L2 on EBNA with polypeptides P27, P2O, P62 and Rabbit anti-polypeptide antibody raised against P89 (receptor of the present invention) It was assayed using

VBS buffer y-(120mM barbitol pH 7, 3, 144mM NaC 1,2,5mM MgCl2 and 0.75mM CaCl! i) 1:1 in 50+d of each rabbit antibody diluted to Incubate (contact with fixed cells) for a predetermined time sufficient for immune reaction. (maintained). Negative control slides were treated with normal rabbit serum in the same manner. I understood.

In the above incubation, a portion of the anti-polypeptide antibody was attached to the immobilized Wl-L2. immunoreacted with EBNA present in l-. Remove unbound antibody by washing with VBS. was removed, leaving only the EBNA-receptor immunoreaction product on the slide.

Anti-polypeptide antibodies conjugated to EBNA were first diluted 1:IO in VBS. Lumott Complement (Tavo, Burlingham, CA) was added to this complement on the slide. Incubate for sufficient time (30 minutes) for L-nocebuter to bind to L-nocebuter. Therefore, it was detected.

The slides were then washed with VBS to remove binding to the rabbit anti-polypeptide IgG. 3° with removed complement Fluororescein-labeled goat anti-guinea pig csC-labeled anti-complement antibody: Opel Lab La!・Antigen-antibody-complement complex using Cochrane Hill, Leeds, PA) A body was detected. Add 50 rnl of indicated antiserum diluted 120 in VBS to each slide. The cells were incubated at 20° C. for 30 minutes as described in Section 16 on the board. Unbonded groove resistor Guinea pig C1 was washed off the slide with VBS. The immune reaction products are then Visual observation was made using a fluorescence microscope.

J1 circular dichroism spectroscopy Conformational properties of polypeptides were determined by immunoassay of polypeptides with human anti-EBNA antibodies. were tested to explain the secondary structure that may be necessary for the epidemic response. polypeptide It was dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/ml. The spectrum is Digital Equation Corporation I 1102 Computer (Jiki Interfacing with Tarui Cubment Co., Ltd. Automated Cariso 61 Spectrovolarimeter (Carly Instruments) , Applied Physics, Inc., Monrovia, California). It was captured using The average of 10 successful scans for each peptide is shown in FIG.

K, sample source and serology Serum samples used in the present invention were obtained from various sources. One set for these tests Serum was obtained from 13 patients with EBV-induced IM. 13 people have positive antibodies for IM. Equine cells in 10 of the patients were previously identified using the differential adsorption tube test. Ta. Human-like antibodies were detected in two patients despite testing with different methods I didn't. One patient received a single sample taken during the acute phase of his illness. The horse had non-diagnostically low levels of equine cell agludinin detected in all cases. child of patients were considered to be anthropophilic antibody negative for the purpose of this study. Serological data for 3 people A number of heterophile-negative patients showed evidence of progression of primary EBV infection.

from 2 of 10 heterophile-negative patients and 2 of 3 heterophile-negative patients. Data have been previously published [Horwitz et al., Am, J., Merl, 63 :947-957 (1977)). In this study, EBV serology tests were as follows: Commercialization of IgG and IgM antibodies against VCA on previously frozen serum samples. Commercially available tests (Litton Bionetics, Charles, South Carolina) The method was performed by direct immunofluorescence method (Ston).

Another group of sera was used for EBV-IM and CMV-1M tests. child In the group of samples, one patient had positive EBV-■M and then normal The patient showed the typical appearance of a mediocre clinical recovery. In the acute phase of the disease, he received anti-EBN Developing high titers of antibodies against VCA without antibodies against A (l・2) (IgM=I.80, VCA IgGl: 1280). 174 days after onset By B, IgM anti-VCA antibodies are no longer detectable and anti-EBNA is 76-Il1 It was arranged in time for the day. Antibodies against CMV! complement fixation in continuous serum. It was not detected (I.8). The second patient (D, S,) is probably an early acute patient. had cytomegalovirus (CMV) infection (CMV-IM) and after 4 months - next E I had a BV infection. This patient received CMV macroglobulin (1:256), complement fixation showed a 4-fold increase in antibodies against anti-CMV (CMF-CF) by There was no antibody response to BV-associated antigens. He starts 124 days after the onset of the first disease. In the second stage of the disease, secondary EBV infection is associated with IgM anti-VCA (1:320) and and EBNA were absent early on but occurred characteristically in later development. For VCA The IgM antibodies disappeared with continued testing. During his second illness (EBV-IM) ,CMV? '/l:l glob 'J7 (CMV-IgM) is no longer detectable and this In contrast, complement-fixing antibodies against CMV still showed a stable value of 1:128. It existed.

Serum was also available from 10 other patients whose blood was drawn during CMV-induced mononucleosis. he The acute phase serum of Loglobulin and anti-CMV (CF) were shown. A 4-fold increase or decrease in titer continues. Complement fixation in 6 of 10 patients (6/10) subsequently sampled indicated by the All 10 patients had moderate, constant levels of anti-VCA (IgG). ), anti-EBNA (1:10) and anti-VCA-(IgM) showed serological evidence of old EBV infection without corticosteroids.

Some data from the latter 11 patients were disclosed in an earlier report. (Horwitz et al., Medicine, 65: 124.-133).

for VCA (IgM and IgG), early antigen (EA), and EBNA EBV-related antibodies as well as CMV macroglobulin were detected using standard indirect immunofluorescence (IFA) ) method (Horwitz et al., Medic-ine, 65:124-1 33 (1986), and Henle et al., Human Pathology 5:551-565 (1974)), anti-CMV was also used in the AD-169 strain. Virus (Microbiologjcal^5 associate, Marylan It was also detected by complement fixation using A.

The foregoing describes many variations and modifications that are intended to be illustrative and not limiting of the invention. Anything may be done without departing from the true spirit and scope of the novel concept of the invention. Book There are no limitations regarding the specific polypeptides, antibodies, their compositions and uses shown in the specification. It should be understood that it is not intended to be a

Engraving (no changes to the content) FIG.1 Wavelength (nm) (No changes to the contents of the engraving) Antiserum EBNA CCB B BABCDEFG % anti-peptide activity Engraving (no changes to the content) years Days after onset of symptoms. time Engraving (no changes to the content) Engraving (no changes to the content) Engraving (no changes to the content) patient slap 9G number of days FIG. 7A Engraving (no changes to the content) Patient DB 16M 45-・ number of days FIG. 7B Engraving (no changes to the content) Engraving (no changes to the content) Patient O8 M number of days FIG.9A Engraving (no changes to the content) Patient O8 QG number of days FIG.98 Engraving (no changes to the content) Engraving (no changes to the content) Engraving (no changes to the content) mononucleosis serum EECCCCCCEE Virus serum number Pure IF (no change in content) mononucleosis serum serum number Engraving (no changes to the content) WIL2 extract 082 0827G1 088 087 G31gM mgG Peptide P62 One÷−＋−＋−＋−＋−＋ 12345678911f lG12 eN@Ot+-11’I 0% M 8-t m＞w kai o 1 - ~ ~ -1 -1 Paar 1 Engraving (no changes to the content) Sequential serum testing from 1M patient D, B blood collection mortar Engraving (no changes to the content) Recruitment of sequential serum tests D, S, from CMV/IM patients Heisei Year Month Day 3. Person who makes corrections Relationship to the case: Applicant 5. Date of amendment order: Self-issued international search report

Claims (8)

[Claims] 1. 15 having a sequence corresponding to that of the CMV-encoded polypeptide shown in Figure 1. ~ contains about 40 amino acid residues, and the above sequence has the formula: -Gey-R1-Gey-R2-Gey (in the formula, R1 and R2 are as indicated individually) Aea, Asu, Arg, Gey, Leu, Pr o, Ser and Thr, but R1 and R2 cannot both be Gey ), its pharmaceutically acceptable salts, and its antigens related variants. 2. polypeptide is a) [There is an array] and b) [There is an array] The polyester according to claim 1, which has an amino acid residue sequence comprising a sequence selected from the group consisting of Ripeptide. 3. polypeptide is a) [There is an array] b) [There is an array] and c) [There is an array] 3. The polypeptide of claim 2, having a sequence selected from the group consisting of: 4. a) mixing a biological sample with the polypeptide according to claim 1 to form an immunoreaction mixture; to make sea bream; b) Add the above mixture to the immune reaction between the anti-EBNA antibody in the biological sample and the above polypeptide. for a predetermined period of time sufficient to generate immune reactants accordingly; and C) the presence of said immunoreactant and thereby the presence of anti-EBNA antibodies in said sample. Assay for anti-EBNA antibodies in biological samples characterized by determining the presence of i method. 5. The polypeptide is a) [There is an array] b) [There is an array] and c) [There is an array] 5. The method according to claim 4, wherein the method is selected from the group consisting of: 6. Step c) comprises (i) combining the immunoreactant of step b) with an anti-human IgM enzyme-labeled antibody molecule; (ii) mixing said second immunoreaction mixture to prepare a second immunoreaction mixture; The anti-EBNA antibody that may be present reacts with the labeled antibody molecule to form a labeled immunoreactant. for a predetermined period of time sufficient to generate; and (iii) the presence of said labeled immunoreactant and thereby the presence of said labeled immunoreactant in said sample; The method according to claim 4, comprising the steps of determining the presence of anti-EBNA IgM antibodies. . 7. In separate packages, a) a polypeptide according to claim 1, and b) an indicator indicating an immunoreaction between the polypeptide and a human anti-EBNA antibody; Diagnostic composition in kit form for assaying the presence of anti-EBNA antibodies in biological samples . 8. polypeptide is a) [There is an array] b) [There is an array] and c) [There is an array] and the indicating means is an anti-human IgM enzyme-labeled antibody. Diagnostic compositions.