JPH0292298A - Monoclonal antibody against hiv-constructing protein - Google Patents

Abstract

PURPOSE:To obtain monoclonal antibody specific to HIV protein gp41 by using synthetic peptide consistent with a part of amino acid sequence derived from gp41 as membrane protein of HIV as an immunogen, without using any natural HIV having possibility of infection. CONSTITUTION:In production of the above-mentioned monoclonal antibody, synthetic peptide is administrated an animal other than human, e.g., mammal such as mouse and immunized. Then, splenic cell is taken out as lymphatic corpuscle cell from immunized said animal and subjected to cell fusion with myeloma cell having infinite reproducibility. A monoclonal antibody obtained by said method is not reacted with a virus main body different from the case of using natural virus protein as an immunogen, but reacted with a part of amino acid derived from gp41 as a membrane protein of HIV and bonded to gp41. Therefore, usable as a positive standard of western blotting or EIA, etc., and biohazard in examination is free from care because derived from hybridoma.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a monoclonal antibody used in the analysis of human immunodeficiency virus (HIV), etc., and particularly as a positive antibody with excellent safety in various AIDS test kits. This invention relates to the establishment of monoclonal antibodies that can be used as standards and can also be applied to basic research on HIV.

[Conventional technology]

Acquired immunodeficiency syndrome (AIDS) in 1981
It was first reported in the United States in 2010 (Gottl i
ab, M.S. et al., N.Eng., J.Med.
Mi, 1425 (1981)], the disease is thought to be transmitted by a retrovirus now called human immunodeficiency virus (HIV) (Cof
fin, J., et al., Science, ■
See L697 (1986)].

Conventionally, as a definitive test to detect antibodies against HIV in serum, HIV was subjected to electrophoresis to detect gp120, gp4
1. p24. Fractionate into constituent proteins such as plB, transfer these to a nitrocellulose membrane to create a strip, and react this strip with human serum to perform enzyme immunoassay (EI).
Western blotting is used to perform A). In this method, the serum of AIDS patients, for which the presence of anti-HIV antibodies is known, is inactivated and used as a positive standard (see Bio-Rad's instruction manual (1987)). When using serum-derived antibodies, it is difficult to stably obtain antibodies in large quantities.For this reason, in recent years, mice have been immunized with HIV itself, and after the immunized tile cells are removed, appropriate myeloma ( Many attempts have been made to obtain a large amount of monoclonal antibodies that exhibit a specific binding reaction to HIV by fusing them with all cells (hyperidoma) and culturing the fused cells.
Michael S., C. Fung et al., Blotech
nology, 5.940. (Like 19B?)
There have been reports of monoclonal antibodies against gp120, which is one of the constituent proteins of IIV, and antibodies against p24. By the way, U.S. Patent No. 4.520.11
Publication No. 3 includes information on AIDS and Buri-AIDS (Pro-AI).
DS) It has been pointed out that the reactivity or specificity of antibodies detected in the serum of patients is primarily directed against gp41, a protein that constitutes the envelope antigen of HIV. Therefore, James J, GJang et al., Pro
c, Natl, Acad, Sc! , LISA+JL1.
6159 (1986), investigated the amino acid region of the protein constituting gp4t that has high immunogenicity against antibodies in the serum of AIDS patients.
Peptides with the following 21 sequences constituting amino acids 84-604 have been specified. Arg-11
e-Leu-Ala-Val-Glu-Arg-Tyr
-Leu-Lys-Asp-G l n- G 1 n-
Lue-Lue-G ly-11e-Trp-G Iy
-Cys-3er (Ala-alanine, Arg=arginine.

Aap*Aspartic acid, Cys-cystine, G
1n==glutamine, Glu-glutamic acid, G
ly glycine.

11e-isoleucine, Leu+-leucine, Ly
s = lysine.

Ser, serine, Trp, tryptophan 1↑yr=
In addition, in this report, James J, G, Wang et al. used a synthetic peptide having such a sequence and directly applied it to a reaction vessel, etc., thereby reducing anti-H! ■Detects antibodies specifically and with high sensitivity.

[Problem to be solved by the invention]

'Conventional HIV detection methods are mostly EIA and waste;
Although the virus has become inactive, there is still a risk of infection for testers and others. Moreover, the absolute amount of patient serum tends to be insufficient. In addition, various mouse monoclonal antibodies produced using HIV itself as an immunogen have been reported against gp120 and p24, but antibodies with specificity for g941, which exists in the form embedded in the virus membrane, have not been obtained. Therefore, although gp4t has great importance in investigating HIV infection and its expression, it is the least analyzed and studied among the HIV constituent proteins.

In view of the above problems, the present invention aims to provide a monoclonal antibody specific to the HIV protein gp41.

[Means and effects for solving the problem] In the present invention,
Monoclonal antibodies are produced using a synthetic peptide that is part of the amino acid sequence derived from the HIM membrane protein gp41 as an immunogen, without using any natural HIV that may cause infection.

At that time, a synthetic peptide is administered to a non-human animal (for example, a mammal such as a mouse) to immunize it, and then tile cells are taken out from the animal as lymphocytes, and myeloma cells, which have unlimited proliferation potential, are fuse. Here, the means used for fusion is polyethylene glycol (PEG).
Methods using fusion accelerators such as oheler
G.

and Milstein C., Nature, 495+ (1975)'), and a fusion method using electric pulses (tl.

Zimmermann and J., Vienken;
J, Menb? ane Biol.

■, 165. (1982)).

The monoclonal antibody of the present invention does not react with the virus itself, unlike when a natural virus protein is used as an immunogen.
It reacts with some amino acids derived from the HIV membrane protein gp41 and binds to gp41.

(Example〕 The present invention will be explained based on examples.

First, a synthetic peptide consisting of the following amino acid sequence was used as an immunogen. Arg-11e-Leu-AlaVal
-Glu-Arg-Tyr-Leu-Lys-Asp-
Gin-Gin-LeuLeu-Gly-11e-Tr
p-Gly-Cys-Ser (A1a = alanine.

Arg-arginine, ^sp=aspartic acid, Cy
s = cysteine, Gln-glutamine, Glu-glutamic acid.

cry-glycine, 11e=isoleucine+Leu
=Leucine, Lys/Lysine, 5er-serine *TrpcF liptophan, Tyr-tyrosine, V
(1) Preparation of lung cells First, 100 μg of the above synthetic peptide was administered to an animal using a specific carrier as a vehicle. Specifically, polyvinylpyrrolidone (PVP) was mixed and dissolved with 100μ2, and 6-week-old female BALB/C mice (obtained from Charles River) were immunized by intraperitoneal injection. One month later, the same A booster immunization was performed by intraperitoneal injection, and one month later, the same injection was performed by dispersing it into the 9 tail veins under the back skin, the eyes, etc., and 4 days later, the lungs were removed from BALB/C mice. was prepared. First, the removed lung was D −
M E M (Dulbecco's former 11malE
Shake the lungs several times in the culture medium to wash off excess fat lumps, etc.Next, transfer the lungs to a vetri dish containing D-MEM culture medium 10-1, cut them into about 4 pieces, and use forceps to remove the lungs. The lymphocytes were pushed out. A lymphocyte suspension was prepared by filtering the D-MEM culture medium containing lymphocytes through a stainless mesh to remove tissue lumps.

(2) Preparation of fused cells and PEG solution The myeloma used for fusion was mouse myeloma cell P3x63-AJI 8.
653 (ring ATCC number CRL-1580) was used. Large-scale culture @ (150d) Ni-grown Shitami Eloma 5X10? Transfer the cells to a centrifuge tube and centrifuge at 1200r, p, m for 5 minutes to collect the cells, which are then added to serum-free D-
After suspending one width of MEM culture medium, 2x10" of lung cells
Mix the above lymphocyte suspension containing
Prepare for fusion by centrifuging at po+ for 5 minutes.

Next, polyethylene glycol (PEG) used for fusion
) The solution was prepared as follows. PEG (Merck 5972
7) Place 1 g in a glass test tube, cover it with an aluminum stopper, autoclave it, cool it to 60°C, add 11 d of D-MEM culture solution warmed to 45°C, stir, and incubate at 45°C. Stored in a warm bath.

(3) Fusion work Remove as much of the supernatant of the centrifuged fused cells as possible, and
EG1 solution lId was added dropwise with a pipette over a period of 1 minute. During this time, the centrifuge tube was shaken vigorously to remove the cell sediment.Next, the centrifuge tube was continuously shaken and stirred for 1 minute to promote fusion.D-MEM culture solution IIdl was added during the next 1 minute.
After dropping, the pipette was replaced and another 1 d was dropped.

After gradually diluting PEG in this way, D-MEM solution 8II! PEG is added dropwise over 2 minutes.
The fusion work was completed by diluting the

(4) Culture of hybridoma The cell suspension in the centrifuge tube after fusion is heated at 1200 rpm.
After centrifuging for 5 minutes at .88μg/ym.
(containing 0.176 μg/d of aminopretene) was suspended in 30 d of 96 microplates of 100 μ2 each.
Dispense 5% CG into each well of the plate! Cultured in an incubator at 37°C. On the 4th day of culture, add 10% of HAT medium.
0μ2 was added to each well, and further on the 7th day of culture and 1
Day 00: Replace 100μi of each well with fresh HAT medium. At around the 115th day of culture, when the hybridomas grown in each well formed a colony, the specificity for the synthetic peptide as an immunogen was examined by screening.

(5) 96-well flat bottom plate for screening ELISA (Nunc: 4
-68667) and inject the synthetic peptide into each well.
Aliquots of IIg were dispensed and allowed to stand at 37°C for 1 hour to solidify.
Fill each well with 0. Washed three times with OIMIJ acid buffer (PBS) and further washed with OIMID buffer containing 3% BSA. Add 100 μl of GIMPBS to each and leave to stand at 37"C for 1 hour, then add 0.01 GIMPBS containing 0.05% Tween20.
Washed three times with MPBS, pH 7.5. In this way, 100 uj was added to each well of the culture plate on which a predetermined amount of synthetic peptide was immobilized. Next, the culture supernatant of each was transferred and left to stand at room temperature for 1 hour.
Wash 4 times with Tween20-PBS solution and 5%
0.0 containing Tween20. OIM PBS.

Diluted 1000 times with P117.2 and diluted it to 100
pl was dispensed into each well. After standing at room temperature for 1 hour, it was washed 4 times with Theen20-PBS solution, and then 1.5 μl/d of orthophenyl diamine (OPD) was dissolved in 0.1 M citrate buffer, pH 5.5, and %
1 μm of hydrogen peroxide was added to each well, and 100 μl of the solution was dispensed into each well. Leave it in a dark place at room temperature for 15~
React for 30 minutes, add 50μ of 2N sulfuric acid! was added to each well to stop the color reaction, and the absorbance of each was measured at 490 m using a microplate reader (Btotec, EL-310). Such indirect ELIS
For wells in which the presence of antibodies reactive with the synthetic peptide was confirmed by method A, hybridomas were taken out in descending order of the measured value, transferred to a 24-well culture plate, and cultured in the same manner as described above.

(6) Cloning The positive hybridomas selected in this way were purified by growing single cells in a 24-well plate for 2 weeks using the conventional soft agar double layer method, and removing the grown clones. Furthermore, by appropriately repeating the same screening as described above, a hybridoma producing a monoclonal antibody specific to the antigenic peptide was established.

Table 1 shows the legal name of the established hybridoma and each immunoglobulin class determined by the Ophthalony method.

Table 1 (7) Production and Purification of Monoclonal Antibodies 10 7 cells of hybridomas established intraperitoneally were administered to mice syngeneic to those used for immunization. The administered hybridoma formed an ascites tumor in about 15 to 20 days, and a large amount of ascites containing antibodies with high antibody activity was obtained from the peritoneal cavity.

Next, the collected ascites was subjected to cation exchange chromatography to separate antibody components.

FIG. 1 shows a fraction pattern obtained by analyzing each adsorbed fraction obtained by elution during purification of 226 of the hybridomas shown in Table 1 by absorbance at a wavelength of 280 degrees. The horizontal axis shows the time since ascites was added, and the vertical axis shows the absorbance. In addition, in the figure, peaks corresponding to each adsorption fraction are numbered 1 to 6, respectively.

Figure 2 shows the results of analyzing each peak part 1 to 6 of the fraction pattern shown in Figure 1 by 5O3-PAGE polyacrylamide electrophoresis. These are the results obtained using the hybridoma stock solution. Comparative examination of FIG. 2 revealed that peak 3 contained antibodies.

(8) Properties of monoclonal antibodies FIG. 3 shows concentration response curves obtained for each dilution series of the culture supernatants of each hybridoma shown in Table 1 showing the reaction with synthetic peptides. The hybridoma is lXl
From the culture solution that grew to 0' cells/d or more, 5 cells were added to D-MEM.
A 3-fold dilution series was prepared using a diluted solution containing %FBS, and each toopx was transferred to the same ELISA plate as used for screening and reacted.

The horizontal axis of each graph in Figure 3 shows a stepwise dilution series,
The vertical axis shows the absorbance of the reaction solution in each dilution series (wavelength 49
0 nm). The symbol ・ in the figure represents the result when using an ELISA plate with a synthetic peptide immobilized.
The symbol O represents the result when using a similar ELISA plate prepared by omitting only the step of adding synthetic peptide as a control. All reaction curves show specificity for the synthetic peptide, but the three immunoglobulin IgG strains, 2E6.3Ell and 4Hll, do not react with BSA blocked on the well surface, and no nonspecific adsorption is observed. In contrast, other 1 gM strains also showed weak adsorption to BSA.

Figure 4 shows the above ELIS using synthetic peptide as a variable.
This is the result of A. That is, by creating a 2-fold dilution series from the synthetic peptide LOug/d and immobilizing it on a plate using 100 μl of each dilution series, the amount of solid phase in each well is gradually adjusted to 1, 0.5, 0.25. .0.125
．． 0.0625.0.03125゜0.0156a 1
/ well. Pour 100μ of various hybridoma supernatants into such solid-phase wells! React at wavelength 490
The absorbance was determined by nm.

The horizontal axis of the figure is the solid phase amount of the synthetic peptide, and the vertical axis is the absorbance. From the results shown in the figure, all of the seven bilidoma supernatants measured quantitatively reacted with the solid-phase synthetic peptide.
In particular, the 1.0 strains of 3E11.2B6 and 4H11 showed highly sensitive quantitative performance.

FIG. 5 shows the results of a known Western blotting method for examining the specificity of HIV to various proteins. r Imwunobl for Western blotting
The band pattern was examined by using ot As5ay J (Bio-rad, catalog number 197-1001) as a kit and making it correspond to each sample. The kit consists of a strip in which images of each HIV component protein electrophoresed into fractions in order of molecular weight are transferred onto nitrocellulose. In addition, goat anti-mouse IgG&M was applied to the secondary antibody against the mouse-derived antibody of the present invention, and other reagents and usage methods were according to the contents of the kit. Hypuridoma culture supernatant and ascites of IgG strain were used, and ascites from 3F5 was used as an example from 1gM strain. In addition, the three samples on the right are two samples, one with lower reactivity and one with higher reactivity than AIDS patient serum as a positive standard, and a fresh culture medium (90% D-MEM and 10% FEM) as a negative standard.
BS mixed solution), the number on the vertical axis is the molecular weight.

As can be seen from Figure 5, 2E6 binds to the fraction with a molecular weight of 41-43,000 (gp41) in both the culture supernatant and ascites, 3E11 binds only weakly to ascites, and no binding reaction was detected for 4H11. There wasn't. On the other hand, 3F5 of 1gM strain
Nonspecific adsorption was observed over the entire length, and there was no specificity for gp41. In this way, even in the same IgG strain, 22
It can be seen that only antibody No. 6 strongly binds to gp41.

Figure 6 shows the recognition site in the synthetic peptide that the 2E6 antibody reacts with and the t! I
I! These are the results of investigating the reaction inhibiting properties of the recognition site. A 3-fold dilution series was made using PBS, PH7,2 containing 20% goat serum for patient serum and normal human serum.Next, as in the ELISA plate described above, Lag The dilution series of patient and normal human serum was dispensed at 100 pe/well into each of two rows of a microplate immobilized with each synthetic peptide. After 1 hour of reaction, the liquid was discarded and 0. OI
After washing three times with MPBS, 2E6 hybridoma culture supernatant was evenly added at 100 d/well and allowed to react for 1 hour. Here, each plate has two rows of diluted patient serum or normal human serum treated in the same way.Next, the same washing as above is performed three times, and then patient and normal human serum are washed three times. For the two rows of identically treated dilution series of human serum wells, one row contains goat anti-mouse IgG that reacts with 2B6 antibody, and the other row contains goat anti-mouse IgG that reacts with anti-HIV antibody in patient serum. Goat anti-human (goat anti-human) IgG was added as a peroxidase-labeled secondary antibody, and the mixture was allowed to react at room temperature for 1 hour. After the reaction, each well was washed in the same manner as in the above-mentioned ELISA, and a substrate reaction was performed to show the absorbance at 490 n-. 6th
The horizontal axis of the graph in the figure is the dilution series of patient or normal human serum.
The vertical axis represents absorbance.

The symbol ・ in the figure is the result of reacting the above ELISA plate with patient serum and then reacting with 2E6, and the symbol O
This is the result of reacting the above ELISA plate with normal human serum and then reacting with 286. In addition, the solid line in the figure shows the results from wells to which goat anti-mouse IgG was added as a secondary antibody, and the dotted line shows the results from wells to which goat anti-mouse IgG was added as a secondary antibody.
The results are shown for wells in which . From FIG. 6, it was found that 2E6 was bound to the synthetic peptide both when the reactive site of the synthetic peptide was blocked with anti-HIV antibody in patient serum and when the reactive site was not blocked by normal serum. This indicates that the anti-HIV antibody and the 2E6 antibody in the patient's serum bind to the synthetic peptide through different reaction sites.

In other words, the monoclonal antibody of this example does not interfere with the reaction between the anti-HIV antibody and the synthetic peptide.

Table 2 shows the results of confirming the reactivity of the monoclonal antibody group of this example with inactivated HIV. For 0 reactions, a commercially available ELISA kit for human serum screening (Pasteur, product number 7D224) was used. Using. This kit is a 96-well microplate consisting of two types of wells: wells in which the AIDS virus is directly immobilized and wells in which the AIDS virus is not immobilized. First, each monoclonal antibody group of this example, AIDS patient serum with different reactivity, and D-MEM culture solution (containing 10% serum) as a control were added to the two types of wells and reacted. As is clear from Figure 0, the determination was made using the same mouse reagents as in the ELISA before Figure 7, without using the reagents from this kit for human serum as reagents after the secondary antibody.
All of the patient sera showed virus specificity by reacting with virus-positive wells and not with virus-negative wells, whereas the mouse monoclonal antibody of this example was particularly sensitive to 2E6゜3.
The IgG strains of Ell and 4 Hll each showed negative results in the two types of wells. This is these IgG
This means that the strain's monoclonal antibodies do not bind to HrV itself. On the other hand, other IgM strains reacted in the two types of wells, and no specificity for HIM was observed.

Due to the specificity shown in Table 2 and above, the monoclonal antibody of this example can be used for Western blotting, EIA, etc. as a positive standard antibody that specifically recognizes gp41, an HIV constituent protein. Furthermore, since this monoclonal antibody is derived from mouse hybridomas, there is no need to worry about biohazards caused by the use of patient serum, which is currently a concern in laboratories. can eliminate the shortage of

FIG. 7 is a schematic diagram of an immune reaction using the monoclonal antibody of this example. As shown in the figure, the monoclonal antibody of this example binds the synthetic peptide of the immunogen at a reaction site different from that for the anti-HIV antibody to form a complex, so the monoclonal antibody in this complex is placed in a reaction vessel etc. If immobilized, anti-HIV in synthetic peptides
The sites that react with antibodies are not hidden; in other words, when a synthetic peptide is immobilized directly, the entire peptide, including the sites that react with anti-HIV antibodies, are not hidden (compared to the fact that they are attached heterogeneously). The peptide is uniformly immobilized.Therefore, the monoclonal antibody of this example can be used in known agglutination tests and E.
By applying it to IA, efficient and stable anti-HIV
Antibody detection can be performed.

Note that the present invention is not limited to the above-mentioned embodiments, and can be modified as appropriate. For example, the immunogen may include a synthetic peptide having the above-mentioned 21 amino acid sequence and a peptide having another amino acid sequence. You may also use In addition, in this example, a specific carrier such as PVP was used as a vehicle when administering the immunogen, but immunization may also be carried out with an appropriate adjuvant (for example, complete Freund's adjuvant), or mice may be used for fusion. Myeloma cells P3 x 63
- Although Ag8.653 was used, various other known myeloma cells may be used.

In addition to the soft agar double layer method, the known limiting dilution method and the like can be used for cloning.

[Effects of the present invention] Since the monoclonal antibody of the present invention reacts with gp41, which is an H[vl protein, it can be used as a positive standard for Western blotting or EIA, and since it is derived from a hybridoma, it can be used as a positive standard for biological tests during testing. There is no need to worry about hazards.

Furthermore, since it does not react with HIV itself, it can be effectively used to detect and analyze the destruction status of the virus, the exposure status of GP41, etc.

[Brief explanation of drawings]

Figure 1 is a graph showing the fraction pattern obtained by fractionating the hybridoma named E6 among the hybridomas shown in Table 1 by DEAE column chromatography. Figure 2 is a graph showing the fraction pattern obtained in Figure 1. A photograph of a 5DS-PAGE polyacrylamide gel electrocardiogram of each peak part of the pattern, Figure 3 is a graph showing the results of ELISA reaction with synthetic peptides using various dilution series of culture supernatants of various hybridomas, Figure 4 is Graph showing the results of ELISA reaction of the culture supernatant of each hybridoma against each dilution series of synthetic peptides, Figure 5 is a photograph showing the results of Western blotting with culture supernatant of various hybridomas, ascites fluid, and AIDS patient serum, Figure 6 The figure is a graph showing the reaction inhibitory effect of the antibody produced by hybridoma 2E6 on the reaction between the synthetic peptide and AIDS patient serum. Figure 7 shows the anti-HIV antibody immobilized with the synthetic peptide via the monoclonal antibody of this example. Anti-HI in detection method
FIG. 2 is a schematic diagram showing the binding state of V antibody.

Claims (1)

[Claims] 1. A synthetic peptide that matches part of the amino acid sequence derived from the membrane protein gp41 of the human immunodeficiency virus HIV is immunized into a non-human animal, and produced from a fused cell of the animal's splenocytes and myeloma cells after immunization. A monoclonal antibody against an HIV constituent protein, which is characterized by not reacting with the HIV body but reacting with the membrane protein gp41.