JPS6037428B2 - Antibodies and their production methods - Google Patents

Description

[Detailed description of the invention]

The present invention provides a method for producing an antibody from an antigen consisting of a novel peptidose protein complex, and the antibody has strong cross-reactivity with both neighboring glucagon and similar glucagon-like substances (glucagon in the gastrointestinal tract, GLI). In addition, the cross-reactivity rate of the resulting antibodies is constant, and therefore accurate quantification of total glucagon in the blood and, therefore, the above-mentioned GLI
The present invention relates to a new glucagon antibody that allows accurate quantification of glucagon antibodies, and a method for obtaining the glucagon antibody with good reproducibility. Traditionally, glucagon and GLI, which are types of physiological total victory hormones, are particularly involved in bran absorption metabolism, and therefore, by quantifying the glucagon concentration in the blood, it is possible to diagnose various pathological conditions such as diabetes. It is well known that this is possible. However, research on the above-mentioned hawk glucagon has been actively conducted in the past, and various antibodies have been developed that enable accurate quantification, that is, antibodies that specifically react with human glucagon. They succeeded in producing the specific antibody with good reproducibility using an antigen using the 18-29 or 19-29 peptide chain of Fuji glucagon as a hapten (Japanese Patent Application Laid-Open No. 53-9932). However, G.L.I.
Almost no research has been conducted on O.
Antibodies that specifically react with LI are still completely unknown.
The present inventors have been conducting research on various glucagon antibodies (AOS) in the process of elucidating the role of glucagon in sugar metabolism and its relationship. In other words, the non-specific antibody does not react to the same degree with the above-mentioned glucagon and GLI, and the degree of cross-reactivity varies greatly (the dilution slopes for both are not parallel), so the The expressed GLI can be far different from the actual GLI amount depending on the dilution factor, and the total glucagon value measured when the same blood acid is measured with different antibodies varies greatly depending on the antibody and cannot be compared between measurements at all. It was something. That is, even if the neighboring glucagon value measured by the neighboring glucagon-specific antibody is subtracted, it is impossible to accurately quantify the total CLI, and the dilution slope of each non-specific antibody with respect to GLI is unique to that antibody, and other Non-specific antibodies could not be used at all. In view of the above-mentioned current situation, the present inventors further conducted various studies, and as a result, it happened that 1-26 peptide homoserine of deglucagon and (
or) Armpit glucagon 1-26 peptide homoserine
Antibodies obtained using specific antigens using lactone peptides as haptens have complete cross-reactivity with both knee glucagon and GLI, and therefore total glucagon measured using the antibodies The values are Satoshi Glucagon and GLI
When subtracting the total glucagon value measured with the porcine glucagon-specific antibody from this value, an accurate GLI value can be quantified, and the above-mentioned complete cross-reactivity is always reproducible from the above-mentioned antigen. They discovered that antibodies with specific characteristics could be obtained and published them. The present invention was completed based on this new knowledge. That is, the present invention utilizes Satoshi glucagon and gastrointestinal glucagon (GL
is-Ser-Gin-GIy-Th, the corresponding antigenic determinant having the planar structural formula is-Ser-Gin-GIy-Th.
r-Phe-Thr-Ser-Asp-TM-Ser-
Lys-Tyr-Leu-Asp-Ser-Arg-A
rg-AIa-Gin-Asp-Phe-Val-Gi
Porcine glucagon 1-26 peptide homoserine represented by n-Trp- and u-homoserine [la] and the planar structural formula is-Ser-Gin-GIy-Thr-
Phe-Thr-Ser-Asp-TM-Ser-Ly
s-Tyr-Leu-Asp-Ser-Arg-Arg
-An antibody characterized by being in any of knee glucagon 1 to 26 peptide homoserine lactone represented by AIa-Gin-Asp-Phe-Val-Gin-Trp-Leu-homoserine lactone [lb], and The manufacturing method thereof, that is, the above planar structural formula [la] and [lb
] At least one of the peptides represented by is a hapten, which has the general formula 〇HC1(CH2)n1CH○
[B] In the presence of a dialdehyde represented by the formula n is an integer of 1 to 5, a vetide protein complex obtained by reacting with a protein to be used as a complex is administered to a clear mammal to generate The present invention relates to the above-mentioned method for producing an antibody, which comprises collecting an antibody that produces an antibody. The representation of the above-mentioned peptides used as haptens herein is in accordance with the method of representing amino acid residues by abbreviations in the amino acid nomenclature adopted by IUPAC. In the method of the present invention, it is essential to use a peptide represented by the above formula [la] and/or [lb] as the hapten. The peptides are those in which homoserine or homoserine lactone is bonded to the 1-26 peptide chain of human flank glucagon, and mixtures thereof.For example, by applying a normal peptide chemical reaction to human flank glucagon, Easy to prepare. More specifically, a formic acid solution of brom cyanide may be added to a formic acid solution of total glucagon to cause a reaction, and after the reaction, gel filtration or the like may be performed. Also, the above general formula

The dialdehyde represented by [0] acts as an intermediary to bind the above-mentioned hapten to the protein used as a carrier, and specifically includes malonaldehyde, succinaldehyde, glutaraldehyde, adipaldehyde, etc. can be used. Further, as the protein used as a carrier, any conventional protein conventionally used in the production of this type of antigen can be used. Representative examples include horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, horse serum globulin, bovine serum globulin, rabbit serum globulin, and human serum globulin. The antigen used for producing the antibody of the present invention is produced by reacting the above-mentioned hapten and protein in the presence of a dialdehyde. The above reaction is carried out in an aqueous solution or an ordinary buffer having a pH of 7 to 10, preferably a HZ 8 to 9 buffer, at a temperature of 0 to 400C, preferably around room temperature, and the reaction is completed in about 1 to 24 hours. Typical buffer solutions used in the above process include the following. 0.2N sodium hydroxide-0. is boric acid - 0. is potassium chloride buffer, 0
．． or sodium carbonate-0. Gborate-0.2 potassium chloride buffer, 0.05M sodium tetraborate-0. Gboric acid - 0.05M sodium chloride buffer, 0.1M potassium dihydrogen phosphate - 0.05M sodium tetraborate buffer In the above, the proportions of the hapten, dialdehyde and carrier can be determined as appropriate, but usually the carrier It is preferable that the hapten be used in an amount of 5 to 2 M sonic moles, preferably 10 to 13 sonic moles, and the dialdehyde in an amount of 3 to 2 M sonic moles, preferably 5 to 1 "sonic moles. An antigen consisting of a peptide-protein complex in which a carrier and a hapten are bound is obtained.The antigen obtained after the reaction is easily isolated and purified by conventional methods such as dialysis, gel filtration, and fractional precipitation. In addition, the antigen can be preserved by a conventional freeze-drying method.In preparing the antibody of the present invention using the antigen obtained above, the antigen is administered to a mammal according to a conventional method, and the antigen can be stored in vivo. A method for collecting antibodies can be adopted. There are no particular restrictions on the mammalian animals used for antibody production, but it is usually preferable to use rabbits or guinea pigs. When producing antibodies, the antigen obtained by the above method is used. Dilute the prescribed amount of the solution with physiological saline to an appropriate concentration and add Freund's auxiliary solution (
CompleteFreund'sAdiuuant)
A suspension may be prepared by mixing the suspension with the following, and the suspension may be administered to mammalian animals. For example, the above suspension is injected subcutaneously into a rabbit (9 times of 0.5 to 2 times as the amount of antigen), and then 2 to 10 times each time every 2 weeks.
Immunization may be accomplished by administering the drug for several months, preferably for 4 to 6 months. Antibodies are collected by collecting blood from the immunized animal at a time when large amounts of antibodies are produced after the final administration of the suspension, usually 1 to 2 weeks after the final administration, and centrifuging the blood, followed by separating and collecting the serum. It is done by doing. In particular, the method of the present invention has the advantage that glucagon antibodies with a sufficiently high titer and high sensitivity can always be obtained stably and reproducibly based on the specificity of the antigen used. As mentioned above, the antibodies thus obtained enable the quantitative determination of glucagon using methods such as RIA, which are in demand in the field, and are useful for the diagnosis of various pathological conditions involving total glucagon and gastrointestinal glucagon, including diabetes. Useful. Hereinafter, reference examples and examples will be given to explain the present invention in more detail, but the present invention is not limited thereto. Reference example 1 Human glucagon (manufactured by Sigma, beef/pork glucagon) 60 parts of 70% formic acid is dissolved in 5 parts of 70% formic acid, 1M of 70% formic acid solution of 1.4 parts of N is added thereto, and the mixture is heated at room temperature. After the reaction was completed, Sephadex G was applied to Sephadex G.
-25 (developing solvent: acetic acid) to obtain a mixture of 1 to 26 peptide chains of knee glucagon - homoserine and 1 to 26 peptide chains of deglucagon - homoserine/lactone mixture 40/9. . The resulting peptide mixture is then lyophilized. Reference Example 2 30 females of the peptide mixture obtained in Reference Example 1 above and 40 females of bovine serum albumin (already A) were inoculated. 1 IM borate buffer (HZ=8.5). Add gglutaraldehyde solution 5' dropwise. The reaction mixture was allowed to react at room temperature for 6 hours. The resulting reaction mixture was dialyzed at 4° C. for 2 hours (dialysate physiological saline), and the dialyzed fluid was lyophilized to obtain 71mc of white powder antigen (peptide-plaque A complex). Example 1 After dissolving the antigen (complex) 7 imitations obtained up to Reference Example 2 in five randomly selected rabbits (1 to V) in 1.8' physiological saline, Freund's auxiliary solution 2 was added. The suspension prepared by adding .7 secretions was added to the

[Administer subcutaneously, and after 2 weeks, administer the same amount subcutaneously. Thereafter, a separately prepared suspension (3 doses of antigen, 3 doses of physiological saline [and 3 doses of Freund's auxiliary solution)] was administered in the same manner at 2-week intervals for 3.5 months to immunize the test animals. do. Ten days after the final administration, blood is collected from the test animal and centrifuged to collect antiserum to obtain each of the antibodies 1 to V of the present invention.
<Measurement of titer> The titer of the obtained antibodies 1 to V is determined as follows. That is, the above antibodies were dissolved in physiological saline at 10, 1, 1 p, respectively.
diluted to 1:1 and 1:1 to 100 muku each, 12
51-glucagon 100 ml and 0.05 M phosphate buffer (pH 7.4) (0.25% BSA, 0.1% NaN)
3 and 0.01 MEDTA) Added 300 mountains and 4
00 for 48 to 72 hours, and the resulting conjugate of antiserum and 1251-glucagon was separated from unreacted 1251-glucagon by the dextran-activated charcoal method and centrifugation (4°C, 15 minutes, 300 Cape m). Then, the radiation is counted and the binding rate (%) of the antiserum to E251-glucagon at each dilution concentration is determined. The final dilution ratio of the antiserum at which the binding rate (%) is 50%, ie, the antibody titer, is shown in Table 1 below. Table 1 <Measurement of sensitivity and cross-reactivity> In this test, the ratio of labeled glucagon to unlabeled glucagon bound to a fixed amount of glucagon antibody corresponds to the ratio of the respective glucagon concentrations in the solution, and the concentration of labeled glucagon is Unlabeled glucagon (glucagon to be measured) when held constant
This is based on the principle that as the concentration of glucagon increases, the amount of bound labeled glucagon [B' that binds to the glucagon antibody decreases, and the amount of free labeled glucagon B' that exists free in the solution increases. It is something that Satoshi glucagon (standard glucagon, concentration l) was used as a test sample.
op yu/gu ~ 10000 uryu/desk) and qutGL1
(Concentration 0.203-40 skin data lyophilized product/Kenny's method [Kenny, A.J,, J, CIin.
955)] is used. Also, as labeled glucagon, side 1-glucagon (1000 p
m) is used. The above-mentioned glucagon sample or qutGL
200 fg of the I sample, 1251 glucagon, 200 f of each of the antibodies 1 to V of appropriate titers obtained in Example 2, 200 glucagon and trasylol (manufactured by Bayer, 100 glucagon)
0KIU) was mixed and incubated at 4°C for 48 to 7 hours. The radiation of bound labeled glucagon leg and free labeled glucagon 'F) was counted by the dextran charcoal method, and the radiation of each antibody used was counted. Assuming that the binding rate (Bo) corresponding to the titer is 100%, determine the percentage of bound labeled glucagon [B-] at the concentration of each test sample. The dilution slope in the total glucagon sample and the dilution slope in the sample rule toLl as the equivalent of the neighboring glucagon are shown in FIGS. 1 to 5. Each figure shows each of the above-mentioned slopes obtained using antibodies 1 to V, respectively. In each figure, the vertical axis represents binding % (B/BOXloo), and the axis represents armpit glucagon concentration (p/box). and gutGLI concentration (muta/
secretion). Furthermore, in each figure, curve A indicates total Glucagoso, and curve opening indicates LiutGLI, respectively. Also, the above figure 1~
From FIG. 5, the sensitivity of the antibodies 1 to V of the present invention when the cross-reactivity and binding % between neighboring glucagon and toLl is 50% is calculated as the total glucagon concentration n, as shown in Table 2 below. Become. Table 2 As is clear from Figures 1 to 5 and Table 2 above, according to the present invention, four out of five randomly selected demons showed complete cross-reactivity to armpit glucagon and ■tGLI. It can be seen that antibodies showing specific characteristics can be obtained, and the reproducibility is extremely high. In addition, these antibodies have good sensitivity and, as mentioned above, have complete cross-reactivity, so their use allows for the use of bait tG.
It is clear that it allows accurate quantification of LI.

[Brief explanation of the drawing]

FIGS. 1 to 5 are graphs showing the cross-reactivity of antibodies obtained by the method of the present invention with glucagon and gutGLI. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Claims (1)

[Scope of Claims] 1. An antibody derived from a mammal having substantially complete alternating reactivity with both pancreatic glucagon and gastrointestinal glucagon, the corresponding antigenic determinant of which has the planar structural formula H-His-S.
er-Gln-Gly-Thr-Phe-Thr-Se
r-Asp-Tyr-Ser-Lys-Tyr-Leu
-Asp-Ser-Arg-Arg-Ala-Gln-
Asp-Phe-Val-Gln-Trp-Leu-pancreatic glucagon 1-26 peptide expressed by homoserine-
Homoserine and planar structural formula H-His-Ser-Gln
-Gly-Thr-Phe-Thr-Ser-Asp-
Tyr-Ser-Lys-Tyr-Ler-Asp-S
er-Arg-Arg-Ala-Gln-Asp-Ph
e-Val-Gln-Trp-Leu-pancreatic glucagon 1-26 peptide expressed by homoserine lactone-homoserine lactone. 2 Mammals have the planar structural formula H-His-Ser-Gln-Gly-Thr-Phe
-Thr-Ser-Asp-Tyr-Ser-Lys-
Thr-Ler-Asp-Ser-Arg-Arg-A
la-Gln-Asp-Phe-Val-Gln-Tr
Pancreatic glucagon 1 expressed as p-Leu-homoserine
26 peptide-homoserine and planar structural formula H-His-
Ser-Gln-Gly-Thr-Phe-Thr-S
er-Asp-Tyr-Ser-Lys-Tyr-Le
u-Asp-Ser-Arg-Arg-Ala-Gln
-Asp-Phe-Val-Gln-Trp-Leu-
Pancreatic glucagon 1-2 expressed as homoserine lactone
6 Peptide - At least one type of peptide selected from homoserine lactone is used as a hapten, and this has the general formula O
An antigen consisting of a peptide-protein complex obtained by reacting with a protein as a carrier in the presence of a dialdehyde represented by HC-(CH_2)n-CHO [in the formula, n represents an integer of 1 to 5]. The antibody according to claim 1, which has been immunized with the antibody. 3 Planar structural formula H-His-Ser-Gln-Gly-Thr-Phe
-Thr-Ser-Asp-Tyr-Ser-Lys-
Tyr-Leu-Asp-Ser-Arg-Arg-A
la-Gln-Asp-Phe-Val-Gln-Tr
Pancreatic glucagon 1 expressed as p-Leu-homoserine
26 peptide-homoserine and planar structural formula H-His-
Ser-Gln-Gly-Thr-Phe-Thr-S
er-Asp-Tyr-Ser-Lys-Tyr-Le
u-Asp-Ser-Arg-Arg-Ala-Gln
-Asp-Phe-Val-Gln-Trp-Leu-
Pancreatic glucagon 1-2 expressed as homoserine lactone
6 Peptide - At least one type of peptide selected from homoserine lactone is used as a hapten, and this has the general formula O
A peptide-protein complex obtained by reacting with a protein as a carrier in the presence of a dialdehyde represented by HC-(CH_2)n-CHO [in the formula, n represents an integer of 1 to 5] is administered to a mammal. A method for producing an antibody, which comprises administering the antibody and collecting the produced antibody.