JPS60149971A - Preparation of antigen - Google Patents

Abstract

PURPOSE:To obtain an antibody having perfect cross-reacting property from an antigen in good reproducibility, by using peptide selected from peptide-homoserine represented by a predetermined plane structural formula as a hapten and reacting said hapten with protein being a carrier in the presence of predetermined dialdehyde. CONSTITUTION:This antigen comprising a peptide-protein composite is prepared by a method wherein at least one of peptide selected from pancreatic glucagon 1-26 peptide-homoserine represented by plane structural formula I and pancreatic glucagon 1-26 peptide-homoserine lactone represented by plane structural formula II, both of which are prepared, for example, by adapting usual peptide chemical reaction to human panceatic glucagon, is used as a hapten and this hapten is reacted with protein being a carrier, for example, horse serum albumin in the presence of dialdehyde represented by formula III such as malonaldehyde or succinealdehyde acting as a medium for bonding said hapten to protein being the carrier.

Description

Detailed Description of the Invention: i*1(7)iul91 The present invention provides a method for producing an antigen consisting of a novel peptide-protein complex, and more specifically, a method for producing an antigen consisting of a novel peptide-protein complex, and more specifically, a method for producing an antigen consisting of a novel peptide-protein complex. It shows strong cross-reactivity with both glucagon and GLI), and the cross-reactivity rate of the obtained antibody is constant, making it possible to accurately quantify total glucagon in the blood and, by extension, to accurately quantify the above-mentioned GLI. The present invention relates to a method for producing an antigen that provides a new glucagon antibody that is capable of producing a glucagon antibody.

Traditionally, pancreatic glucagon and GLI, which are types of physiological pancreatic homomons, are particularly involved in sugar absorption and metabolism. It is well known that it will be possible to diagnose the condition. However, much research has been conducted on the above pancreatic glucagon, and antibodies that allow accurate quantification, that is, antibodies specific to human pancreatic glucagon, have been developed. Various reactive antibodies have been developed, and the present inventors also succeeded in producing the specific antibody with high reproducibility from an antigen using the 18-29 or 19-29 peptide chain of pancreatic glucagon as a hubten (Japanese Patent Application Laid-Open No. However, little research has been conducted on GLI, and there are many unknowns about its structure and its relationship with sugar metabolism.
Antibodies that specifically react with I are still completely unknown.

Problems to be Solved by the Invention The present inventors have been conducting research on various glucagon antibodies (AGS) in the process of elucidating the role and relationship of glucagon in sugar metabolism, The known various antibodies that react, that is, non-specific antibodies, do not react to the same degree with pancreatic glucagon and GLI, and the degree of cross-reactivity varies greatly (the dilution slopes for both are not parallel). GLiffi, expressed as an amount equivalent to, is
Depending on the dilution ratio, the actual GL lit could be far from the actual value, and the total glucagon value measured when the same plasma was measured using different antibodies varied greatly depending on the antibody and could not be compared between measurements at all. That is, even if the pancreatic glucagon value measured with a pancreatic glucagon-specific antibody is subtracted, accurate quantification of total GLI is impossible, 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, by chance, a peptide consisting of 1-26 bebutide homoserine of pancreatic glucagon and/or 1-26 bebutide homoserine lactone of pancreatic glucagon was discovered. In addition to successfully creating a specific antigen as a hapten, the antibody obtained using the antigen has complete cross-reactivity with both pancreatic glucagon and GLI,
Therefore, the total glucagon level measured using this antibody is
It corresponds to the total value of pancreatic glucagon and GLI, and therefore, when subtracting the pancreatic glucagon value measured with a pancreatic glucagon-specific antibody from this value, an accurate GLI value can be quantified, and the above-mentioned complete We have discovered that antibodies with significant cross-reactivity can be obtained.

The present invention, which is a method for solving the same problem, was completed based on this new knowledge. That is, the present invention provides at least one type selected from pancreatic glucagon 1-26 peptide-homoserine represented by the planar structural formula 8 formula % and pancreatic glucagon 1-26 peptide-homoserine lactone represented by the planar structural formula % formula %. It is characterized by using a peptide as a hapten and reacting it with a protein as a carrier in the presence of a dialdehyde represented by the general formula 0% () [wherein n represents an integer of 1 to 5]. The present invention relates to a method for producing an antigen consisting of a peptide-protein complex.

The designation of superpeptides used as hubtens herein is in accordance with the designation of amino acid residues by abbreviations in the amino nomenclature adopted by IUPAC.

In the method of the present invention, the above formula [1a] is used as the hub ten.
It is essential to use a peptide represented by and/or (1b). The peptide is a type of human pancreatic glucagon.
~26 peptide chains bound to homoserine or homoserine lactone, and mixtures thereof, and can be easily prepared, for example, by applying a common peptide chemical reaction to human pancreatic glucagon. More specifically, a solution of bromic acid in formic acid is added to a solution of pancreatic glucagon in formic acid, and the reaction is carried out, followed by filtration of the glucagon.

Further, the dialdehyde represented by the above general formula (II) is
It acts as an intermediary to bind the above-mentioned hapten and the protein used as a carrier, and specifically, malonaldehyde, succinaldehyde, glutaraldehyde, adibaldehyde, 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, lattice serum globulin, rabbit serum globulin, and human serum globulin.

The antigen of the present invention is produced by reacting the above hapten and protein in the presence of dialdehyde. The above reaction is carried out in an aqueous solution or a normal buffer solution having a pH of 7 to 10, preferably 1) a buffer solution of 88 to 9, at 0 to 40° C., preferably around room temperature, and the reaction is completed in about 1 to 24 hours. As typical buffer solutions used in the above, the following can be exemplified.

0.2N sodium hydroxide-0.2M boric acid-0.2M
Potassium chloride buffer, 0.2M sodium carbonate-0.2M boric acid-0.2M potassium chloride buffer, 0.05M sodium tetraborate-0.2M boric acid-0,
05M sodium chloride buffer solution, 0.1M potassium dihydrogen phosphate-0.05M sodium tetraborate buffer solution. In the above, the proportions of hapten, dialdehyde and carrier can be determined as appropriate, but usually the hapten is added to the carrier. The amount is preferably 5 to 20 times the mole, preferably 10 to 15 times the mole, and 3 to 20 times the dialdehyde, preferably 5 to 10 times the mole. Through the above reaction, an antigen consisting of the peptide-protein complex of the present invention, in which a carrier and a hapten are bonded via dialdehyde, is obtained. After completion of the reaction, the antigen can be easily isolated and purified by conventional methods such as dialysis, gel filtration, and fractional precipitation. The antigen can also be preserved by conventional freeze-drying methods.

In producing antibodies using the antigen obtained above, a conventional method can be employed in which the antigen is administered to a mammal and the antibodies produced in vivo are collected.

There are no particular restrictions on the mammal used for the production of the antibody, but it is usually desirable to use rabbits or guinea pigs. For antibody production, a predetermined amount of the antigen obtained above was diluted with physiological saline to an appropriate concentration, and 70% Indian supplementary solution (Complete Freund'5Adjuvan) was added.
t) to prepare a suspension, which may be administered to a mammal. For example, the above suspension is injected subcutaneously into rabbits (0.5 to 21M times as the amount of antigen), and then every 2 weeks,
Immunization can be achieved by administering for up to 10 months, preferably 4 to 6 months. Antibodies are collected by collecting blood from the immunized animal after the final administration of the suspension, when a large amount of immunized bodies are produced, 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 above method 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.

The antibodies thus obtained enable the quantitative determination of glucagon by methods such as RIA, which is in demand in the field, as described above, and can be used in the diagnosis of various pathological conditions involving pancreatic glucagon and gastrointestinal glucagon, including diabetes. It is useful for etc.

Reference examples and examples are given below to explain the present invention in more detail, but the present invention is not limited thereto.

Reference Example 1 Human pancreatic glucagon (manufactured by Sigma, Beef Bork Glucagon) 60mA was dissolved in 70% formic acid, 1ml of 70% formic acid solution of 1.43M BrCN was added thereto, and the reaction was stirred at room temperature for 24 hours. After the reaction was completed, the gel was filtered using Sephadex G-25 (layered solvent 0.
2M acetic acid) to obtain 40 ml of a mixture of 1-26 peptide chains of pancreatic glucagon-homoserine and 1-26 peptide chains of pancreatic glucagon-homoserine lactone.The resulting peptide mixture is then lyophilized.

Example 1 3011 of the peptide mixture obtained in Reference Example 1 above! If and bovine serum albumin (BSA) 401 (J) were added to 15 mQ of 0.1 M borate buffer (pH = 8.5), 0.2 M glutaraldehyde solution was added dropwise. The reaction mixture was stirred at room temperature. Stir and react for 6 hours.

The resulting reaction mixture is dialyzed at 4° C. for 24 hours (dialysate physiological saline), and the dialyzed fluid is freeze-dried to obtain the antigen of the present invention (peptide-BSA complex) 71+o in the form of a white powder.

Antibody Production Example 1 Five randomly selected rabbits (■ to V) were given an antigen of the present invention (complex>7ma) obtained in Example 1 after dissolving it in 1.8 μm of physiological saline, and then adding 70 μm of the antigen to 5 randomly selected rabbits (■ to V). Add 2.7 liters of auxiliary liquid to W4.
One dose of the 1FJ suspension was administered subcutaneously to each rabbit, and after 231 hours, the same amount was further administered subcutaneously. From now on 23fl
Separately prepared suspensions (antigen 31111,
3 mQ of physiological saline and Freund's auxiliary solution 311i11
> is administered in the same manner for 3.5 months to immunize test animals. After 10 days from the final administration, blood is collected from the test animal and centrifuged to collect antiserum to obtain each antibody.

Measurement of potency> The titer of the obtained antibody ~V is measured as follows.

That is, the above antibodies were dissolved in physiological saline at io and io2゜103, respectively.
．． Diluted 10' and 105 times, 100 μQ each of these.
125■-glucagon 100μQ and 0105M phosphate buffer (pH 7.4) (0.25% B5A 10.1%
>300μ containing Na N3 and 0.01M EDTA
Q was added and incubated at 4°C for 48 to 72 hours, and the resulting conjugate of antiserum and 1251-glucagon was separated by dextran-activated charcoal method and centrifugation (4°C, 15 minutes,
30001) l) from unreacted 125■-glucagon, its radiation is counted, and the binding rate (%) of the antiserum to 1251-glucagon at each dilution concentration is determined. The final dilution ratio of the antiserum at which the binding rate (%) is 50%, that is, 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 labeled glucagon concentration 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 (F) that exists free in the solution increases. It is something that was given.

Pancreatic glucagon (labeled glucagon, concentration 1) was used as a test sample.
0pil/m? ~i 00000 no/m, ) and gut GLI (concentration 0.203 to 400 uCJ lyophilizate/+11111, Kenny's method [to enny,
A, J,, J.

Cl1n, Endocrinol, Hatabl,
15.1089-1105 (1955)) is used. Also, as labeled glucagon, 1
25I-glucagon (100OOCpffl) is used.

200μQ of the above test glucagon sample or 17tlt GLI sample, 125■ - 200μQ of each of the antibodies of appropriate titer obtained in Production Example 2 of glucagon 200μQ1 antibody - 200μQ and trasylol (manufactured by Bayer, '100
After mixing 100 μQ of 0'K I Ll ') and incubating at 4°C for 48 to 72 hours, the radiation of bound labeled glucagon (B) and free labeled glucagon (F) was counted by the dextran charcoal method. The binding rate (BO) corresponding to the titer of each antibody used is set as 100%,
Bound labeled glucagon (B) at each test sample concentration
Calculate the percentage. Dilution slope in the pancreatic glucagon sample and gutGLI as the equivalent of the pancreatic glucagon sample
The dilution slopes in FIGS. 1 to 5 are shown in FIGS. Each figure shows the above-mentioned slopes obtained using antibodies ① to V, respectively, and the vertical axis in each figure represents the binding % (B/Bo
0), and the horizontal axis is pancreatic glucagon concentration (PAL system) and 01
lt GLI concentration (μq/−). Also, in each figure, the curve (a) represents pancreatic glucagon, and the curve (b) represents! 1lu
tGLI are shown respectively.

Also, from Figures 1 to 5 above, pancreatic glucagon and ΩLlt
Antibody engineering when cross-reactivity and binding % with GLI is 50% ~V
When the sensitivity is expressed as pancreatic glucagon concentration (ng/red), it is as shown in Table 2 below.

Table 2 As is clear from Figures 1 to 5 and Table 2 above, the use of the antigen of the present invention completely inhibits pancreatic glucagon and gutGLI in 4 out of 5 randomly selected rabbits. It can be seen that antibodies exhibiting cross-reactivity can be obtained and that 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 put
It is clear that it allows accurate quantification of GLI.

[Brief explanation of the drawing]

FIGS. 1 to 5 are graphs showing the cross-reactivity of antibodies obtained from antigens obtained by the method of the present invention with pancreatic glucagon and putGLI. (Above) The procedural amendment was filed on February 18, 1985, Patent Application No. 165519 2, Invention F)
Name Antigen Production Method 3, Relationship with the Amendment Case Patent Applicant 4, Agent Tsuru Hill, 10 Sawa, 2 Hirano-cho, Higashi-ku, Osaka Telephone: 06-203
-0941 (Representative) January 29, 1985 As attached to the attached sheet

Claims (1)

[Scope of Claims] ■ At least one selected from pancreatic glucagon 1-26 peptide-homoserine represented by the planar structural formula % formula % and pancreatic glucagon 1-26 peptide-homoserine lactone represented by the planar structural formula % formula % One type of peptide is habten, and it is characterized by reacting it with a protein used as a carrier in the presence of a dialdehyde represented by the general formula 0 formula % (in the formula, n represents an integer from 1 to 5). A method for producing an antigen consisting of a peptide-protein complex.