JPS6224743B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antigen for immunological measurement of estriol-3-glucuronide and a method for producing the same. More specifically, the present invention relates to a novel method for synthesizing a hapten-protein-bound antigen to obtain a specific antiserum for estriol-3-glucuronide. Estriol is a type of estrogen
Free type, conjugated type, etc. are known. Of these, the free form exists in the blood but does not normally appear in the urine.
Excreted as glucuronidated or sulfate-conjugated forms. Among these, the physiological significance of glucuronide-conjugated forms has recently attracted attention, and estriol, which appears in the blood and urine of pregnant women.
It has been pointed out that it is necessary to more accurately understand fetal placental function by differentially measuring 3-glucuronide and estriol-16-glucuronide. Conventionally, estriol in body fluids has been measured by hydrolyzing bound estriol to form a free form, extracting it, and then using a combination of colorimetric methods using the Kober reaction, fluorescence methods, or immunoassay methods. Ta. However, these measurement methods that require hydrolysis are complicated, and although the total amount of conjugated and protein-bound erythriol can be determined by subtracting the amount of free erythriol from the total amount of estriol, It was almost impossible to measure the fraction of glucuronide-conjugated type and sulfate-conjugated type, as well as the fractional measurement of estriol-3-glucuronide and estriol-16-glucuronide among the glucuronide-conjugated type. The present invention relates to a novel method for synthesizing a hapten-protein-bound antigen for producing a specific antibody necessary for immunologically measuring estriol-3-glucuronide in body fluids, and relates to a method for producing a hapten-protein-bound antigen using the antigen. By using such antibodies, it becomes possible to specifically measure estriol-3-glucuronide in body fluids without being influenced by coexisting similar substances. Radioimmunoassay, which has recently attracted attention as a method for measuring hormones in body fluids, determines the binding ratio of a hormone labeled with a radionuclide and an antibody against the hormone to determine the amount of antigen added to the reaction solution, that is, the amount of unlabeled hormone. This method takes advantage of the fact that analytes change depending on the temperature, has high sensitivity, and can measure a large number of samples, so it is widely used in clinical laboratories. However, its specificity is subject to much debate. In other words, since there are generally many substances in body fluids that have a similar structure to the substance to be measured, the antibody used for measurement should be one that reacts only with the substance to be measured, that is, has high specificity and low cross-reactivity. things are required. If an antibody with a large binding constant and high specificity for a substance to be measured is prepared, a radioimmunoassay for measuring the substance to be measured has almost been established. As mentioned above, when measuring estriol in body fluids using conventional methods, a hydrolysis procedure is generally required, but in radioimmunoassay, an antibody that specifically binds to estriol glucuronide is prepared. This eliminates the need for hydrolyzing operations and allows specific,
Since high-sensitivity measurement is possible, today there is a method for producing specific antibodies for immunologically measuring not only estriol glucuronide but also various steroid hormone conjugates without hydrolyzing them. Research is underway to establish an immunological measurement method to determine what kind of substance should be used as an antigen for producing specific antibodies. As one example, a so-called hapten-protein binding antigen was synthesized in which BSA was bound to the carboxyl group of the glucuronic acid moiety of estrogen-3-glucuronide. Although a method for measuring 3-glucuronide has been reported, the antibody has low specificity and exhibits cross-reactivity with free estrogen and estrogen-16-glucuronide, so it has not given satisfactory results.
(P.Samarajeewa et al: Biochem.J. 151 , 369~
376, 1975) In addition, in the case of a hapten-protein binding antigen in which BSA is bound to the C ₂ or C ₄ position of carboxyphenylazoestriol-16-glucuronide using the carbodiimide method, the antigen can be prepared using the antigen. The antibodies produced showed relatively low cross-reactivity to free estrogens of less than 2.2% and showed improved specificity, but still insufficient, and this insufficient specificity was explained by adding BSA to the benzoyl group. At the time of binding, the carboxylic acid of the glucuronic acid moiety is also
It is presumed that this is due to BSA binding.
(JRSoares et al: FEBS Lett, 61 , 263~
266, 1976) In addition, BSA is the C _6th place of estrone sulfate.
(T.Nambara et al: J.Steroid
Biochem. 13 , 1075-1079, 1980), a method for binding BSA to the _C2 or _C4 position of estrogen D-ring glucuronide (T.Nambara et al: J.Steroid
Biochem. 9 , 785-790, 1978, J.Pharm.Dyn.
1 , 55-59, 1978), all antibodies produced using hapten-protein-bound antigens show high specificity for hapten, and therefore are located far from the binding site of hapten and BSA. It is understood that a portion of the hapten molecule serves as an antigenicity-determining site, and that the specificity of the antibody is directed toward the antigenicity-determining site. In the case of estrogen-3-glucuronide, this means that the antigen with a carrier protein bound to the _C6 position of the B _ring is more effective than the antigen with a carrier protein bound to the _C6 position of the B ring, compared to the antigen with a carrier protein bound to the C2 _{or C4} position adjacent to the C3 position of the A ring. It has been shown that this method is more advantageous in producing antibodies that recognize the A-ring structure better. However, at present, a hapten to obtain a specific antiserum against estriol-3-glucuronide by conjugating BSA to the _C6 position of estriol-3-glucuronide.
There are no reports on methods for synthesizing protein-bound antigens. From this perspective, the present inventors conducted an intensive search for hapten-BSA conjugates with the aim of producing highly specific antibodies that can be used when immunologically measuring estriol-3-glucuronide. As a result, the present invention has been completed. The present invention aims to prevent BSA from binding to the glucuronic acid site of the A ring of estriol-3-glucuronide, and to bind the BSA to the _C6 position of the B ring of the steroid skeleton.
This article relates to a method for synthesizing a hapten-BSA conjugate containing BSA. The substance described in claim 1 synthesized based on the present invention, that is, 6-oxoestriol-3-glucuronide-6-
Antibodies produced using (O-carboxymethyl)oxime-BSA conjugates can be obtained using estriol-3-glucuronide-
It became clear that this antibody has particularly excellent recognition of the A-ring structure of steroids compared to antibodies produced using BSA conjugates, and that the specificity is further improved, leading to the completion of the present invention. Antibodies produced according to the present invention are particularly suitable for use in radioimmunoassays of estriol-3-glucuronide, but are not limited to radioimmunoassays. It can also be used in reactions and aggregation inhibition reactions. The present invention provides 6-oxoestriol-
Regarding the method for synthesizing 3-glucuronide-6-(O-carboxymethyl)oxime-BSA conjugate, one of the additional features of the present invention is that the glucuronic acid moiety of estriol-3-glucuronide is The purpose is to remove the protecting group in the final stage of the synthesis process, that is, in the process described in claim 2 (d). The novel steroid compound used as an antigen for measuring estriol-3-glucuronide according to the present invention has the chemical structural formula: It is a 6-oxoestriol-3-glucuronide-6-(O-carboxymethyl)oxime-BSA conjugate represented by The novel steroid compound according to the present invention has the following structure, i.e., chemical structural formula 6-oxoestriol-16,17-
Diacetate is converted into methyl (2,3,4-tri-O-
6-oxoestriol-16,17-diacetate-3- by reacting with acetyl-1α-bromo-1-deoxy-D-glucopyranoside) uronate
Process of forming glucuronide-triacetate methyl ester (2), chemical structural formula 6-oxoestriol-16,17-
Diacetate-3-glucuronide triacetate methyl ester was dissolved in O in the presence of sodium acetate.
-Process of reacting with carboxymethylhydroxylamine hydrochloride to form 6-oxoestriol-16,17-diacetate-3-glucuronide-triacetate methyl ester-6-(O-carboxymethyl)oxime (), chemical structural formula 6-oxoestriol-16,17-
Diacetate-3-glucuronide-triacetate methyl ester-6-(O-carboxymethyl)oxime is reacted with BSA to produce 6-oxoestriol-16,17-diacetate-3-glucuronide-triacetate methyl ester-6-
(O-carboxymethyl)oxime-BSA conjugate process, chemical structural formula 6-oxoestriol-16,17-
A step of partially hydrolyzing diacetate-3-glucuronide-triacetate methyl ester-6-(O-carboxymethyl)oxime-BSA conjugate (), combining the above steps A, B, C, and D It is manufactured by Next, examples of the present invention will be shown. Example 1 Commercially available compound represented by chemical structural formula (), 6
-Oxoestriol-16,17-diacetate
After dissolving 300mg in 50ml of toluene, cadmium carbonate
After adding 300mg of methyl (2,3,4-tri-O-acetyl-1α-
Bromo-1-deoxy-D-glucopyranoside)
After adding 300 mg of uronate and refluxing for 14 hours, the mixture was subjected to silica gel column chromatography (hexane/ethyl acetate 1:1). Next, it was subjected to preparative TLC (hexane/ethyl acetate 3:2), and the fraction with an Rf value of 0.23 was eluted with ethyl acetate, concentrated under reduced pressure, and then recrystallized from methanol, yielding 105 mg of compound () as colorless needle-shaped crystals. obtained as. Analysis value of colorless acicular crystalline compound () mp 230° ~ 231.5° [α] ²⁵ _D -55.9° (C = 0.47, CHCl ₃ ) Elemental analysis value C: 59.70, H: 6.14 Chemical formula C ₃₅ H ₄₂ O ₁₅ Calculated values for C: 59.83, H: 5.98 NMR (CDCl ₃ ) 0.87 (3H, s, 18−CH ₃ ) 2.04, 2.10 (12H, 3H, eachs, 16−, 17−,
2'-, 3'-, 4'-OCOCH ₃ ) 3.72 (3H, s, COOCH ₃ ) 4.25 (1H, m, 5'-H) 4.95 (1H, d, J=7Hz, 1'-H) 5.30 (5H, m, 16β-, 17α-, 2′-, 3′-,
4′-H) 7.22 (2H, m, aromatic H) 7.60 (1H, d, J=2Hz, 4-H) Dissolve 80 mg of colorless needle-like crystalline compound () in 18 ml of methanol/ethyl acetate (5:1) O-carboxymethylhydroxylamine hydrochloride 190mg
and add 1.0 ml of 10% sodium acetate solution, 35
After stirring at °C for 12 hours, water was added to the reaction mixture and extracted with ethyl acetate. After washing the extract with water and drying, the solvent was distilled off and the residue was recrystallized from ether to obtain 60 mg of compound () as colorless needle crystals. Analysis value of colorless acicular crystalline compound () mp 183° ~ 185° [α] ²⁰ _D -58.6° (C = 0.47, CHCl ₃ ) Elemental analysis value C: 55.29, H: 5.69, N: 1.89 Chemical formula C ₃₇ Calculated values for H ₄₅ O ₁₇ N・11/2H ₂ O C: 55.36, H: 6.03, N: 1.74 NMR (CDCl ₃ ) 0.82 (3H, s, 18−CH ₃ ) 2.02 (15H, m, 16−, 17―, 2′―, 3′―4′―
OCOCH ₃ ) 3.75 (3H, s, COOCH ₃ ) 4.20 (1H, m, 5'-H) 4.70 (2H, s, = NOCH ₂ -) 4.95 (1H, d, J = 7Hz, 1'-H) 5.05 ~5.40 (5H, m, 16β-, 17α-, 2'-,
3'-, 4'-H) 6.95 (1H, dd, J=8, 2Hz, 2-H) 7.20 (1H, d, J=8Hz, 1-H) 7.50 (1H, d, J=2Hz, 4 -H) Dissolve 60 mg of colorless needle-like crystalline compound () in 2.4 ml of dimethylformamide (hereinafter abbreviated as DMF), and add 80 μl of tri-n-butylamine and 18 μl of isobutyl chloroformate under ice cooling.
After 30 minutes, add water-containing DMF containing 192 mg of BSA under ice cooling.
alkaline solution (H ₂ O3.6ml―DMF4.8ml―1N
Add NaOH0.2ml) and adjust the pH to 7.0-7.5, then at 4℃.
After stirring for 15 hours, the resulting reaction solution was dialyzed against cold water for 23 hours and then freeze-dried to obtain 275 mg of the hapten-BSA conjugate (). After dissolving 275 mg of hapten-BSA conjugate () in 50 ml of water, the solution was adjusted to pH 12.0 to 12.5 with 5N NaOH and stirred at 25°C for 18 hours. Next, the mixture was dialyzed against cold water for 22 hours and then freeze-dried to obtain 232 mg of compound (). The number of moles of hapten bound per mole of BSA in compound (), ie, 6-oxoestriol-3-glucuronide-6-(O-carboxymethyl)oxime-BSA conjugate, was determined by UV method to be 34 moles. Example 2 Creation of antibodies (rabbit immunization) 6-
Oxoestriol-3-glucuronide-6
-(O-carboxymethyl)oxime-BSA conjugate antigen was immunized as follows. Specifically, 2 mg of the antigen was dissolved in 0.5 ml of sterile physiological saline, and this was made into an emulsion using 0.5 ml of Freund's Complete Adjuvant, and this emulsion was subcutaneously administered to the shoulder blade and several footpads of rabbits. Thereafter, the same administration was carried out every two weeks for two months. One week after the last dose (fifth dose), 10
ml of blood was collected, centrifuged at 3000 rpm for 10 minutes, and the resulting antiserum was stored at -20°C. Before use, thaw the antiserum and add BSA0.06
%, bovine-γ-globulin 0.05% at a dilution rate of 1:3000 using 0.05M borate buffer (PH8.0) to prepare a standard curve. Measurement procedure Add estriol-3-glucuronide-6 to the sample containing estriol-3-glucuronide.
Add ^7-3H (approximately 7500 dpm) and 0.25 ml of antiserum, and incubate at 4°C for 1 hour. Next, 0.25 ml of 50% ammonium sulfate solution was added, and after standing at 4°C for 20 minutes, centrifugation was performed at 3000 rpm for 15 minutes, and 0.2 ml of the resulting supernatant was taken and radioactivity was measured. About cross-reactivity 6-oxoestriol-3-glucuronide-6-(O-carboxymethyl)oxime-
To examine the specificity of the aforementioned antisera obtained from rabbits immunized with BSA conjugates, we used 32 different steroids to determine their cross-reactivity with estriol-
Table 1 shows the amount of 3-glucuronide-6,7- ³ H bound to this antiserum determined from the standard curve. The values in the cross-reactivity column of Table 1 are the reciprocals of the reading ratios of various steroids relative to the 50% inhibition amount of unlabeled estriol-3-glucuronide as a percentage.

【table】

[Table] From the results in Table 1, the specificity for estriol-3-glucuronide using the antibody based on the antigen using the novel compound of the present invention is extremely high compared to other methods, and the cross-reactivity is low. It is clear that it is being suppressed.

Claims (1)

[Claims] 1. Chemical structural formula 6-oxoestriol-3-glucuronide-6-(o-carboxymethyl)oxime-BSA conjugate (wherein -NH-BSA represents bovine serum albumin residue, G is represents a glucuronyl group represented by Same below. ) is an antigen for measuring estriol-3-glucuronide. 2 A Chemical structural formula 6-oxoestriol-16,17-
Diacetate () is converted into methyl (2,3,4-tri-o-acetyl-1α-bromo-1-deoxy-
D-glucopyranoside) reacted with uronate6
-Oxoestriol-16,17-diacetate-3-glucuronide-triacetate methyl ester (), (b) Chemical structural formula (In the formula, G′ is represents a glucuronyl group represented by Same below. ) 6-Oxoestriol-
16,17-Diacetate-3-glucuronide-triacetate methyl ester () was reacted with o-carboxymethylhydroxylamine hydrochloride in the presence of sodium acetate to produce 6-oxoestriol-16,17-diacetate-3-glucuronide. -Triacetate methyl ester-6-(o-
(c) Chemical structural formula 6-oxoestriol-16,17-
Disetate-3-glucuronide-triacetate methyl ester-6-(o-carboxymethyl)oxime () was added to bovine serum albumin (hereinafter referred to as
6-oxoestriol-16,17-diacetate-3-glucuronide-triacetate methyl ester-6-(o-
(carboxymethyl)oxime-BSA conjugate (2) Chemical structural formula 6-oxoestriol-16,17-
Diacetate-3-glucuronide-triacetate-6-(o-carboxymethyl)oxime-
Chemical structural formula characterized by partial hydrolysis of BSA conjugate () A method for producing 6-oxoestriol-3-glucuronide-6-(o-carboxymethyl)oxime-BSA conjugate () shown by