JPH04254760A - Serum amyloid a reference substance and setting method thereof - Google Patents

Abstract

PURPOSE:To obtain a serum amyloid A(SAA) reference substance for immunological measurement which can be used as a marker for inflammation, etc., and its setting method. CONSTITUTION:SAA is refined in an associated form with a high-density lipoprotein (HDL-SAA) and used as an SAA reference substance for immunological measurement. The HDL-SAA has such advantages that the HDL-SAA has less difference in reactivity with the SAA contained in the sample, is better in water solubility, can be more easily refined, and so on, as compared with pure SAA. In addition, by deciding the SAA content of the HDL-SAA by utilizing a cataphoretic analysis method, the standard substance composed of the HDL-SAA can be set.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to amyloid A in serum.
(hereinafter abbreviated as SAA) relates to a standard substance used for immunoassay. SAA is amyloid protein A that is deposited in tissues in certain types of amyloidosis.
(hereinafter abbreviated as AA protein) is considered to be a precursor protein of
It is a serum protein with a molecular weight of approximately 12,000. [ Journal of Clinical Investigation (J.C.
lin. Invest. )53:1054-1061,
[1974] In recent years, it has been revealed that the serum level of SAA increases in inflammatory diseases other than amyloidosis, and it has been evaluated as a sensitive inflammatory marker. (Clinical Examination 32:2, P168, 1988)

00
02]

Problems with the Prior Art In fields such as clinical testing, immunological assays are often used to easily measure protein substances. SAA is no exception; RIA and EL
ISA [Scandinavian Journal of Immunology (Scand. J. Immunol.) 18: P3
29, 1983], immunoturbidimetry [Marker Proteins in Inflammation (Marker Pr.
3:
P157, 1986], there are many reports regarding immunoassay methods. The standard substances used in these measurement methods were prepared by diluting pure SAA or adding it to serum. However, when measurements are performed using the standard materials obtained in this way, they often show values that are significantly different from the actual values. This difference is due to SAA in serum
This is expected to be due to the fact that SAA exists in a form different from that of pure SAA, or that SAA is denatured during the purification process. Furthermore, when originally insoluble SAA is dissolved using a protein denaturant or the like, changes in the immunological reactivity of SAA are unavoidable. In order to obtain more highly purified SAA, complicated steps are required and the yield tends to be low. Therefore, there is a need for a standard substance that can be prepared by simple operations and that does not cause the above-mentioned problems in immunological measurements.

On the other hand, conventionally, for example, Japanese Patent Application Laid-Open No. 62-2161
Currently, standard materials are set as appropriate for each report, as in the publication, and it has been difficult to compare measurement results between different measurement methods. Therefore, the above-mentioned literature (M
arkerProtein in Inflammat
ion3:P157, 1986), there is a need for standard materials that can be applied to a wide range of immunological assays. Standard materials that are used in such a wide range are required to be easy to prepare and handle, but no SAA standard material that satisfies these conditions is known yet. JP-A-61-191697 discloses peptide fragments useful as antigens for SAA measurement. However, it goes without saying that this peptide fragment has a molecular weight different from that of SAA in serum, and it is unsuitable as a standard material because it only has reactivity with a specific antibody.

0004

SUMMARY OF THE INVENTION An object of the present invention is to provide a standard material for SAA that satisfies the following conditions. 1) There should be no difference between the actual value and the value observed when pure SAA is used as a standard material. 2) It can be obtained with simple operations. 3) Applicable to a wide range of measurement methods. 4) Be water-soluble.

[0005]

[Means for Solving the Problems] The present invention provides serum amyloid A purified in a form in which SAA is associated with high-density lipoprotein (hereinafter referred to as HDL) (hereinafter referred to as HDL-SAA).
Serum amyloid for immunoassay, in which the serum amyloid A content in serum amyloid A associated with high-density lipoproteins is determined by electrophoretic analysis together with a pure serum amyloid A of known protein content. A. Standard material setting method”.

[0006] Conventionally, highly purified SAA was used as a standard, but in the present invention, SAA is collected as an HDL fraction from serum, ascites, etc. containing a large amount of SAA, and is not purified further. It is used as a standard material in the following conditions. It is known that SAA exists in serum in association with HDL (Scand. J. Immu.
nol. 14: P201, 1985), and there are no known examples of its use as a standard material in immunoassays. This is because it was thought that the amount of SAA protein could not be determined if contaminant proteins such as HDL were present. In particular, since HDL has lipid molecules on its surface, it exhibits a different reactivity from proteins such as BSA. Therefore, it is difficult to determine the protein amount of SAA in HDL-SAA using known protein assay methods (such as the Lowry method) using BSA as a standard. The present inventors have arrived at the present invention by discovering that it is possible to determine the amount of SAA protein even in the presence of contaminant proteins by using techniques such as electrophoretic analysis.

HDL-SAA used in the present invention does not necessarily need to be highly purified; it is sufficient to remove components that interfere with SAA assay. Specifically, for example, SAA
SAA was determined by SDS-PAGE etc. using the pure product as a control.
If the content is to be determined, it is sufficient to purify to such an extent that SAA can be obtained as a single band. In this case, starting materials similar to those used to obtain conventional pure SAA products, such as ascites, plasma, and serum rich in SAA, can be used. HD from these materials by ultracentrifugation
Collect the L fraction. (J.Clin.Invest.3
4: P1345, 1955, etc.)

[0008] At this stage, other apolipoproteins are present, so further purification steps such as delipidation, gel filtration, and ion exchange chromatography are essential in order to obtain pure SAA. However, in the present invention, the HDL-SAA obtained here is used as it is as a standard substance. To determine the amount of SAA protein present in HDL-SAA, techniques such as SDS-PAGE can be applied. S
In DS-PAGE, SAA is
Since it is possible to analyze the amount of SA in HDL-SAA compared to pure SAA whose protein content has been measured in advance,
The protein amount of A can be assayed. In this way S
A secondary standard can be set by using HDL-SAA, in which the protein amount of AA has been determined, as a primary standard and measuring the SAA content of a pool of SAA-positive sera. The SAA standard material according to the present invention has a concentration that allows accurate determination of the protein amount of SAA, that is, at least 10 μg/ml if the protein amount is assayed by SDS-PAGE.
A higher concentration is required. Considering that the SAA standard according to the invention is used as a primary standard, the lower limit of its concentration should be set at least at the highest concentration of the secondary standard (approximately 50
It is preferable to set it to a value of about 1 μg/ml or more. The present invention will be explained in more detail below based on Examples.

[0009]

[Example] 1. Purification of HDL-SAAI Rheumatism patient serum containing high concentration of SAA-I [SAA content 309 μg/
ml Yamada et al.'s method (Clinical Examination 32: P167, 1988
) 76 ml was used as the starting material, and the upper layer with a specific gravity of 1.23 g/l was first collected by ultracentrifugation. Next, the specific gravity was adjusted to 1.063 g/l, ultracentrifugation was performed again, and the lower layer was collected. After concentrating this fraction with polyethylene glycol, Sepharose CL6B column 1.6 x 85c was used to increase the concentration of HDL-SAA.
m (manufactured by Pharmacia), 0.5M salt,
Elute with 0.01M Tris-HCl buffer pH 8.6 containing 2mM EDTA, follow the absorption at 280nm, collect 2ml of the peak fraction, and collect HDL-SAA.
I got I. This HDL-SAA was transferred to an agarose film (
Electrophoresis was carried out by applying a cholesterol coloring reagent (Polymo Film System, Nippon Shoji Co., Ltd.)
When the color was developed, it was proved to be a highly pure HDL fraction. Furthermore, apo-AI, apo-AII, apo-CI
Western blotting was performed using antibodies against lipoproteins I, apo-CIII, and SAA, and it was confirmed that the SAA band consisted of a single protein.

2. Purification of HDL-SAAII Rheumatism patient serum-II containing high concentration of SAA (SAA content 132
HDL-SAAII (2 ml) was obtained from 76 ml (0 μg/ml) by the same operation as in Example 1.

3. Determination of the protein amount of SAA in HDL-SAA 3-1. Purified SAA Prior to determining the protein amount of SAA, purified SAA was first obtained by the following method. Using 1 liter of patient ascites pool (118 μg/ml) containing a large amount of SAA as a starting material, first collect the upper layer with a specific gravity of 1.23 g/l by ultracentrifugation.
Next, the lower layer with a specific gravity of 1.063 g/l was collected, degreased with methanol/ether (1:2) under cooling, and then coated with a Sephadex G-200 column (6M urea, 0.5% Tweed).
0.01M Tris-HCl buffer pH containing en 20
8.6) and then passed through a column bound with anti-apoAI, apoCIII, and human serum albumin antibodies to remove contaminant proteins using Sepharose 4B (Pharmacia) activated with bromcyan in the usual manner. 1l
31 mg of purified SAA was obtained from patient's ascites. Purified SAA showed a single band at a molecular weight of 12,000 by SDS-PAGE, and did not react with other apolipoprotein antibodies. In addition, the amino acid sequence is from the N-terminus to Ser
Phe Phe Ser Phe Leu Gly
Glu Ala Phe Asp Gly Ala A
rg Asp Met Trp Arg Ala Ty
r, and from a database search, a report by Doulette et al. [Biochem. 27: P
1677, 1988] was found to be identical to forms II and IV lacking the N-terminal Arg.

3-2. Determination of protein content of SAA Purified SAA thus obtained (6M urea, 0.5% Tween20
, 0.01M Tris-HCl buffer)
As a standard, it was determined by the Lowry method to be 31 mg.
(yield 26.3%). SDS-PAGE (15
% gel), several serial dilutions of the above purified SAA and HDL-SAA obtained in Example 1 were run on the same gel. SA protein stained using CBB using conventional method
The A band was measured with a densitometer (ACD-250DX, manufactured by Atto), and a purified SAA concentration curve was created from the integrated concentration value to determine the protein amount of HDL-SAA. The results are shown in Table 1. The analysis was performed three times.

[Table 1]

[0013] The protein amount of HDL-SAA was confirmed by the Lowry method, and the HDL obtained by SDS-PAGE was
We also attempted to determine the protein amount of SAA from the composition ratio of - and SAA, but since it was difficult to accurately measure the protein amount, purified SA
A method was adopted to determine the protein amount of SAA relative to A. This is thought to be because HDL has lipid molecules on its surface and exhibits a different reactivity than BSA.

4. Evaluation of HDL-SAA as a standard material according to the present invention It was confirmed by the latex agglutination reaction method and ELISA that HDL-SAA, whose protein amount was determined in Example 3, can actually be used as a standard material for other SAA measurement methods. confirmed. 4-1. SAA latex agglutination reaction reagent After mixing equal amounts of SAA obtained in Example 3 with Freund's Complete Adjuvant and thoroughly emulsifying it,
The limbs were immunized. Immunization was performed every two weeks, and after four months, a portion of the blood was collected and the obtained antiserum was fractionated with 40% ammonium sulfate, dialyzed, and concentrated to obtain an anti-SAA antibody (0.01 mg/ml). This anti-SAA antibody was physically adsorbed on polystyrene latex (average particle size 0.109 μm, Sekisui Chemical Co., Ltd.) at 56°C for 1 hour, washed with 0.1M HEPES buffer, and finally adjusted to a latex concentration of 1%. Dispersion medium (0.1M HE containing 5% sucrose, 5% choline chloride)
A reagent for SAA latex agglutination reaction was obtained by suspending it in PES buffer (pH 7.4).

4-2. SA by latex agglutination reaction method
Measurement of A Using the reagent for latex agglutination reaction prepared in Example 4-1, HDL-SA according to the present invention was determined by the following method.
The reactivity of A with SAA in serum was compared. That is,
Examples 1, 2, and 1 using rheumatism patient serum (hereinafter abbreviated as provisional standard) containing a certain amount of SAA as a standard
HDL-SAAIII, IV, derived from four different patient sera, purified in the same manner as above, and the SAA content was determined.
A latex agglutination reaction was performed for V and VI. According to this experiment, SAA in the provisional standard and HDL-
Confirm the difference in reactivity between HDL-SAA and HDL-SAA due to differences in starting materials by comparing the value obtained by latex agglutination reaction with the protein amount of SAA obtained by SDS-PAGE. can do. The conditions for the latex aggregation reaction are as follows. Sample volume: 20μl (if diluting, use 50mM HEP
(Using ES buffer pH 7.4) Latex reagent amount: 300 μl Reaction temperature: 37°C Photometry conditions: Measure the absorbance increase rate at 585 nm for 50 seconds Measuring instrument: LA2000 (manufactured by Tama Seiki)

[0016]
As the results are shown in Table 2, the values measured by latex agglutination reaction were always about 1.8 times higher (that is, the value of the temporary standard was 1.8 times higher), and there was a good correlation between the two. Admitted. A high correlation between the two indicates that there is no difference in reactivity to the latex agglutination reagent.

[Table 2]

4-3 Using the anti-SAA antibody obtained in Example 4-1, the same sample as in Example 4-2 was measured by the EIA sandwich method. The operation is as follows. Anti-SAA antibody 0.
01mg/ml (0.05M NaHCO3-Na
After sensitization, 3% BSA (0.01M PBS) was dispensed into a 96-well microtiter plate.
) to form a solid phase. On the other hand, the same antibody was labeled with POD using a conventional method to obtain a labeled antibody. The same provisional standard as in Example 4-2 and 100 μl of sample (1% BSA for dilution)
(Using 0.01M PBS) was dispensed into each well.
After reacting for 1 hour at °C, the mixture was discarded and the wells were
Wash 3 times with 1M PBS and then add POD labeled antibody 100ml.
µl was dispensed and reacted at 37°C for 1 hour. After the reaction is complete, discard the reaction solution, wash with PBS, and add 0.84mM3,3
50 μl of ',5,5'-tetramethylbenzidine (0.1M citrate buffer pH 6.0) and 50 μl of 2mM hydrogen peroxide (same buffer solution) were added at the same time for 30 minutes at 37°C.
Allowed to react for minutes. After stopping the reaction by adding 3.6N sulfuric acid, the absorbance at 450 nm and 600 nm was measured using a micro autoreader MPRA4 (manufactured by Tosoh, trade name), and the SAA concentration was determined with respect to a provisional standard. The results are shown in Table 3.

[Table 3] Similar to Example 4-2, a good correlation was observed between the two, and it was confirmed that there was no major difference in reactivity to EIA reagents.

[0018]

Effects of the Invention As confirmed in Example 4, HDL-SAA according to the present invention shows no difference in immunological reactivity between it and SAA present in the specimen, and is suitable as a standard substance. Furthermore, purification of HDL-SAA is simpler and more economically advantageous than obtaining pure SAA. In addition, HDL-
Since SAA has better water solubility than SAA, there is no need for solubilization using protein denaturants, etc., and it can be used as a standard substance in a wide range of immunoassay methods.

Claims (2)

[Claims] Claim 1: Serum amyloid A standard material for immunoassay, purified in a form in which serum amyloid A is associated with high-density lipoproteins. Claim 2: Serum amyloid A for immunoassay, in which the content of serum amyloid A in serum amyloid A associated with high-density lipoproteins is determined by electrophoretic analysis together with a pure serum amyloid A of known protein content. How to set up standard materials