JP2541759C - - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a solid phase or a solid phase, wherein a water-soluble glycoside-type surfactant is added to an incubation medium. The present invention relates to a method for detecting an analyte, which comprises contacting the analyte with at least one specific receptor to be obtained, and a reagent suitable for the method. 2. Description of the Related Art Methods for detecting an analyte in a sample are known which comprise contacting the analyte with a specific receptor which is or can bind to a solid phase.
The detection method usually corresponds to a heterogeneous method. In the case of immunoassay, as another method, for example, a sandwich method, an indirect method, and a competitive method are known. In the sandwich method, an antibody is bound to a carrier, and a test solution is added. As a result, the specific antigen present in the test solution binds to the antibody. Next, a specific labeled antibody corresponding to the antigen-antibody complex or a portion of the complex is added and binds to the complex. Then, the amount of the antigen can be calculated using the labeled antibody. In the indirect method, the antigen is bound to a carrier material. The test solution is added to this,
As a result, antibodies specific to the carrier-bound antigen and present in the test solution react with the antigen. When labeled antiglobulin is added, such antiglobulin binds to the antigen-antibody complex, so that the amount of the unknown antibody in the test solution can be determined. In the competition method, one partner of the immune response is bound to a carrier material. Then, a solution containing the other partner of the immune response present in an unknown amount and a known amount of the labeled other partner of the immune response is added. Both the labeled and unlabeled immune response partners compete for the binding site of the carrier-bound immune response partner. In all these alternatives, it is not necessary for the wall-bound receptor to be directly bound to the solid phase. A second wall-bound receptor that specifically binds the first receptor and binds the first receptor to a solid phase as the test method proceeds can be used. As a result, a single-step or multi-step test method can be adopted. If the analyte is a nucleic acid, the method comprises a hybridization wherein one receptor, in this case a nucleic acid complementary to the nucleic acid to be detected, is bound to a solid phase or is capable of binding. It is a test. Further, a method of taking another form in this case is known to those skilled in the art. Known heterogeneous methods for detecting all these analytes are not normally involved in specific reactions, but have a positive effect on the results of the heterogeneous method,
It is necessary to add proteins, polysaccharides and / or detergents. Heterogeneous methods lack accuracy due to non-analyte-specific interference, so-called "matrix effects", "background", or "non-specific binding" among those skilled in the art. . A solid-phase immunochemical test is described in DE-A 36 38 767, in which lactoferrin, fetal calf serum and polyoxyethylene-20-sorbitan monolaurate (Tween ^™ 20) are added. ing. Tween 20 is 0.01%
When added to the incubation solution at higher concentrations, the receptor bound to the carrier material may dissociate, such that the dissociated receptor has a negative result on the detection method. In this way, non-ionic block copolymer-type surfactants with an HLB value greater than 20, such as Tetronic ^™, are used to avoid non-specific interference in immunoassays in the heterogeneous phase. Used in -A0215457. Such surfactants are advantageous in that they do not dissociate the wall bound receptor as strongly as with Tween 20. These block copolymer type surfactants are not uniform in composition, and homologous molecules are distributed with some width. The composition of the surfactant varies depending on the contents of the batch. Therefore, a new test must be performed for each batch for its suitability for each detection method in order to avoid non-specific interference. As a result, it has been found that some batches cannot be used in the past. A further disadvantage of the commonly used surfactants is that toxic and carcinogenic starting materials, such as ethylene oxide, are used to prepare the surfactants. Ethylene oxide-propylene oxide block copolymer type surfactants are additionally difficult to biodegrade. Therefore, an object of the present invention is to find a method for detecting an analyte and to find a reagent suitable for preventing non-specific binding. The reagents need to be free from batch-dependent variability, or very little, if any, and should be compatible with the environment. The object of the present invention is to provide at least one of the following: a water-soluble glycoside type surfactant added to an incubation medium; It has been achieved by a method for detecting an analyte which comprises contacting a specific receptor. Furthermore, the present invention relates to a reagent for detecting an analyte, comprising at least one specific receptor bound or capable of binding to a solid phase, and further comprising a water-soluble glycoside-type surfactant. Surface active glycoside-type surfactants have been known as raw materials for detergents for more than 50 years, and are described for example in the production of lauryl glucoside in Austrian Patent No. 135333. Production method of alkyl polyglycoside (APG) is DE-A3723826
It is described in. Such an APG is commercially available, for example, under the trade name Plantaren ^™ . Glycoside-type surfactants are synthesized by transglucosylation via alkylglucoside. In the synthesis of known glycoside-type surfactants, precisely defined starting materials leading to well-defined products can be used. The batch-dependent variations found in the case of block copolymer surfactants are not noticeable in this case to the extent that they cause interference. Well-defined glycoside-type surfactants, such as Biochemistry 19 , 4108-41
Octyl-D, which can be prepared by more time-consuming synthetic methods according to No. 15 (1980)
-Glucopyranosides are also preferred. While APG is a mono-, di-, and trisaccharide, these compounds represent a truly single substance. According to the present invention, a glycoside-type surfactant is understood as the reaction product of a sugar and a fatty alcohol as follows. That is, aldose or ketose such as glucose, fructose, mannose, galactose, xylose, or ribose is considered as the sugar component. Glucose is particularly preferred. Aliphatic as well as aromatic alcohols are considered as fatty alcohols. The fatty alcohol must be selected so that the glycoside-type surfactant is still readily water-soluble. As the aliphatic alcohol, those having an alkyl group having 4 to 18 carbon atoms are preferably used, and an alkyl group having 6 to 8 carbon atoms is particularly preferable. As the aromatic alcohol, if desired, it is preferable to use a group having an aromatic ring substituted with a group that makes the aromatic alcohol water-soluble, for example, —OH, —OCH ₃ , or —SO ₂ . Examples of aromatic alcohols include benzyl alcohol, salicyl alcohol, and β-phenylethanol. Benzyl alcohol is particularly preferred. Hexylglucoside, octylglucoside, and benzylglucoside, or mixtures thereof, have proven to be particularly preferred glycoside-type surfactants. By adding these glycoside-type surfactants, at least one specific receptor is bound to the solid phase, or unnecessary reactions in the method of detecting an analyte that can be bound are suppressed, and at the same time, Desorption of the bound specific receptor will not occur. Moreover, glycoside-type surfactants are not affected by a wide range of batch-to-batch variations, so that similar results are achieved between batches. All known analytes, such as haptens, antigens, proteins, antibodies or nucleic acids can be detected by heterogeneous methods. The sample containing the analyte to be detected can be, for example, blood, serum, plasma, secretion, cerebrospinal fluid, urine, or a tissue product. The method according to the invention achieves very good results in plasma and serum and is thus particularly suitable for the detection of analytes in such plasma and serum. When no surfactant according to the invention is used, unwanted strong side reactions are frequently observed in plasma. As a result, measurement results can vary widely between plasma and serum samples. The method of the present invention can be used for all types of plasma. It can also be used for plasma to which EDTA has been added for the purpose of stabilization or for plasma to which heparin or citrate has been added. The method can be performed as a competitive method, as an indirect method, or as a sandwich method. Depending on each method, the specific receptor is a hapten or antigen in the case of a competitive or indirect method; an antibody or a fragment thereof in the case of a sandwich method; and a nucleic acid in the case of a hybridization test. Alternatively, it is understood as a nucleic acid fragment. In this context, the specific receptor may be bound directly to the solid phase or may be bound to the solid phase during processing. If the specific receptor can be bound only during processing, the receptor preferably comprises a specific receptor, for example an antigen, antibody or conjugate of a nucleic acid and an S1 substance. At that time, the S2 substance having the ability to specifically bind to the S1 substance is bound to the solid phase. Antigen-antibody, hapten-antibody, biotin-avidin or streptavidin, protein-antiprotein, protein A-immunoglobulin, hemoglobin-haptoglobin, or enzyme-substrate are preferred as the S1-S2 substance pair capable of specifically binding. Biotin is preferably used as the S1 substance, and streptavidin or avidin is preferably used as the S2 substance. Water-insoluble carriers to which one or several specific receptors are bound or can be bound should be used as solid phase within the meaning of the invention. Solid phases include latex particles, beads, tubes, and microtiter plates made of various plastics materials, such as polystyrene and absorbent or porous materials. Such solid phases are known to those skilled in the art. The types and methods of labels ultimately used to detect complexes of the specific receptor and analyte bound to the solid phase are known to those skilled in the art. All of the usual labeling reagents may be used, for example, radioactive labels, enzymes, fluorescent or chemiluminescent substances. The water-soluble glycoside-type surfactant can be added to all of the solutions used for the measurement. The glycoside-type surfactant can be added even during the preparation of the test sample. Such a surfactant is preferably added to the reaction buffer, or added corresponding to the reaction buffer in a two-step method or a multi-step method. The buffer may further comprise, in addition to buffer substances and specific receptors which may be present in the buffer, stabilizing additives such as proteins, polysaccharides, preservatives and other customary additives. The water-soluble glycoside-type surfactant is used in an amount of 0.1 to 2% based on the weight of the whole reaction preparation. A concentration of 0.5-1% is preferred. A reagent comprising a specific receptor bound or capable of binding to a solid phase and comprising a water-soluble glycoside-type surfactant is used to carry out the method. The reagent may also contain other conventional components. For example, buffers, proteins such as bovine serum albumin or IgG; and / or preservatives may be present. In addition, the reagent may include a conjugate of the label with the specific receptor, or in a competition test, a conjugate of the label with the analyte or an analog of the analyte. When an enzyme is used as a label, the reagent further includes a detection system for enzyme activity. The reagent preferably comprises, as glycoside-type surfactant, APG based on hexylglucoside, octylglucoside or benzylglucoside, or a mixture thereof, or a similar well-defined single substance. Glycoside-type surfactants are present in the reagents at a concentration of 0.1 to 2% by weight of the total reaction preparation. Use of the methods and reagents of the present invention allows for improved recovery of analyte detection in a heterogeneous phase. In addition, undesired side reactions, such as, for example, nonspecific attachment of the conjugate to the solid phase, are suppressed without increasing the dissociation of the bound reaction partner. The problems that arise when using samples that exhibit undesirable side reactions, especially plasma samples, are eliminated. The variation between different batches is
It is minimized by the clear and more uniform composition of the APG and the clear composition of glycoside-type surfactants produced according to Biochemistry 19 , 4108-4115 (1980). A further positive effect is that the glycoside-type surfactant is well or very well matched to the environment. EXAMPLES The present invention will be described by the following examples. Example 1 Preparation of Hexylglucopyranoside 400 g of 1-hexanol was heated to 110 DEG C. together with 3.6 g of p-toluenesulfonic acid in a 2 liter multi-neck flask equipped with a stirrer and a distillation apparatus. Then a suspension of 300 g of anhydrous glucose in 300 g of 1-hexanol was added to the above preparation in small portions, ie in four portions. After the first addition, reduce the pressure (3
00 mbar) and the reaction water formed was distilled off quickly. When all of the first addition was dissolved, the second addition was performed. The remaining additions were treated similarly. Immediately after the last addition, only the hexanol was distilled and the preparation was aerated and adjusted to pH 8 with sodium ethylate. This was followed by distilling off hexanol at 80 ° C. under high vacuum. The oily residue was dissolved in 7 liters of water and extracted with two 1 liter portions of ethyl acetate. The aqueous phase was evaporated and subsequently lyophilized. 275 g of a pale yellow substance were obtained. Example 2 Preparation of benzylglucopyranoside 800 g of benzyl alcohol and 3.6 g of p-toluenesulfonic acid were heated to 110 DEG C. in a 3 liter multi-neck flask equipped with a stirrer and a distillation apparatus. Then, a suspension of 300 g of anhydrous glucose in 500 g of benzyl alcohol was added to the above preparation in small portions, ie in four portions. After the first addition, reduced pressure and no
The water of reaction formed was quickly distilled off. When the glucose was clearly dissolved, the following portions were each added. Immediately after the last portion was added, only the benzyl alcohol was distilled off, and immediately the preparation was aerated and the catalyst was neutralized with sodium ethylate. This was followed by distilling off excess benzyl alcohol at 80 ° C. under high vacuum. The oily residue was dissolved in 2 liters of water. After 8 hours, excess benzyl alcohol separated at the bottom of the vessel. The aqueous phase was decanted and extracted once with 400 ml of ethyl acetate. The aqueous phase was further concentrated and subsequently freeze-dried. 360 g of a pale yellow substance were obtained. [Example 3] The effect of various surfactants on the calibration curve was examined by using, for example, Enzymun test ^™ LH manufactured by Boehringer Mannheim GmbH.
It measured using. The tests for the detection of such luteinizing hormone (LH) were performed according to the instructions for use. Tween 20, hexylglucoside, or benzylglucopyranoside at a concentration of 0.005% to 1% in an incubation buffer (40 mmol / l phosphate buffer pH7)
.4). Table 1 shows the effect of Tween 20 on the calibration curve in the enzyme test LH. When the concentration of Tween 20 was 0.5%, the slope of the calibration curve was 60 to 70% or less of the slope of the calibration curve when no surfactant was added. Table 2 shows hexyl glucoside, which is a glycoside surfactant in the present invention,
Alternatively, the effect of the addition of benzylglucopyranoside on the calibration curve is shown.
Even with the addition of 0.5% surfactant, the slope of the calibration curve is reduced only by 5-10% compared to the control without surfactant. Example 4 The effect of various batches of surfactants on the calibration curve and the recovery in human serum were measured using Enzyme Test LH as an example. Different Pluronic F68 batches were used in the state of the art and the results obtained were compared with different batches of benzyl glucoside. Detergent was added to the Enzyme Test LH incubation buffer as a precedent at a concentration of 0.5%.
Recovery was tested in various human sera. A calibration curve was prepared for each batch using the standard product af. each
The concentration of LH was read from the absorbance of a human serum sample using such a calibration curve. It can be seen from Table 3 that the calibration curve differs significantly depending on the Pluronic F68 batch used. On average, the recovery in human serum is the highest between these batches
16% different. It can be seen from Table 4 that the calibration curves do not differ significantly when using different batches of benzylglucoside. With these batches, the recovery differs by only 4%.

Claims (1)

Claims: 1. A method for detecting an analyte, comprising contacting the analyte with at least one specific receptor bound or capable of binding to a solid phase. A water-soluble glycoside-type surfactant characterized by adding non-analyte interference to the incubation medium by adding a surfactant to the incubation medium.
A method for detecting an analyte, wherein the surfactant is a reaction product of a sugar and a fatty alcohol which is a low molecular compound . 2. The method according to claim 1, wherein the glycoside surfactant is hexyl glucoside, octyl glucoside, or benzyl glucoside, or APG based on hexyl glucoside, octyl glucoside, or benzyl glucoside, or a mixture thereof. Item 7. The method according to Item 1. 3. A reagent for detecting an analyte containing at least one specific receptor bound to or capable of binding to a solid phase, comprising a sugar and a low molecular weight compound.
A reagent for detecting an analyte, further comprising a water-soluble glycoside-type surfactant that is a reaction product with a fatty alcohol . 4. The method according to claim 1, wherein the glycoside surfactant is hexyl glucoside, octyl glucoside, or benzyl glucoside, or APG based on hexyl glucoside, octyl glucoside, or benzyl glucoside, or a mixture thereof. Item 7. The reagent according to Item 3.