JPS63503569A - Preparation for immunological measurement and method for producing the preparation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Preparations for immunological assays and methods for producing the same The present invention provides a solid carrier and a method for producing the same. An immunoassay preparation comprising two combined ligands, and a method for producing this preparation Regarding the law.

Immunological assays can be performed on blood fractions, secretions, tissue extracts, plant sap, etc. anti-oxidants in complex liquids such as bacterial cultures, food extracts and similar liquids. used for qualitative and quantitative measurements of single components such as molecules or antibodies. Ru.

Antibodies bound to solid surfaces have been used in many such measurements. 'fJ Kh are capable of binding to a specific antigen, such as isotopes, enzymes, Fluorescent dyes are used.

For the measurement, equilibrating amounts of the solid surface-bound component and the soluble component are Used with signs.

When performing solid surface-based measurements, the initial binding to the surface or Surface coverage forms an essential part of the method and all fundamental properties of this measurement method are: In particular the purity of the active ingredient in the coating mixture and the ingredients bound to the surface and subsequent immunization. It depends on the strength of the bond between the specific soluble auxiliary components in the chemical measurements.

The purity or purity of other components in the coating mixture that are simultaneously deposited on the solid surface by the coating mixture In terms of relative amounts, these components are Conflict with minutes.

Since it is difficult to actually obtain a specific component with high purity, most of the bonding positions are located elsewhere. It will be dominated by the components of

This means that these other components may be present in soluble form in subsequent immunoassays. shows the non-specific properties of the adsorbent and eliminates the inaccuracy and background noise of the measurement method. This further includes the risk of increasing the

Binding between surface-bound antibodies and antigens subsequently added in soluble form The use of surface-bound antibodies for measurements is related to the strength of this binding. The ability of this antibody, expressed in terms of affinity, is decisive for subsequent processing steps. It means something.

Selection of antibodies with high affinity is not possible using known immunological specific techniques. In reality, it is extremely difficult.

Additionally, there are differences in antibody type between polyclonal and monoclonal antibodies. It is conclusive because it The affinity distribution or binding of polyclonal antibodies is different As with animal preparations, this may vary from formulation to formulation.

Monoclonal antibodies always have the same affinity, and this affinity is often insufficient, i.e. average or low affinity. Monoclonal antibodies also have a large It is extremely expensive to form and manufacture in large quantities.

In performing immunoassays, the purity and binding strength of the ligand are dependent on the processing time, It is critical to other characteristics of the measurement method, such as accuracy and sensitivity.

Use of immunological complexes for the production of antigens or antibodies bound to solid supports has already been described in European Patent Publications m80.108 and Kan80,109. has been done.

The latter publication states that the immunological complexes formed in vivo are first dissociated and then transformed into a solid state. It is described that analysis can be performed by binding to a carrier. formed in the body Immunological complexes of pathogens are highly heterogeneous mixtures, and in this mixture there are many There are many antibody specificities for many components that exhibit immunological responses.

Furthermore, these immunological complexes involve a large number of biologically important serum proteins, including It is known that serum complement factors are bound to this protein.

The contents of all these immunological mirrors formed in vivo are largely unknown. There is no unit.

Their production in pure form can be achieved, for example, by precipitation with low concentrations of propylene glycol. This is done by methods such as binding CIq or protein A to lees or solid supports. be called.

The purpose of the present invention is to analyze the composition of immunological complexes formed in vivo. Rather, the objective is to provide a sufficiently quantitative immunochemical measurement means.

The immunological precipitate of the present invention has one specific titer. Serum contamination has been identified in many steps during the in vitro manufacture of antibodies and in addition Measurements are made to prevent adhesion of complement factors and the like.

European Patent Publication No. 80.108 describes immunological complexes bound to solid supports. Detection of antigens by body components has been described, and antigens and antibodies are no longer considered immunological. It does not have the form of a complex and is separately bound to the carrier.

In contrast, the present invention provides an incomplete method for immunological precipitates that is different from binding to a solid support. A distinct group of small immunological aggregates with high antibody activity is proposed for dissociation. Served.

This method results in a significant increase in the binding capacity of the surface for co-antigens. Ta.

As a result of the progressive dissociation test, extreme pH values i.e. pH 2,5 and p) 112 ．． Incompletely dissociated immunological aggregates were found near 0, making it difficult for immunoassays to It has been shown that there is an optimum size in relation to the use of

These immunological aggregates are superior to free and all-dissociated antibodies. Indicates the functions provided.

The present invention selects immunological aggregates with respect to both size and average antibody avidity. Describe how to select.

The pH conditions used for dissociation described in the Examples of the present invention may vary from one measurement system to another. The individual p required for the measurement system.

Does not form a general prescription for adjustment.

Animal species and molecular size are important parameters; some immunization methods It always has a low binding property and almost a tendency to form an immunological precipitate, which is optimal for the purposes of the present invention. It is possible to obtain antibodies that rarely have antibodies.

In summary, effective and immunologically distinctive purification of antibodies makes the method more expensive. It is concluded that Therefore, place and ion exchange chromatography Antibody preparations purified by physical/chemical methods such as .

Such formulations do not contain the main component globulin, but contain numerous locations on the solid surface. It still contains 95-99% of the inactive, irrelevant antibody molecules.

In many immunoassays, these inactive antibody fractions are isolated from the solution used. This eliminates undesirable and non-specific adsorption of associated molecules. This therefore means that the background signal increases and reduces the signal to noise ratio.

As already mentioned above, the immunologically distinctive properties of components for use as ligands Purification, if possible at all, is a very expensive operation.

In particular, the immunologically specific purification of antibodies with high affinity and binding properties is extremely This is difficult due to the strong antigen-antibody binding. Cleavage of these bonds occurs in many types of Requires the use of extremely strong dissociation means known to have a denaturing effect on the components. Essential.

In solid-phase immunosorbent systems, insolubilized antigen is often removed during the elution process. Antibodies, on the other hand, lose their activity. This means that the corresponding antibody Adsorbents can be used only a few times or not at all for the purification of means.

In liquid phase systems, dissociated antigens and antibodies are separated by known physical-chemical separation methods. Mutual separation often involves many problems.

The aim of the invention is to overcome such shortcomings and disadvantages of the known methods. , and this purpose, as stated in the preamble of claim 1, involves the use of two ligands. and by the immunological preparation comprising a solid carrier bound to This is achieved by the manufacturing method of immunological preparations as described in the sample, and the above preparations are that the two ligands are an antigen and a corresponding specific antibody; Two ligands are defined in vitro obtained from antigen solutions and animal-derived antibodies. The method for producing the above-mentioned immunological preparation is characterized in that it originates from an immunological precipitate. Antigens and antibodies from defined immunological precipitates are used as antigens and antibodies and that the immunological precipitate is in the form of small and highly active immunological aggregates. It is characterized in that the bond to the solid support is in a partially dissociated form.

This results in a formulation that is simple, inexpensive, and highly advanced for use in immunoassays. This provides a method to obtain highly purified ligands, while at the same time eliminating heterogeneous bond strengths prior to coating. unambiguous binding, independent of the composition of the starting antibody. The reproducible selection of potent antibodies is now possible.

In addition to this, a significant increase in the binding capacity of a surface for specific antigens, on the other hand, Significantly reduces the processing time for epidemiological measurements and increases the accuracy of this measurement method. Melt.

This is due to the increased density of specific ligands on the surface in question.

The present invention will be described in more detail below by describing each step of the measurement method.

Precipitation, which is a molecular complex of an antibody and its corresponding antigen, from a soluble antigen and an antibody. The formation of the immunological precipitate is carried out by a generally known precipitation reaction.

Antigen here refers to proteins, nucleic acids, polysancharides, small molecule antigens, and cells. Antibody refers to an immunological compound or immunoglobulin of a species of animal.

The concept of antibodies includes polyclonal as well as monoclonal antibodies. monoclonal antibodies require the use of two or more clones to achieve precipitation. Frequently requires reactive products.

The optimal reaction is one in which the antigen as well as the antibody is used quantitatively in the formation of the immunological precipitate. It is found at or near the "equilibrium point" where

The optimal ratio between antigen and antibody is determined, for example, by weighing the washed precipitate or nitrogen determined by the quantification of A simple method is to measure the turbidity of milky precipitates, or by visual inspection.

Separation of insoluble immunological precipitates formed in the liquid phase can be achieved by simple centrifugation and This is done by washing several times. The ability of the complex to adsorb cofactors leads to contamination. However, contamination can be reduced by adding a cofactor-consumer prior to the actual precipitation. can do.

Partial blocking of cofactor binding can be achieved by addition of EDTA .

Dissociation of immunological precipitates can be performed at high temperatures, high ionic strength, high pH, low pH, specific ions ( antibodies and competing molecules (e.g. chaotropic ions) and competing molecules. and the corresponding antigen that can partially dissociate the bond between it and its corresponding antigen. It means sound.

This dissociation occurs at high temperatures, at high or low pH, and at specific temperatures such as chaotropic ions. It is carried out by the influence of ions and competing molecules. According to the analysis results, the current For this purpose, dissociation under high pH was shown to be most successful.

At this stage, the selection of small immunological aggregates containing antibodies with high avidity is also possible. It is done.

Preliminary dissociation under relatively mild dissociation conditions ensures that only the weakest antibody binding is dissociated and then after centrifugation at low G values, soluble antibodies, antigens and immunological The aggregate is decanted.

Pelleft consists primarily of a medium of highly associated immunological precipitates; can be partially separated by effective dissociation means and have high antibody activity. It can be used to coat surfaces in the following steps in the form of small immunological aggregates. can.

This process involves the covalent and non-covalent attachment of components, i.e. antigens or antibodies, to a solid support. It consists of being connected by both binding forces.

What these methods have in common is that the physico-chemical environment in which they are carried out provides binding of the antigen and the corresponding antibody to the surface of the immunological assembly in parallel with the antigen and antibody. It is possible to maintain a partially dissociated state for fixation.

Attachment of molecules to solid supports is achieved by both covalent and non-covalent forces. be exposed.

The solid surface can be of a plastic, e.g. polystyrene-like nature; or can be composed of different types of polymeric materials such as cellulose .

Supports can also be based on surface structure or chemically reactive groups introduced onto the surface. Other substances that have the property of being adsorbed or bound to molecules or components based on It can also be made of substances.

Example 1 HPL in serum from female patients (human placenta > lactose) Structure of ELISA that competes with Gen).

This measurement method is based on the relationship between human placental lactogen and sheep anti-HPL-antibody. It is based on the camping of immunological precipitates made.

After camping, HPL, like anti-HPL, is reactive with soluble auxiliary components. . Enzymes are used as indicators.

In the assay, a patient sample containing an unknown amount of HPL is purified and bound to an enzyme. ) is mixed with a certain amount of IPL.

After mixing, a certain volume is placed in a micro titer tray (micr) made of polystyrene. partial dissociation of the titre tray (NUNC immunoplate) The HPL-anti-HPL immunoprecipitate was then transferred to the well coated well.

The enzyme-bound HPL and the patient's liver are then coated with anti-) on the plastic surface. IPL - competes for accessible binding sites to antibodies and binds depending on their relative concentration. The HPL concentration in the combined patient sample is inverse to the insolubilized enzyme activity.

With this technology, the total operating time required for measurements can be reduced to less than 60 minutes. discovered. Reaction times can be further reduced using fully automated equipment. I can do it.

According to a similar measurement method, it is possible to measure 0.5 to 20 micrograms/m1. , the method of the present invention described provides an extremely wide measurement range compared to other methods. Served.

It also provides higher sensitivity than other methods, although this may not be clinically desired. Ru.

The camping operation of the above-mentioned HPL-anti-HPL complex will be described below.

■, Formation of immunological precipitate An antigen mixture consisting of crude HPL-containing placenta extract was obtained as follows.

One part of the placenta was placed in 500 ml of saline buffer containing 0.1 M phosphate at pH 7.4. Homogenization was carried out for 5 minutes in the presence of water.

Solid pieces of tissue were removed by centrifugation at 1000 G for 5 minutes. Cell functional substances etc. was removed by further centrifugation at 50,000 G for 15 minutes. 1O in the final concentrate Added n+M EDTA.

To find the optimal ratio of antigen to antibody, various volumes of antigen were added as shown below. The formulation was mixed with various volumes of antibody formulation to perform a simple flocculation serum reaction.

Garth magnetic 1 2345 Crude placental extract 50 100 200 400 1000 (μl) Single species antibody 1000 400 200 100 50 (μl) Formation of milky precipitate observed The maximum distance between antigen and antibody corresponding to the first glass with a visible precipitate An appropriate ratio was selected.

■0 minutes away Separation of the immunological precipitate from non-reactive material was achieved by four centrifugations (10 (min) and washing with IM NaCl aqueous solution.

■0 selection Pre-dissociation and weak (bound antigen-antibody complexes were dissolved in 0.1M NaO at pH 11.0) Strongly binding antibodies were selected by removal with H-borate buffer.

Separate the undissociated immunoprecipitate by centrifugation at 10,000 G for 5 minutes. did.

Partial dissociation of strongly bound immunological precipitates can be achieved by reducing these precipitates to pH 12.0-1. 2.2 0. IM: Suspend in NaOH-borate buffer for 15 minutes at room temperature. It is done by. Furthermore, pH 11.4 and 0. IM Na0) I-borate When diluted with buffer, the final protein concentration is approximately 1 microgram/++/! Nana Ru. Adjust the pH to 11.6 and leave at room temperature for 15 minutes and the coating mixture is ready. Complete.

■0 surface treatment Polystyrene microtiter tray (NIJNC F 16 module, high binding) It was used. Add 200 microliters of coating mixture to each microtiter well. Add to the water and keep warm at room temperature for 4 to 16 hours in a moisture-proof box. did. The depression was filled with pH 2 with 0.02% Tween 20 added. , 3 in 0.01M citrate buffer, then 0.1% NaN3 and Wash twice with OIM 3x buffered saline solution supplemented with 0.02% Tween 20. Ta.

As a result, the coated microtiter tray was ready for use.

Example 2 The above example describes the configuration of a competitive measurement method.

The advantage of the Campling method is that it can also be used in the sandwich method (double antibody method). Ru. In this assay, the immune activity on the surface, which is bound to the corresponding antibody, is The presence of antigen must be considered.

It is believed that molecule X in serum should be quantified. This molecule is two or several Clarified Fragment XI.

Enzymatically cleaved into X2 and X3. Presumably, these fragments are from known isolates. can be separated from each other by law, so that one specific antibodies may also be obtained.

Open the sandwich method by forming an immunoprecipitate between Xl and anti-Xl. These can then be attached to a carrier. Either X2 or X3 A specific antibody against a specific antibody can also be used as a second labeling agent, which is supplied by combining it with a labeling agent. can be done.

Antibodies against X2 or X3 will not react with insolubilized X1-antigen.

Example 3 Construct both competitive and sandwich immunoassays using current technology; The antigen moiety rather than the antibody was used as the primary ligand. However, the dissociating agent New facts will come to light in cases where antigens are affected, but international investigation reports LLII-2・-1^-+a1i@'IN・PCT/DK87100074

Claims (5)

[Claims] 1. An immunoassay preparation consisting of a solid carrier to which two ligands are bound, and the above-mentioned the two ligands are an antigen and a corresponding specific antibody; defined immunological precipitates obtained in vitro from antigen solutions and antibodies from animals. An immunoassay preparation characterized by being derived from sediment. 2. Antigens and antibodies derived from defined immunoprecipitates are used as antibodies; and the immunological precipitate contains small and highly active molecules during binding to the solid support. Claims characterized in that they exist in partially dissociated form in the form of immune aggregates. A method for producing the formulation according to item 1. 3. The immunological precipitate is purified using commonly known immunologically specific methods. A method for producing a preparation according to claim 2, characterized in that: 4. The immunological precipitate is previously dissociated and the immunological precipitate has weak binding strength. A readily soluble part consisting mostly of free antigen and antibody and a mostly tightly bound part. The latter is separated into poorly soluble parts consisting of tightly associated immune aggregates with is used for subsequent binding to a solid support. is a method for producing the formulation according to item 3. 5. The component coordinated to the solid carrier is defined in claim 1, 2, 3, or 4. immunization of an antigen in a sample, characterized in that said manufacturing method is used as described. Preparation of the preparation according to claim 2, 3 or 4 used for chemical detection Construction method.