JPS604938B2 - Immunological reagents and their use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to immunological reagents that can enhance the range of immunoprecipitation reactions.

More particularly, the present invention relates to immunological reagent solutions that can be utilized in various immunological assay methods. The present invention also relates to immunological assay methods, particularly those involving the reaction of an antibody with an antigen to produce an antibody-antigen complex, the insolubilization of which can be substantially achieved by the use of the reagents of the present invention in the assay method. will be increased. There are numerous immunological assay methods with a wide range of methodologies that require immobilization of as many antigen-antibody antibodies as possible in one step or another in the assay and for different purposes.

For example, insolubilization of fin bodies can be performed using electrophoresis analysis, enzyme assay,
It plays an important role in illustratively important conventional immunological assay techniques such as radioimmunoassay (RIA) and turbidimetric assay, and many studies are developing methods to increase the insolubilization of complexes to improve these assays. directed towards. Typically, insolubilization of the reaction product allows isolated reaction products or unreacted reactants to be analyzed separately to provide meaningful diagnostic assays, as well as unreacted immunological reagents contained in a particular assay. Necessary for isolating immunological reaction products from the body. For example, it is already known for immunological methods, including nephelometric analysis, to dilute the sample with a polyethylene glycol polymer solution before taking the warm layer and densitometry readings of the sample.

Polyethylene glycol improves nephelometry by increasing the concentration of suspended particles, thus improving the analysis, but due to inadequate test range or sensitivity due to an unsatisfactory concentration of suspended particles in the sample. The problem remains that satisfactory analysis of many biological components cannot be performed. A broad object of the present invention is to significantly increase the insolubilization of antibody-antigen molecules beyond that obtained with polyethylene glycol alone, whether nephelometric or other quantitative methods are then used. The object of the present invention is to solve the aforementioned problems by providing improved immunological reagents that are effective in increasing the concentration of suspended particles in a sample to allow analysis of biological components that are not possible. The present invention provides an immunological reagent comprising at least a polyethylene glycol solution, wherein the reagent comprises an aqueous solution of a mixture of polyethylene glycol and another non-ionic surfactant, and wherein the aqueous solution has a concentration of about 3 to 6% by weight during use. %
and within such range from about 0.7 to 1.
Calculated HLB value (hydrophilic-lipophilic balance value) in the range of 7
It is characterized by having the following.

Importantly, for example in immunological assays, "in use"
If not, the aqueous solution may have a mixture concentration of 3% or less and 6% or more, but is nevertheless adjusted prior to use or "in use" to have a concentration within the range of about 3-6%. It is useful for purposes of the present invention to provide a concentration of the mixture that Therefore, by the term "in use" as used in the additional claims, if the concentration range is outside the specified range of 3-6%, as long as it is adjusted to these limits during or before the assay procedure; It is our contention that the mixture includes reagents that do not necessarily need to be initially in the range of about 3-6%. The inventors also provide an immunological assay method comprising a reaction between a second antibody and an antigen to produce an antibody-antigen body, which method - characterized by the use therein of a reagent of the invention which substantially increases the extent of insolubilization of the antibody rust.

Conveniently, the reagents are introduced into the test medium for carrying out nephelometric, enzymatic, nebulization or radioimmunoassays.

For example, when a turbidimetric assay is introduced to analyze biological components in the form of antibody-antigen complexes, the suspended particles of reagents and biological components are first warmed up and then the turbidimetric analysis is performed. be done.

According to this method, the light source is made to pass through the liquid sample so that the light beam is directed through the suspended particles. When these light rays strike particles, they are scattered or diffused at an angle, such as at right angles, from the light source and then received by the photovoltaic cell. This scattered light is directly proportional to the amount of particle concentration and is in turn converted into an electrical signal that is thus accurately measured on the instrument face. An example of a suitable instrument for nephelometric analysis is the Philando Laser Nephelometer PDQTM.
(Hyland serNephelometer
PDQTM Hyland Borat
Amine cofluorocolorimeter (A
minco - FI side mcolorime r, Amer
icanlnstmment Company)
F) (Aminco-Bowman Spectrum
Auto-Analyzer (Auto AM1yzer, Technico) with oto-fluorometer (SPF) and attached fluoronephelometer
rlnstr Yamaments Corporation).

Using these turbidimetric principles and devices, clinical technicians can detect, for example, immunoglobulin 1gG, 1gA, 1gM, transferrin, complement C3, haptoglobin, Q, mono-antitrypsin, 8-lipoprotein, albumin, Q2-macroglobulin, Q. Accuracy of low concentrations of a wide variety of special proteins such as monoacid glycoproteins and various other biological components such as triglycerides, lipoproteins, and ciliated gonadotropins. quantification can be performed.

The most important advantages obtained from the use of the reagent according to the invention are {a} the incubation time is significantly reduced and {b} the large concentration of insolubilized body particles reduces the test area and sensitivity with the resulting improved test area and sensitivity. This is what can be obtained from the warm calendar time of time.

These advantages apply to any immunological assay that benefits from enhanced immobilization of antigen-antibody bodies at some point during the assay. Advantages also apply to immunoassays that rely on the insolubility step of antigen-antibody antibodies at some point during the assay procedure. As previously noted, the aqueous solution of the reagent in use contains about 3-6% by weight of a mixture of polyethylene glycol and a nonionic surfactant;
It should also have a calculated HLB number in the range of about 0.7 to 1.7.

The HLB number is a well-established measure of the hydrophilic-lipophilic balance (hence the "HLB") of a given surfactant.

The HLB system for surfactant identification was developed by Atlas Chemical Company (A
tras Chemical Industries, Inc.
．． ) and the Guide to the Use of Atlas Surfactants and Sorbitol in Cosmetic and Pharmaceutical Products.
SuMactants and Sorbitol
in CosmeticaikePharmaceutic
aIProd (1965), pages 28-36, which is incorporated herein by reference. Each surfactant is assigned an HLB number. The lower the HLB number, the more lipophilic the surfactant, while the higher the number, the more hydrophilic the surfactant. Methods for establishing the HLB value of any given surfactant are well known and include, for example, the Atlas HLB system "The Atlas HLBS".
$tem (Code LD-97)'', published by Atlas Corporation. The HLB values of many surfactants are also widely clarified in the literature, especially those published by the manufacture of the surfactants in question. The HLB value of a surfactant formulation, such as is present in the reagents of the invention, is a function of the concentration of each surfactant in the formulation, thus making the concentration of an individual surfactant the HLB value of the formulation.
Must be taken into account when calculating the B value.

The HLB value of a formulation is calculated by multiplying the specified HLB value of each surfactant present in the formulation by its concentration in the composition in question, according to recognized methods. For example, if the reagent of the invention contains 1% by weight polyethylene glycol (HLB-20) and 30.5% HLB-20,
When containing 3% by weight of nonionic surfactant with B, the HLB value of the reagent is calculated as follows: 0.01
×20 -0.2 0.03 × 30.5 = 0.915 Total 1.115... HLB value of the reagent The HLB value of the specified reagent falls within the range of 0.7 to 1.7 of the reagent of the present invention Based on the amount of each ingredient in the formulation, use the published HLB value of the ingredient in question or use the HLB value determined by the atlas method
Calculations such as those described above can be easily performed using the LB value.

The present invention provides that a mixture of a nonionic surfactant and polyethylene glycol is
~6% by weight and ■ The calculated HLB value of the reagent is simultaneously within the range of 0.7 to 1.7, intended for use in conjunction with polyethylene glycol of a wide variety of nonionic surfactants. It is something to do. In use, the polyethylene glycol-nonionic surfactant mixture is part of the reagent.
%, or the reagent has an HLB of about 0.7 to 4.
If the antigen-antibody compound has a high concentration, it becomes difficult to insolubilize the desired amount of antigen-antibody rust required for a successful immunoassay. Also, if the mixture is at a concentration greater than 6% of the reagent, or if the reagent has an HLB value greater than 1.7, other proteins than the desired antigen-antibody antibody may be insolubilized, thereby increasing the selectivity of the assay. damage and render the results meaningless. Of course, for various reasons, the reagent solution may not contain the requisite 3-6% polyethylene glycol and non-ionic surfactant mixture, or do not have the requisite HLB value of 0.7-1.7. It can be prepared and sold commercially.

Poor storage properties may be one such reason.
However, as noted above, such solutions may, at some point during their use in immunological assay techniques,
or earlier, in the range of 3-6% and about 0.7-1.7
of . Adjustment is required to HLB value. For this reason, the required 3
Reagents that do not contain ~6% of said mixture or HLB values of about 0.7-1.7, but which can be easily adjusted to these values during or before the immunological test procedure, are those reagents. is the concentration and H required during the assay procedure itself.
Even outside of the specific parameters of the LB range are within the scope of the present invention. For example, commercially available reagent solutions have specific parameters of concentration and HLB value of about 3-6% and 0.7-1
．． In order to bring the solution up to a calculated HLB value of 7,
Each requires dilution, concentration, or other adjustment steps before performing an immunological assay.

In such circumstances, commercially available solutions would still fall within the scope of the present invention. In this way, the reagent, e.g.
The mixture can be commercially available at a concentration higher than 6%, which is diluted to a range of 3-6% at some appropriate point during or before the immunoassay procedure. Similarly 0.7-1.7
have a calculated HLB number outside the range of 0.7
It is possible to commercialize reagents with the requirement that the reagent solution is somehow changed at or before some suitable point during the immunoassay procedure to bring about a calculated XLB range of ~1.7. can. In some cases the reagent is diluted to the appropriate level with the antiserum used in the test -- while in other cases it is diluted with the sample, and in some cases with both the sample and the antiserum. be able to.

Importantly, at some point during the immunoassay procedure, at the same time or prior to the universal antigen-antibody reaction, the requisite levels of combined polyethylene glycol and nonionic surfactant of 3-6% and 0.5% are added. A requisite HLB value of 7 to 1.7 must be provided to the reagents of the invention in order to function properly during the assay procedure. The point in the assay procedure at which these essential parameters must be provided can vary depending on the particular procedure involved. For example, for nephelometry, it is convenient to prepare and commercialize the reagents of the invention with polyethylene glycol and nonionic surfactant concentrations greater than 6% and HLB values outside the range of 0.7 to 1.7. It is. Then, immediately before use in nephelometric analysis, the reagents were
Dilute with the antiserum used in the analysis to give a concentration of polyethylene glycol and nonionic surfactant of ~6%, preferably about 4%, and a calculated HLB value ranging from 0.7 to 1.7. The relative proportions of polyethylene glycol and nonionic surfactant in the mixture are highly dependent on the surfactant used and can be varied within wide limits.

Illustratively, the mixture contains about 10-9% by weight polyethylene glycol and 10-9% by weight nonionic surfactant. Preferably, the mixture contains about 15-85% polyethylene glycol and 15-85% nonionic surfactant. One or more different forms or types of polyethylene glycol can be used as the polyethylene glycol component of the mixture.

Generally, the polyethylene glycol polymer used is
about 200 to 10,000, preferably about 4,000 to 600
It has a molecular weight of 0. These materials are, for example,
CARBOWAX 4000 or 600 from Union Carbide
0, Dow Chemical Company
It is available as PEG 4000 or 6000 from 1Company. A molecular weight range of about 4000 is particularly preferred. The nonionic surfactant component of the mixture is
It can be any nonionic surfactant that produces a calculated HLB number in the reagent solution of 0.7 to 1.7, and preferably about 0.7 to 1.3 at a mixture concentration of 3 to 6%.

To illustrate, the HLB value of the nonionic surfactant itself can range from about 10 to 30 or more. A preferred nonionic surfactant is a block copolymer of ethylene oxide and polyoxypropylene. This particular polymer and its preparation are described in US Pat. No. 2,674,61. These block and block copolymers are generally prepared by condensing ethylene oxide with a polyoxypropylene polymer and can be represented by the structural formula below. Day ○ (CH2C ratio ○) a (C Tsuru CHCH20) b (
CH2C&○)cH For purposes of this invention, these block copolymers contain at least 50% ethylene oxide in the molecule, as described in the same patents in U.S. Pat. and a polyoxypropylene hydrophobic group molecular weight of at least 950. Examples of such suitable block copolymers include Wyandotte Chemical Corporation.
There are F-38 and F-68 Pluronic ■ polyols commercially available from ).

F-Shigi contains 80% polyoxyethylene hydrophilic units in the molecule, and 95 polyoxypropylene hydrophobic groups.
It has a molecular weight of 0. F-38 is a preferred nonionic surfactant. F-68 contains 80% polyethylene hydrophilic units in its molecule, but its hydrophobic groups have a molecular weight of 1,750. The total molecular weights of these two Pluronic II polyols are 4750 and 8750, respectively. Pluronic L-125 has also been found to be useful. A further description of these polyols can be found in the Wyandotte Chemical Co. paper "meP1monicGrid, 6th edition"
, which is incorporated herein by reference. For Pluronic nonionic surfactants,
about 20-4% by weight polyethylene glycol and about 8% by weight polyethylene glycol
Mixtures comprising block copolymers of 0 to 6 weight percent ethylene oxide and polyoxypropylene polymers are generally preferred.

A highly preferred reagent solution is about 1 part by weight of polyethylene glycol having a molecular weight of about 4000 and Pluronic F.
Contains about 3 parts by weight of -3 phantom material.

This solution can be prepared in a saline solvent (e.g. 0.9% NaCI) above or below the desired level of 3-6% and then adjusted as needed to the desired level of 3-6%. Ru. The solution is prepared as an 8% solution of the polymeric polymer compound in saline, which is then diluted with antiserum (see Examples 1 and 5) or other suitable diluent prior to use in the immunoassay procedure. The reagent thus diluted contains approximately 1% polyethylene glycol and 3% F-38. The HLB value of 1.115 can be calculated as follows: 1 PEG: 0.01×2○*
=○． 23% F-38: 〇. 〇3×30.5**=
〇． 915 total 1-115* From the literature, the HLB of PEG is 20** From the literature, the HLB of F-38 is 30.5
A wide variety of other non-ionic surfactants are available at concentrations of 0.7-1.
It can also be used to supplement polyethylene glycol in the reagent solution of the present invention if it provides the desired HLB value of 7.

For example, BASF Wyandotte Co., Ltd.
dotte Corporation)*, and can be obtained from
on TetroniC ■ Series No.
nionicSmnectanG”, BASF W scale and
otteBrochure” and U.S. Patent No. 29795
No. 28 (statements of the above-mentioned publications and patents are cited in the specification)
Nonionic surfactants of the Tetmnic ■ series described in
Those classified as 0&1107, 1307 and 1508 have been found to be quite suitable. Tetronik II products are based on ethylene diamine and have the following general formula: The molecular weight of the Tetronik II series can vary from about 1,650 to over 26,000.

A more preferred Tetronic ■ surfactant is about 15,000
Molecular weight in the range of ~27,000 and H of about 20-30.5
It has an LB value. The proportion of polyethylene glycol to Tetronic surfactant in the mixture can vary widely, as described above. Other nonionic systems that are useful are also the pLURAFAC series of products published by BASF-Wyandotte,
Especially pull-luff attack ■17R8 2 balls & D25 A38
and those identified as A39.

Plura Hook ■Products are ``TechnicalDa
taonTypicalPhysical Prope
rties of PLURAFAC ■Nonion
ic Su actans'', BASF Wy
It is a straight chain, primary aliphatic oxyalkylated alcohol as described in detail in ``Andottebrochure''. Preferred Plura Fac ■ products are approximately 10-20 H
It has an LB value and can be used in widely varying ratios to polyethylene glycol. Other suitable nonionic surfactants include the glycerin monostearate and alkylaryl sulfonate systems. The preferred glycerin monostearate is the trademark Arlacel 1
65 (ARLACEL■165) (HLB:11.0)
Atlas Industries, Inc.
Inc. ), while typical useful alkylaryl sulfonate salts are also available from the same company under the trademark G-3
300 (HLB=11.7). The foregoing description of nonionic surfactants is merely illustrative.

The reason for this is that when used in immunological assay procedures approximately 3
A reagent containing a mixture of ~6% polyethylene glycol and a surfactant has a calculated HLB value of approximately 0.7 to 1.7, based on the values of the individual components present in the solution, and is desired. producing the effect of increasing the insolubilization of the desired antigen-antibody complex to the extent desired to improve the assay, without insolubilizing components that are not present or, optionally, without fatally interfering with the assay procedure. This is because the provision of reagents falls within the scope of the present invention. Of course, one or more surfactants can be present with the polyethylene glycol, subject to the requisite mixture concentration and HLB value of the solution.

To select a nonionic surfactant for use in the present invention,
Also, one must use a reagent solution that can provide a clear solution at least when the reagent solution is first used in an immunological assay procedure.

This ensures that the reagents do not interfere with test results or cause unspecified precipitation of biological sample components or biological reagents used in the assay system. The reagent system of the present invention is useful in many conventional immunotherapy systems that, at one time or another, require a step to insolubilize the antigen-antibody bodies generated during the assay procedure by antigen-antibody reactions, for whatever reason. It finds widespread use as an improvement in scientific assay methods.

Such assay methods are well known to those skilled in the art and need not be described in detail here. The applicability and effectiveness of the present invention in these various assay methods, and the details of how to use the reagents of the present invention in such procedures, will be apparent to those skilled in the art from the description of this specification, and in this way, these properties can be understood. Don't repeat yourself too much here. For example, the reagents of the present invention can be used to enhance any system that relies on the precipitation of antigen-antibody bodies to produce a clear superluminescent solution for photoluminescence or colorimetric detection. The reagent can also be used to remove interfering substances found in biological fluids or reagents (eg, lipids, salts, and extraneous proteins) for turbidimetric, enzyme, or other assay systems. This is facilitated by the removal of the reactants from the reaction solution in order to wash and resuspend these reactants. especially,
The reagents of the present invention can be utilized to increase the relative concentration of insoluble antigen-antibody compounds and to increase the test range and sensitivity of a given assay system.

These principles can be applied to quantitative light scattering from immune bodies by turbidimetry, which is currently the preferred embodiment of the present invention. The sardine assay using the reagents of the present invention is particularly useful in the analysis of various immunoglobulins. However, there are other techniques such as, for example, radioimmunodiffusion (RID), enzymatic analysis, and various types of pneumophoresis techniques, such as immunoelectrophoresis (IEP), reverse electrophoresis (CEP), and electroimmunodiffusion (EID).
Other test systems based on precipitating antibodies can utilize these reagents. The reagents of the invention can be used, for example, to enhance immunological assays that rely on the primary antigen-antibody co-precipitation interaction commonly used in radioimmunoassays (RIA).

This enhanced insolubilization property of the present invention can be utilized in a variety of ways to attach antibodies or antigens to inert particles for use as carriers in RIA, nephelometric, and crab light assays in ways that are well understood by those skilled in the art. Various radioimmunoassay techniques have been employed to quantify relatively small concentrations of biological components found in body fluids.

To produce a Labelert immunizing antigen-antibody fin, an isotopically labeled antigen or antibody is reacted with the corresponding antigen or antiserum. These collar bodies are then usually combined with an insoluble carrier,
It must be made insoluble by precipitation reagents or other known techniques. Free or unreacted labeled antigen or antibody can be removed by washing techniques. The radioactive concentration in the precipitated fin bodies is then quantified by gamma method or liquid scintillation counting. An example of an instrument used for this type of analysis is an auto gamma counter (Auto Gamma Counter).
oGammaCohadater,NhadalearChicag
o, Inc. ). The reagents of the present invention are useful in enhancing the essential insolubility phase of the complex, thus improving analysis. Example 7 details the use of reagents of the invention in radioimmunoassay procedures. Enzyme technology also
It is employed to quantify small concentrations of biological components found in body fluids.

The enzyme-labeled antigen or antibody is reacted with a specific antibody or antigen to generate an immunolabeled antigen-antibody body.
These collars are made insoluble by insoluble carriers, precipitation reagents, or other techniques. Free or unreacted enzyme-labeled antibodies or antigens are removed by washing techniques. The bound or reacted enzyme can react with a suitable substrate to produce a colored or luminescent solution that can be measured spectrophotometrically or spectrophotometrically. An example of an instrument useful for this purpose is the Beckman DB Spectrophotometer.
kman DB Spectrophotometer
, Beckman Instment Company)
and Aminco-BbWman Specification
Fluorometer, Anericanlnst
mentCompany). The reagents of the present invention are also useful for enhancing the essential cell insolubility step, thus improving analysis. The usefulness of reagents in nephelometric analysis has been studied in detail as described above, and in Example 5
Further exemplified in

It is now clear to those skilled in the art that the improved immunological reagents of the present invention have versatile utility in the field of immunological assay procedures.

Using improved reagents, clinical technicians can use immunoglobulin 1gG, 1gA, 1gM, transferrin,
Complement C3, paptoglobin, Q, anti-trypsin, 8-
lipoproteins, albumin, Q2-macroglobulin, Q, monoacid glycoproteins, and various other biological components such as triiodothionine (T3), thyroxine (T4), triglycerides, ciliated gonadotropin lipoproteins, and the like. Accurate quantification of many specialized proteins over a wide range of concentrations can be performed, as are many other components whose quantification would benefit from the enhanced immobilization effect produced by the present invention. In most applications that find utility with the present invention, the desired biological test components or components are added to the aqueous reagent solution of the present invention, e.g.
~25°C) for a predetermined period of time, such as about 1 hour, and then read on suitable instrumentation, such as a nephelometer in the case of nephelometric analysis.

Sample results are compared to control samples to quantify unknown concentrations. Next, examples of the present invention will be described.

Example 1 Polyethylene glycol 25 with a molecular weight of 4000
A reagent solution was prepared by adding parts by weight to parts by weight of Pluronic F-3875, and then the mixture was dissolved in normal saline (0.9%
Up to 8% by weight of the mixture was dissolved in NaCl).

This solution can be diluted, for example with antiserum, to a 4% mixture concentration (HLB ratio - 1.115) as in step 3 of Example 5 and used directly in the immunoassay procedure, or it can be used until immediately before use. It was possible to retain 8% of the morphology. Example 2 Polyethylene glycol with a molecular weight of 40
Example 1 was repeated except that an equivalent amount of 0.00 material was substituted.

Example 3 Example 1 was repeated except that an equivalent amount of F-plaque was replaced with Pluronic F-68.

Example 4 F-38 was added to Tetronic 707, 90 &
1107, 1307, 1508
R & 2 balls & D2 fu A Satoshi and A39: Pluronits 4k L125, Aracell 165; and G-300
Example 1 was repeated except that 0 was substituted.

Example 5 The immunological reagents prepared in Examples 1-4 were used in separate turbidimetric assays for immunoglobulins (1 gA, 1 gG, 1 M), complement C3, and transferrin, and each assay was performed as follows. 1:1 Control standards for each of the biological components (of previously known assays) were added 1:10 in saline.
Diluted to 0.

2 unknowns were then similarly diluted 1:100 in saline.

3 Pre-diluted antisera against 1gG, 1gM, 1gA, C3 and transferrin were diluted 1:2 with 8% immunological reagents from Examples 1, 2, 3 or 4, respectively (as the case may be) and Mixed by inversion to yield a 4% concentration of polyethylene glycol and nonionic surfactant in solution and a calculated HLB between 0.7 and 1.7 in all cases.

4 0.45r Millipore filter (Millipore
fiMr).

5 Appropriately labeled blanks, controls #1, #2,
A series of test tubes (IQ port x 75 side disposable culture tubes) were prepared for #3, #4, #5, #6 and unknown.

6. To each tube was added 1' of the diluted mixture of antiserum and reagents prepared in step 3.

7 Added 25r to appropriate tubes for 1gG, 1gA and transferrin, control and unknown diluent (1gG, 1gA and transferrin).
100 for gM and C3).

8 corresponding to a suitable blank tube containing 25 or 100 A of the brine.

9 Mix the test tube by inverting it and then bring it to room temperature (20-25
q0) for 1 hour.

10 Sample blanks were prepared as in step 3 except that (optionally) the 8% solution was diluted with saline instead of antiserum. Adjusted by using.

Blanks were placed in the same labeled tubes as in the previous steps (5 and 6). 11 Identical control and sample volumes were added to each tube as before (step 7).

12 Laser/nephelometer/PD all tubes
QTM (Laser Nephelometer P
The relative % light scattering was read on a DQTM (Hyland Laboratories).

13 The blank was read and electronically subtracted from the reaction value by the meter.

14 Control results were plotted on linear graph paper as relative % light scattering versus control concentration.

15 The unknown value was determined by reading from the control curve.

The immunological reagents of the invention used in this example exhibited substantially greater antigen-antibody keying, greater linearity over a wide range, and greater sensitivity than reagents containing only polyethylene glycol.

Example 6 In this example, experiments are performed to further support that the reagent obtained in Example 5 above exhibits wide range linearity and great sensitivity.

Solutions of polyethylene glycol alone, a nonionic surfactant alone, and a combination of polyethylene glycol and a nonionic surfactant dissolved in an aqueous salt solution were prepared.

Each solution was then subjected to the following tests. 'a' The solution was diluted to a 1:1 volume per antiserum containing the optimal concentration of specific antibodies against the antigen being tested.

As a result, in the case of the mixture of polyethylene glycol and nonionic surfactant of the present invention, a solution containing about 3 to 6% by weight of the mixture and having an HLB value of 0.7 to 1.7 is obtained;
In the case of polyethylene glycol alone or nonionic surfactant alone, * indicates a solution containing sufficient polymer to equal the total amount of polymer in the mixture of polyethylene glycol and nonionic surfactant of the present invention. Become. {
Solution 25 of the solution containing a known level of the desired antigen was mixed with the previous solution W of 1 bird.

This was repeated for several antigen solutions with varying known antigen levels. Blanks were prepared in the same manner except that no antigen was added. 'c' Next, the obtained solution 'b} and the blank were transferred to the laser nephelometer PDQ described above.
TM and incubated at room temperature for ten minutes.

'd} The % light scattering of the blank and solution was measured and recorded.

The values taken were automatically corrected against the blank. If % light scattering was read more than once for each sample, it was averaged. As a result, numerical values of various nonionic surfactants were determined.

The experimental results are shown in Tables 1-5 and Figures 1-5. Each data item represents the range of insoluble antigen-antibody bodies measured by the range of light scattering observed with a nephelometer. Table 1 shows that the polyethylene glycol of the present invention is
% by weight and 3% by weight of Pluronic F-38,
A solution with a total polymer content of 4% by weight is shown.

．． Table 1 First, the insolubilization range of the antigen-antibody compound produced by 1% by weight polyethylene glycol (PEG) alone was measured over a wide range of concentrations {1'.

Next, for the same range of 1 gG concentration, the insolubilization range of the antigen-antibody body produced by 3% by weight of Pluronic F-Shigeru alone was measured. The measurements of '11 and {2) were then added together to make a 4 wt.% mixture of these two polymers, 1 wt.% polyethylene glycol and 3 wt.% Pluronic F-38. 3'.
Next, the insolubilization range of the antigen-antibody antibody produced by the mixture of 1% by weight polyethylene glycol and 3% by weight Pluronic F-38 of the present invention was actually measured.
4'.

Therefore, when comparing the calculation side 3' and the actual measurement value [4}, the actual measurement value '41 increases linearly to a greater extent. As those skilled in the art will appreciate, it is significant that the relationship between % light scatter and concentration of the antigen or immunospecies being analyzed is linear.

The larger the linear range, the wider the detection range of the assay. Thus, the presence of the 1% by weight polyethylene glycol and 3% by weight Pluronic F-total combination mixture of the present invention during the immunoreaction significantly improves the immunodetection range of the assay. In particular, in the case of polyethylene glycol alone [1] and Pluronic F-spot alone ■, the detection value is small even at 9/d' of 1 g G37.5, but with the mixture ■ of the present invention, it can be detected with 1 g GIO female 'd'. Only a small amount is required. Furthermore, since the average light scattering percentage of the nephelometer can be increased to a larger value, there is an advantage that the sensitivity and reproducibility of the assay can be improved. Similarly, different combinations of polyethylene glycol and other polymers are shown in Tables 2 to 5 and Figures 2 to 5.

In Table 2, Table 3, Table 4, Table 5, and Tables 2 to 5, similar to Table 1, the measured value [4] gave a more linear result than the calculated value '3'.

This is particularly noticeable in the 9/9 range where 1 gG is 600 to 1200. Example 7 This example describes the use of immunological reagents of the invention in a radioimmunoassay procedure for the quantification of human thyroid stimulating hormone (HTSH).

A sample of the immunological reagent of the present invention was prepared by mixing 8.4' of a 5% solution of polyethylene glycol in 0.9% salt aqueous solution with 6% Pluronic F-hydrogen nonionic surfactant 20%. .

The radioimmunoassay was performed as follows: Rabbit anti-daily TSH of 0.060' absorbed in human ciliary gonadotropin (HCG) was injected at various strengths of 0.200'.
HTSH standard.

0.050' phosphate buffer, pH 7.4, was then added to the mixture, and the mixture was then incubated at 37°C for 2 hours.

At this point, 0.100' labeled 1125'
of HTSH was added and the mixture was further flooded at 37° C. for 3 hours. The previously prepared reagent of the invention 1.0 was then added and the mixture was incubated at room temperature for 1 hour. Due to dilution of the reagents upon addition to the mixture, the concentrations of polyethylene glycol and F-38 were 1.4 and 4. a wt% to 1 and 3%, respectively. mix 100
Centrifuged at 0x9 for 10 minutes. If washing of the centrifuged solids is required, the wash solution should be at the same concentration as the reagent of the invention when the reagent was first used in the assay, i.e. 1.
% polyethylene glycol and 3% F-38. The supernatant solution was decanted and the precipitate counted. This procedure is repeated for a variety of different HTSH standards and a standard curve is obtained by plotting the counts in the various precipitates versus the concentration of the corresponding HTSH standard. Once the standard curve is obtained, the assay is performed on the unknown sample using the procedure described above, except that the HTSH standard is replaced by the test sample.

HTSH levels in samples are easily quantified from the location of precipitate counts on a standard curve. Use of the reagent solution of the present invention increases the range of precipitates produced over that obtained from reagents using only polyethylene glycol and results in improved radioimmunoassays. Comparative Example 4% by weight of Pluronic F-3 alone and 4% by weight
The effect of polyethylene glycol alone on antigen-antibody insolubilization was compared.

The results are shown in Tables 6 and 7 and Figures 6 and 7. Table 6 Table 7 From this data, it can be said that 4% by weight of Pluronic F-Shii alone is better in solubilization than 4% by weight of polyethylene glycol alone.

In both Tables 6 and 7, Pluronic F-spots have better linearization of data at skin p/d' of 600 to 1200. Thus, for example, using an aqueous solution containing about 4% by weight of a block copolymer of ethylene oxide and polyoxypropylene polymer, suitable turbidimetric assay results can be achieved without the presence of polyethylene glycol.

This result is unexpected since polyethylene glycol is generally considered to be the best. However, as mentioned above, a mixture of the block copolymer of the present invention and polyethylene glycol is preferred in order to obtain optimal results, since a more linear graph can be obtained.

[Brief explanation of the drawing]

Figures 1 to 5 show each 1gG level (female/
d′) is a graph showing measured values and calculated values of light scattering %. Figures 6 and 7 show 1gG of 4wt% Pluronic F-38 alone and 4wt% polyethylene glycol alone.
It is a graph showing the measured value of the average light scattering % at the level (skin 9/d'). Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

Claims (1)

Claims: 1. An immunological reagent comprising at least a polyethylene glycol solution, said reagent comprising an aqueous solution of a mixture of said polyethylene glycol and another non-ionic surfactant, said aqueous solution being used, 6% by weight of said mixture and having a calculated HLB value (hydrophilic-lipophilic balance value) within such range of about 0.7 to 1.7. target reagent. 2. Even if the concentration of the mixture is not initially in the range of 3 to 6%, the aqueous solution contains an antiserum in order to adjust the concentration within the range. The immunological reagent according to item 1. 3. The nonionic surfactant is (a) a block copolymer of ethylene oxide and polyoxypropylene, (b) a linear primary aliphatic oxyalkylated alcohol, (c) glycerin monostearate, (d) an alkyl Claim 1 consisting of an aryl sulfonate or (e) a polyol as described in U.S. Pat. No. 2,979,528.
The immunological reagent according to item 1 or 2. 4. The polyethylene glycol has a molecular weight of about 4000 to 6000, and the nonionic surfactant is composed of a block copolymer of ethylene oxide and polyoxypropylene,
3. Immunological reagent according to claim 1 or 2, characterized in that the HLB value of the solution is comprised between about 0.7 and 1.3. 5. Claim 1, characterized in that the aqueous solution contains about 20-40% by weight of polyethylene glycol and about 80-60% by weight of a block copolymer of ethylene oxide and polyoxypropylene polymer. or the immunological reagent according to item 2. 6. The concentration of said mixture is about 4% by weight, and said mixture comprises about 25% by weight of polyethylene glycol and about 75% by weight of said block copolymer. Immunological reagents as described in Section. 7. In an immunoassay method involving a reaction between an antibody and an antigen to produce an antibody-antigen complex, polyethylene glycol and other and a nonionic surfactant, said aqueous solution containing in use about 3 to 6% by weight of said mixture and within said range a calculated HLB value in the range of about 0.7 to 1.7. An immunological assay method characterized by using an immunological reagent consisting of at least a polyethylene glycol solution having a hydrophilicity-lipophilicity balance value. 8. The immunological assay method according to claim 7, wherein the reagent is used to perform a turbidimetric, enzymatic, electrophoretic, or radioimmunological assay. 9. Incubating said antigen, antibody and reagents to generate said antibody-antigen complex prior to performing nephelometric analysis by performing light scattering measurements that are a function of minute concentrations of said antibody-antigen complex. An immunological assay method according to claim 7, characterized in that: 10 The antibody-antigen complexes of the insolubilized immunoglobulins are prepared by using the reagents and the antibodies and antigens in the form of immunoglobulins prior to performing nephelometry by performing light scattering measurements that are a function of minute concentrations of the insolubilized immunoglobulins. Claim 7 characterized by incubating to produce
Immunological assay method described in Section.