DK150809B - REAGENT CONTAINING POLYETHYLENE GYLOL FOR IMMUNOLOGICAL EXAMINATIONS - Google Patents

Description

in 150809

The invention relates to an immunological reagent of the kind set forth in claim 1.

There are a large number of immunological assessment methods based on widely different principles, which at one or the other stage of the study and for different purposes make it necessary to precipitate as much as possible of the antibody-antigen complex. For example, precipitation of the complex plays a significant role in such usual immunological assessment methods as electrophoretic analysis, one-zymatic assessments, radioimmunological assessments (RIA = "Radio ^ mmunoAssays") and nephelometric assessments, and much work has been done to develop means to increase the precipitation of complexes in order to improve these methods of investigation. Usually, the complex precipitate is required to isolate the immunological reaction product from the unreacted immunological reagents included in the particular study method, whereby either the isolated reaction product or the unreacted reagents can be analyzed separately to obtain 20 a meaningful diagnostic assessment.

It is known by an immunological evaluation method comprising e.g. a nephelometric analysis, to dilute samples with a solution of polyethylene glycol polymer, before both the incubation of the sample and the measurement on the nephelometric instrument. Admittedly, the use of polyethylene glycol improves nephelometric analysis by increasing the concentration of suspended particles, but the problem remains that a satisfactory analysis on many biological materials cannot be performed due to an insufficient test area or insufficient sensitivity due to an insufficient concentration of suspended particles in the sample portion.

It is an object of the invention to resolve the above-mentioned test by providing an improved immunological reagent which results in a significant increase in the precipitation of antibody-antigen complexes beyond that which has been obtained only by the use of polyethylene glycol. and thus an increase in the concentration of suspended particles in the sample portion, so that it is possible to analyze biological components which have not been able to be analyzed alone using polyethylene glycol, regardless of whether nephelometric or other evaluation methods are used.

The stated object is achieved by means of a reagent which according to the invention is composed as set forth in the characterizing part of claim 1.

Advantageously, the reagent can be introduced into the sample medium for performing a nephelornetric, enzymatic, electrophoretic or radioimmunological assessment.

For example, when If a nephelometric assay method is used to analyze biological constituents in the form of antibody tigen complexes, an incubation of the reagent and the suspended particles of the biological constituents is performed first, after which the nephelometric analysis is performed. In this method, rays from a light source are passed through a liquid sample port, directing the light rays through the suspended particles. When the rays hit the suspended particles, they are scattered or diffused at any predetermined angle, from the light source, and received by photocells. This scattering light is converted into an electrical signal which is directly proportional to the particle concentration, which is then expressed on the scale disk of a measuring instrument.

30 Examples of instruments that can be used for nephelometric analyzes include the following: Hyland Laser Nephelometer PDQ® (Hyland Laboratories), Aminco-Fluoro Colorimeter (American Instrument Company), Aminco-Bowman 3 150809

Spectrophotofluorometer (SPF), and Auto Analyzer II with Fluoronephelometer (Technicon Instruments Corporation) attached.

Using these nephelometric instruments and principles, the clinical technician can make accurate determinations of small concentrations of a number of widely different specific protein substances, e.g. the immunoglobulins IgG,

IgA, IgM, transferrin, complement C3, haptoglobin, αβ-antitrypsin, β-lipoprotein, albumin, β-macroglobulin, αβ-acid glycoprotein, and various other biological components such as e.g. triglycerides, lipoproteins, and human chorionic gonadotropins.

The main advantages obtained by using the reagent of the invention are a) that the incubation times are significantly shortened, and b) that a higher concentration of precipitated complex particles is obtained at shorter incubation times and, as a result, an expanded study area and greater sensitivity.

These advantages are obtained by any immunological assay method which can be improved by increasing the precipitation of the antibody-antigen complex at some stage of the study. The benefit is also obtained by immunological assay methods, which are based on a step of precipitating antibody-antigen complexes at some stage during the assay procedure.

As set forth in claim 1, in addition to containing about 3-6% by weight of the mixture of polyethylene glycol and the nonionic surfactant, the aqueous reagent solution should also exhibit a calculated HLB value of between about 0.7 and about 1.7.

The 30 HLB value is a well-established measure of the hydrophilic-lipo-file balance (hence the term "HLB") for a particular surfactant. The HLB system for the identification of surfactants has been developed by Atlas Chemical Industries, Inc., and is further described on pages 28-36 of the Atlas publication entitled "Guide to the Use of Atlas Surfactants and Sorbitol in Cosmetic and Pharmaceutical Products (1965). Each surfactant is provided with an HLB number. A low HLB number means that the substance is relatively lipophilic ("happy with oil"), while a high HLB number means that the substance is relatively hydrophilic ("happy with water"). The method of determining the HLB value of any particular surfactant is well known and is described in e.g. an Atlas publication entitled "The Atlas HLB System" (Code LD-97).

The HLB value of a mixture of surfactants, as found in the reagent of the invention, is a function of the concentration of each surfactant in the mixture, and HLB value must therefore be taken into account when calculating the mixture's HLB value. the values of the individual surfactants. In accordance with publicly known and recognized methods, the HLB value of the mixture is calculated by multiplying the HLB value of each surfactant in the mixture by its concentration in the particular substance mixture, and then the values thus obtained are added. For example, if the reagent of the invention is e.g. had a content of 1 wt% polyethylene glycol (HLB = 20) 25 and 3 wt% of a nonionic surfactant with an HLB value of 30.5, the HLB value of the reagent can be calculated as follows: 0, 01 x 20 = 0.2 0.03 x 30.5 = 0.915 Sum 1.115 (reagent HLB value)

In order to determine whether a mixture's HLB value is within the range prescribed for the reagent of the invention between 0.7 and 1.7, a calculation of the kind shown above can easily be made on the basis of the amount of each component. in the mixture and using HLB values, which can either be published for the substance in question or found using the Atlas method.

Within the scope of the invention, a variety of widely different nonionic surfactants can be used with polyethylene glycol, provided that 1) the content of the mixture of nonionic surfactant and polyethylene glycol is between 3 and 6% by weight at the time of the reagent. 2) that the calculated HLB value of the reagent at the same time is between 0.7 and 1.7.

If at the time of use the mixture of nonionic surfactant and polyethylene glycol constitutes less than 3% by weight of the reagent, or if its HLB value is less than about 0.7, it becomes difficult to precipitate the desired amount of antibody-antigen complex to the extent required for the successful completion of the immunological assessment. On the other hand, if the mixture constitutes more than 6% by weight of the reagent or its HLB value is greater than about 1.7, other proteins are precipitated in addition to the desired antibody-antigen complex, thereby destroying the discriminability of the assay so that the results are not going to measure the desired sizes.

The relative proportions of polyethylene glycol and nonionic surfactant in the mixture can be varied within wide limits, which largely depends on the surfactant used. Eg. For example, the mixture may contain between 10 and 90% by weight of polyethylene glycol and between 90 and 10% by weight of nonionic surfactant. Preferably, the mixture contains between about 15 and 85% polyethylene glycol and between 85 and 15% non-ionic surfactant.

For the polyethylene glycol content of the mixture, one or more different types or forms of polyethylene glycol may be used. The polyethylene glycol polymer used generally has a molecular weight of between about 200 and about 10,000, preferably between about 4,000 and about 5,000. These materials are commercially available, e.g. in the form of

CARBOWAX 4,000 or 6,000 from Union Carbide, or PEG

4,000 or 6,000 from the Dow Chemical Company. The molecular weight range of about 4,000 is particularly preferred.

The nonionic surfactant component of the mixture can be any nonionic surfactant which will produce in the reagent solution an HLB value of between 0.7 and 1.7, preferably between about 0 , 7 and about 1.3 at a concentration of the mixture between 3 and 6%. For information, the HLB-15 value for the nonionic surfactant itself may be somewhere in the range of 10 to 30 or more.

A particularly preferred nonionic surfactant is a block polymer of ethylene oxide and polyoxypropylene. This particular polymer, and how it is made, is disclosed in the disclosure of U.S. Patent No. 2,674,619. These block polymers are generally prepared by condensing ethylene oxide with polyoxypropylene polymer, and may be denoted by the following structural formula: HO (CH2CH2O) a (CH3CHCH2O) b (CH2CH2O) c H.

Preferably, for use with the invention, these block polymers contain at least 50% ethylene oxide in the molecule and exhibit a polyoxypropylene hydrophobic base molecular weight of at least 950, similar to those described in U.S. Patent Nos. 3,450,502, 3,577,522, and 3,590. 125th

30 Examples of such suitable copolymers are those polyols which are sold under the terms F-38 and F-68 PLURONIC® by Wyandotte Chemicals Corporation. F-38 contains 80% of polyoxyethylene hydrophilic units in the molecule and and the polyoxypropylene hydrophilic base has a molecular weight of 950.

As a non-ionic surfactant, F-38 is particularly preferred.

F-68 also contains 80% polyoxyethylene hydrophilic units 5 in the molecule / but the hydrophobic base has a molecular weight of 1,750. The total molecular weight of these two PLURONIC® polyols is 4,750 and 8,750, respectively. PLURONIC® L-125 has also been found to be usable. A more detailed description of these polyols can be found in the bulletin "The Pluronic Grid", 6th ed., 10 from Wyandotte Chemical Corporation.

When using PLURONIC non-ionic surfactants, it is generally preferred to use a mixture consisting of about 20-40% by weight polyethylene glycol and about 80-60% by weight block copolymer of ethylene oxide and 15 polyoxypropylene polymer. .

A preferred reagent solution contains about one part by weight of polyethylene glycol having a molecular weight of about 4,000 and about three parts by weight of the Pluronic F-38 material. The reagent solution contains approximately 1% polyethylene glycol 20 and 3% F-38. The HLB value of the solution can be calculated as follows: 1% polyethylene glycol: 0.01 x 20+ = 0.2 3% F-38 0.03 x 30.5 ++ = 0.915 1.115

Many widely different nonionic surfactants can be used with the polyethylene glycol in the reagent solution of the invention, provided that at a mixture concentration of between 3 and 6%, they provide an HLB value in the desired range between 0.7 and 1. 7th For example, the TETRONIC® series of non-ionic surfactants, + in the literature, specifies the HLB value of polyethylene glycol as 20 ++ in the literature, the HLB value of F-38 is stated as 30.5 8 150809 available from BASF Wyandotte Corporation and is more particularly, in this company's brochure entitled "Technical Data on Tetronic® Series Nonionic Surfactants" and in the specification to U.S. Patent No. 2,979,528, found to be suitable, in particular those of TETRONIC® 707, 908, 1107, 1307 and 1508. The products designated by TETRONIC® are based on ethylenediamine and have the following general formula:

H (C2H4 °> y (C3H60) χχ ^ <C3H6 °> x (C2H40> yH

N-Ch.-CH_-N

H <C2H40) y (C3H60) x XV1 x <C2H40) yH

10 The molecular weight of the TETRONIC® series fabrics can range from about 1,650 to over 26,000. The most preferred surfactants of the TETRONIC® series have molecular weights ranging from about 15,000 to about 27,000 and HLB values of about 20-30.5. The ratio of polyethylene glycol 15 to TETRONIC® surfactant in the mixture can - as discussed above - be varied within wide limits.

Another group of usable non-ionic substances is the PLURAFAC® product series, also marketed by BASF Wyandotte, in particular those designated PLURAFAC® 20 17R8, 25R8, D25, A38 and A39. The PLURAFAC® products are straight-chain, primary aliphatic oxyalkylated alcohols, which are further described in BASF Wyandotte's brochure entitled "Technical Data on Typical Physical Properties of PLURAFAC® Nonionic Surfactants". The preferred PLURAFAC® products 25 have HLB values of about 10-20 and can be used in widely varying polyethylene glycol mixing ratios.

Other suitable nonionic surfactants include glycerol monostearate. A preferred glycerol monostearate can be obtained from Atlas Industries Inc. under the trade name 30 ARLACEL® 165 (HLB = 11.0).

15080S

9

Of course, more than a single nonionic surfactant may be present with the polyethylene glycol, provided the mixture has the prescribed concentration and the solution has the prescribed HLB value.

In order to select a nonionic surfactant for the practice of the invention, such a substance should be selected which is also capable of ensuring that the reagent solution is ready at least at the time the reagent solution is first used in the immunological evaluation process. The purpose of this is to ensure that the reagent does not interfere with the study results or cause a non-specific precipitation of the components of the biological sample portion or the biological reagents used in the evaluation system.

The reagent system of the invention finds an extensive application for improving a large number of conventional immunologic assessment methods which at some point - for whatever purpose - require the execution of a process step involving precipitation of the antigen-antibody complex. which is formed during the evaluation process by the antigen-antibody reaction. Such methods of assessment are well known to those skilled in the art, and therefore do not need to be discussed further in this specification. The utility and utility of the invention in these various methods of assessment, and the details of such methods, will be readily apparent to those skilled in the art upon reading this disclosure, and it is therefore not necessary to provide exhaustive repetitions of these details and procedures of this disclosure. The reagent of the invention may e.g. is used to enhance the effect of any system based on precipitation of antibody-antigen complexes to form a clear supernatant for fluorescence measurement or colorimetry. The reagent may also be used to remove disruptive substances contained in biological fluids or reagents (e.g., fats, salts, or foreign protein substances) for nephelometric, enzymatic or other assessments. This is accomplished by removing reaction participants.

In most of the areas where the invention can be applied, the biological component (s) to be tested is mixed with the aqueous reagent solution of the invention, after which the mixture is incubated for a predetermined period of time, e.g. about one hour at room temperature (20-25 ° C), after which measurements are made by means of 10 instruments, e.g. a nephelome.ter when it comes to a nephelometric analysis. The results of the measurements on the sample portions are compared with the measurement results from reference portions, so that the unknown concentration can be found.

The invention will now be elucidated in more detail by a series of 15 experimental results.

The experiments were performed to compare differences in the degree of precipitation of antigen-antibody complex, obtained in an immunological reaction performed in the presence of (1) the combination of the invention of polyethylene glycol 20 (PEG) and nonionic surfactant, (2) PEG alone, and (3) the nonionic surfactant alone.

From the data obtained from PEG alone and the nonionic surfactant alone, the expected effect of the combination of PEG and nonionic surfactant was calculated and then compared with the actual observed effect of the combination of PEG and nonionic surfactant. to determine whether the observed effect was consistent with the calculated (or expected) effect or not.

The experiments were carried out as follows:

Solutions of PEG alone, nonionic surfactant alone and the combination of PEG and nonionic surfactant were prepared by dissolving PEG and / or nonionic surfactant in brine. Each solution was then subjected to the following series of experiments: (a) the solution was diluted in a 1: 1 ratio with an anti serum containing the optimal concentration of specific antibody for the antigen to be evaluated; There was thus obtained a solution which, in the case of the combination according to the invention, contained 3-6% polymeric solids and had a HLB value of 0.7-1.7, and which in the case of solutions with PEG alone or nonionic surfactant substance alone contained sufficient polymer in the tested combination of the invention.

(b) 25 µl of a solution containing a given antigen 15 at a known level was mixed with one ml of solution according to (a), and this was repeated for a number of antigen solutions in which the known antigen level was varied ; control portions were prepared in the same way, but without the addition of antibody.

(C) The solution of (b) and the control portion were then placed in a Laser Nephelometer PDQ®, an instrument sold by the Hyland Division of Travenol Laboratories, Inc. of Costa Mesa, California, USA, and then incubated for ten minutes. at room temperature.

(D) The percentage light scattering of the control portion and solution was then observed and recorded. The instrument readout function automatically compensated for the control portion. In the cases where more than one reading of the light scatter was taken for each sample, the 30 results were converted to average.

A large number of different nonionic surfactants have been tested. The test results are shown in subsequent Tables 1-6. Each data set shows the amount of precipitated antigen-antibody complex as measured by the light scattering degree observed with the nephelometer. Certain data from each table are shown graphically in FIG. 1-6 to make the test results more clear, each figure having the same number as the table in question.

Taking Table 1 as an example, the experiment can be explained as follows: Table 1 was set up to examine the following polymer combination which is within the scope of the invention: 1 wt% PEG 3 wt% PLURONIC F-38 10 or a total polymer weight percent of 4.

The degree of precipitation of antigen-antibody complex, generated over a wide range of IgG concentrations of 1% PEG alone, was first measured nephelometrically as expressed by the percentage light scatter. These data are listed in column (1) of Table 1. Subsequently, measurements of the precipitation rate of antigen-antibody complex produced over the same range of IgG concentrations of 3% PLURONIC F-38 were made. These data are listed in column (2) of Table 1. The result, which could logically be expected by combining 1% PEG with 3% PLURONIC F-38 to form a mixture of a total of 4% of the two polymers, was then calculated by adding this data in columns (1) and (2).

The data thus obtained represent the expected or calculated result and are listed in column (3) of Table 1.

The actual degree of precipitation of antigen-antibody complex, produced by the mixture of 1% PEG and 3% PLURONIC F-38, was then determined by experiment. These data are listed in column (4) of Table 1. The expected results (in column (3)) were then compared with actual results 30 (in column (4)) to see if they matched or not.

This comparison is best shown graphically, and for this purpose the data from columns (3) and (4) of Table 1 are graphically shown in FIG. First

In view of FIG. 1, it could immediately be seen that the actual data from the measurements on the mixture of the two polymers 5 differ substantially from the expected results as based on the measurements on the individual polymers.

As to the expected curve (corresponding to data in column (3)), it is obvious that no increase in the precipitation of antigen-antibody-10 complex would be expected in the range 0-37.5 mg IgG / dl, with an increase could only be expected in the range of 37.5-75 mg IgG / dl. The reason for this is evident by examining the data in columns (1) and (2) from which the expected curve was plotted. These data show that the curve for 1% PEG remains essentially flat over the entire range of 0-75 mg IgG / dl, whereas the curve of 3% PLURONIC F-38 is flat only in the range of 0-37.5. Since both the curve for 1% PEG and the curve for 3% PLURONIC F-38 are flat in the range 0-37.5, one would hardly expect the curve for 1% PEG and 3% PLURONIC F-38 to be anything other than flat in 20 this area. Nevertheless, the actual results (column (4)) surprisingly show that this is definitely not the case. The actual curve is only flat in the range of 0-10 mg IgG / dl; then it begins to rise sharply in the range of 10-37.5, where the expected curve is precisely flat.

The significance of these results is that the actual curve of the polymer blend exhibits a greatly increased linearity or curvature compared to the expected curve.

As will be appreciated by those skilled in the art, a linear relationship between the percent light scatter and the concentration of the antigen or immune substance under investigation is of significant importance for a meaningful diagnostic assessment. And the larger the linearity range, the wider the rating range is. In FIG. 1 it appears that the presence of the polymer mixture of 1% PEG

14 150809 and 3% PLURONIC F-38 during the immunological response result in an unexpected, dramatic improvement in the immunological coverage range of the assessment. Using the polymer combination, the smallest detectable amount of IgG was lowered 5 to 10 mg IgG / dl, compared to a minimum detectable amount of as much as 37.5 mg IgG / dl based on the individual contributions of PEG and PLURONIC F-38 separately.

This represents a dramatic increase in the rating coverage area, which is quite unexpected. From these data, it can be seen that the combination of 1% PEG and 3% PLURONIC F-38 produces a desired effect that far exceeds what is expected.

A further unexpected advantage is that the actual curve of FIG. Figure 1 shows higher measurement values with respect to the average percentage light scattering on the nephelometer. Of course, this means a significant improvement in the assessment's sensitivity and reproducibility, and as a result, a more reliable assessment.

Similar and even more dramatic unexpected effects can be observed in the data listed in Tables 2-6. These data clearly show that the combination of PEG with another nonionic surfactant results in an increase in the upper limit of the assessment range from 600 to 1200 mg IgG / dl, compared to what could reasonably be expected by combining the polymers. Thus, in the systems studied, the combination of the polymers results in a synergistic effect.

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Claims (5)

150809 An immunological reagent of the kind containing an aqueous solution of an antibody or antigen and at least one polyethylene glycol, characterized in that it also contains at least one nonionic surfactant, the amount of glycol and surfactant thus selected, that the aqueous solution contains between 3 and 6% by weight of glycol and surfactant and that the calculated HLB (Hydrophil-Lipophil Balance) value is between 0.7 and 1.7 when the reagent concentration is between the indicated values. Reagent according to claim 1, characterized in that the aqueous solution contains an antiserum. Reagent according to claim 1 or 2, characterized in that the aqueous solution is a mixture of polyethylene glycol and a mixture of surfactants comprising a) a block copolymer of ethylene oxide and polyoxypropylene, b) straight chain aliphatic oxyalkylated alcohols, c ) glycerol monostearate, or d) polyols of formula H <c2h40) y (C3H60) χ ^ (c3h60) x (c2h40) yH x N-CH2 ~ CH2-N ^ B (C2H40yC3H60) x ^ <C3H60) X <C2H40) y H Reagent according to claim 1 or 2, characterized in that the polyethylene glycol has a molecular weight of between 4000 and 6000 and that the nonionic surfactant 25 is a block copolymer of ethylene oxide and polyoxypropylene and that the HLB value of the solution is from 0.7 to 1.3 when the concentration of the reagent is in the range of claim 1. Reagent according to claim 1 or 2, characterized in that the aqueous solution contains a mixture consisting of