DE2411884A1 - METHOD AND COMPOSITION OF COMPOSITION FOR ANALYSIS OF BIOLOGICALLY ACTIVE MOLECULES - Google Patents

Description

Method and composition of matter for analysis of biologically active molecules

The detection of low concentrations of a large number of biologically active substances is becoming more and more important in the medical field and in other fields. The ability to detect very small amounts of drugs, antibodies, hormones, toxins, viruses, proteins and the like allows the investigation, diagnosis and manipulation of a wide variety of situations involving biological systems. The amounts of interest are generally in the microgray range or less, in some. Cases even in the order of magnitude of picograms (10 " ^y g). So if one wants to detect small amounts of a certain material, one must have available a detection substance which is particularly specific for the substance of interest and which is detectable and with the amount of the material to be detected provides a related signal.

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.BAD ORIGINAL

There are many advantages to using stable free radicals as jar molecules. Free radicals can be detected in opaque solutions. The -free radical can also be selected so that naturally occurring interfering substances can be eliminated or easily removed Concentrations of free radicals of less than about one ymol / liter with the usual apparatus no signal can prove more. The use of a free radical or a spin marking substance as a detection substance was therefore limited if the concentrations of interest were below one yuMol / liter

There are already several works on the lysis of "bags" (sacsι see definition given below) that contain glucose or enzymes, for example "Complement-Dependent Damage to Liposomes Prepared from Pure Lipids and Porssman HaptenJ," SO Kinsky et al, Biochemistry , Vol. 8, ITr. 10, pp. 4H9-4-158, October 1969} "Immune Daage to a Lipid Model Membrane", SC Kinsky, Annals Lew York Academy of Sciences, Vol. 195, pp. 429-438} and Kataoka et al, Biochem. Et Biophya Aeta, 298 , 158 (1973).

In U.S. Patents 3,453,288, 3,481,952 and 3,489 is the bond τοη Ilektrenen-Ipinresonanz-Markierungssu »- stanzea on biomolecules describes »what is the consentration of the biomolecules (siologically active molecules) by means of "eightuag 4er TerüULeru & fem des IFt-ljektrue" (llektroaemlaramafmetiscke lesomans, sometimes also as ESR or llektrenen-Spim-lesemans seseichnet) one biologically active molecule against another », that is to say identical ■ iolegically active »Melekül, which with another» iol · - gischen substance was converted, can be determined.

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BATH ORIGINAL

U.S. Patent 3,690,854 is an immunological one Analytical method (immunoassay technique) indicated, using a spin-labeling substance as detection substance tied to a hapten. That spin marked Hapten is able to with a free hapten around one Antibodies to compete. The EPR spectrum of a spin-labeled hapten bound to an antibody is, apart from: that of an unbound spin-marked haps.

The invention relates to an immunological method (immunoassay) for the determination of compounds with a determinant or epitope site, ie antigens and haptens, antibodies or complements, and is based on the availability of the antibody and / or complement. "Sacks" are provided in which stable free radicals are trapped in a sufficiently high concentration to provide: EPR spectrum of the stable free radical; furthermore, at least one epitope site is provided, bound to the wall of the "sack ¹¹ ", which is "recognized" by the antibody. The "sacks" are allowed to react with the available antibody, and the "sacks" bound to the antibody are lysed.

The amount of antibody available isn't just going through his, concentratio.n, but also through the presence a connection with an epitope site associated with the Antibody connects, definitely. The lysis of the antibody Bound "bags" can: either without separation with complement or after: the separation of the agglutinated "Bags" by a suitable lysing agent, e.g. a detergent, can be achieved * Then the EPR spectrum of the stable, free radicals released in the solution and with standards for which the individual components used in known concentrations, compared.

The 'present immunoanalysis method for the determination of Connections with epitope sites, antibodies or complements, gives a substantial multiplication factor in that A single event from a "sack" releases a large number of stable free radical molecules, their EPR spectrum is distinguishable from the EPR spectrum of the radicals trapped in the "sack". When the free radical molecules are contained in the "sack", so they are relatively tightly packed, reducing their spectrum through mutual influencing (Spin exchange and / or dipolar broadening) so that a relatively broad, flat spectrum is created. If, on the other hand, the spin marker molecule is free and in a relatively low concentration in solution, a sharp peak is obtained, d.ho the half width of the peak of the free radicals trapped in the "sack" is much larger than that Half width of the peak of the free radicals released in the solution. Therefore you get a greatly enlarged Peak intensity or amplitude between two peaks.

The immunoanalysis according to the invention is based on the use of antibodies that are capable of a certain to "recognize" spatial and chemical structure called the epitope or determinant site. if to a "sack" in which stable free radical molecules are trapped in a relatively high concentration If an epitope site is located, this results in a distribution of "sacs" which are bound to antibodies and which can be lysed by the complement, and "sacs" which are not lysed by the complement.

If the amount of the antibody is a limiting factor, the bound "bags" can either contain the Complement lysed or after an agglutination process

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can be separated and then lysed in any way. If the lysis is carried out by the complement, the amount of the available antibody due to the change in the EPR spectrum of those enclosed in the "bags" Determine free radicals to be the stable free radicals released in solution. According to the agglutination process you can separate the agglutinated "sacks", the "sacks" either in the supernatant liquid or in the Lyse the precipitate and determine the amount of stable free radicals in each or both media. By using standards in which the individual components are present in known concentrations, you can one can determine the amount of the component of interest present.

if the complement is present in a limited amount, the "bags" and the antibody can be used in excess and the amount of complement due to the rate at which the free radical molecules enter the Solution, or based on the total amount of ■ free radical molecules that are released into the solution, determine,,

Before the invention is explained in detail, some are used in the description and in the claims Terms are defined:

1. Antigen: Any substance that is capable of producing an immune response of any kind in one to induce immunologically responsive vertebrate, in particular a stimulation of the formation of specific Antibodies ,, Antigens are substances with a high molecular weight and include, for example, proteins, glycoproteins, glycolipids, polysaccharides, and lipopolysaccharides.

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2. Hapten: I am an incomplete antigen that does not yet have one itself Elicits immune response} but when it comes with an antigenic Material is connected, antibodies are formed which recognize the hapten molecule in a specific way,

3. Epitope: A specific chemical or spatial configuration, which is specifically recognized by an antibody. Antigens and haptens can be one or contain multiple epitope sites, the antigens usually containing a plurality of epitope sites.

4. Sack: A bag-shaped structure made of any material that encloses a volume, a wall has one or more components and has at least one interior compartment, the wall of the Compartments forms a permeability barrier to the outside.

5 · Vesicle: A term used for "bags" with one and more compartments, as used herein Case only for "sacks" with one compartment.

6. Shadows (ghosts): "sacs" obtained from cells are by removing the cell contents by opening the cell membrane, either physically or chemically Ways that have been removed in order to practically completely empty the contents of the cell, whereupon the membrane is resealed or sealed to enclose the material contained in the sealing or sealing solution. The "shadows" are most commonly made from mammalian red blood cells (erythrocytes).

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2A11884

_ 7 -

7. Iiposomes: structures consisting of several compartments, which are obtained when lipids, especially lipid mixtures, by vigorous stirring in the form of a aqueous suspension are dispersed. The walls or membranes form a coherent lipid bilayer (see Kornberg et al, Biochemistry, U), 1111 (1971)).

According to the invention, vesicles, liposomes and shadows are suitable "bags". Suitable membranes can be produced according to the above-mentioned parts of the literature by Kinsky and Kinsky et al and according to the processes described in the present application _e

The membrane walls of the "sack" are different, depending after whether the "sack" comes from a naturally occurring cell or has been synthetically produced (e.g. Liposomes) ο The naturally occurring "sacs" or "shadows" have a membrane that is mainly made up Composed of lipids and proteins in approximately equal proportions is.

The "shadows" or "ghosts" can be made using Erythrocyte cells, which are suspended in a solution with a significantly lower osmolarity, are formed. After centrifuging the lysed cells, the cells are placed in a "resealing solution" with brought suitable osmolarity, the alkali and alkaline earth halides as well as a goenzyme, e.g. adenosine triphosphate, contains. The sealing solution contains a water-soluble stable free radical in a suitable concentration to to generate the desired concentration of free radicals in the "shadow".

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Synthetic "bags" or liposomes can be prepared from a wide variety of lipids, such as phospholipids, glycolipids, steroids, relatively long chain alkyl esters (eg, _o alkyl phosphates) Fettsäureestern (z.Bo lecithin), iPett-r amines, etc. are produced _e are preferably mixtures of fat-like substances are used, in particular a combination of a neutral steroid, a charged amphiphilic or amphipathic compound and a phospholipid ₀

Examples of phospholipids are lecithin, sphingomyelin, dipalmitoyl lecithin, sheep or mutton fraction Ha, Kinsky’s phospholipid (see _above cited literature), etc.

Cholesterol, cholestanol, lanosterol or the like can suitably be used as the steroid.

The charged amphiphilic compound, which normally contains 12 to 30 carbon atoms, is conveniently a mono- or Dialkyl phosphate ester or an alkylamine, e.g. dicetyl phosphate, Stearylamine, hexadecylamine, dilauryl phosphate or similar·

The liposomes can easily be prepared by adding the substances to an aqueous medium, which contains the stable free radicals, whereby the solution is vigorously stirred at moderate temperatures. The liposomes are then freed from the externally adhering spin marking substance by chromatography.

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"Sensitized sacs" can be obtained by various methods. If the "shadows" are formed from naturally occurring cells, the determinant sites may be naturally present in the membrane wall. For active sensitization, which normally requires an amphipathic antigen or hapten, the antigen or hapten is added to the solution at the time the liposome is formed. In passive sensitization, the amphipathic antigen or hapten is added after the liposome is formed, adsorbing it to its surface. Finally, covalent sensitization can be performed using a bifunctional reagent with the same or different functional groups that interacts with the "shadow" or liposome as well as with the antigen or hapten covalently connects. But you can also use a monovalent reagent _o eg carbodiimides

The individual sensitization methods are known per se, e.g. from the literature references cited above and from Golub et al, J. Immunol. IjX), 153-137 (1967).

Depending on the origin of the "sack", its size varies.

When using "shadow" the size (volume) is

of the sack usually about 1 - 80> u, in particular 5 - 20 / rev. In the case of liposomes, the volume of the "sack" is between about 0.1 and 150 / U, in particular between about 1 and 80 / U. It was found that the membrane in general has a thickness of about 100 Å

The concentration of free radical usually depends on the production method and of the form of free radical from ₀ The concentration of the free radical in the "bag"

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- ίο -

should be expediently such that the ratio between the EPR line width of the released free radical molecule and the entrapped free radical molecule is no more than about 0.7, preferably no more than about 0.3. Normally, the concentration of the free radicals in the "bag" should not exceed an osmolarity of 0.3 in the case of nonionic free radicals, while it should generally not exceed an osmolarity of 0.15 in the case of ionic free radicals. In general, the concentration of the free radicals Radicals in the "bag" at least about 0.02 molar, in particular at least about 0.03 molar _o The concentrations are chosen so that the EPR spectrum of the enclosed free radical has a half-width of at least about 3 Gauss (from peak to peak) at a resonance frequency of about 9.2 SHz (gigahertz), usually at least about 3.5 Gauss at 9.2 GHz.

A wide variety of stable free radicals can be used, factors influencing the selection of the stable one Free radicals are important, are water solubility, minimal "leakage" from the bag, strong interactions when enclosed, a sufficiently narrow EPR spectrum at low concentrations and a broad one EPR spectrum at high concentrations and a sufficiently large peak height or amplitude.

Inorganic compounds with ions that provide the best EPR spectrum can be used. For example, Manganese II ions (Mn ⁺⁺ ) can be used. In most cases, however, stable organic free radicals are used, of which the mitroxy free radical is preferred.

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The nitroxide organic radicals usually contain about 6 to 40 carbon atoms.

The free radicals used preferably have a simple EPR spectrum and are water-soluble free ones Hitroxydradicals are particularly suitable and can be cyclic or be acyclic The nitroxide radical has the formula given below:

AWAY
Έ

wherein Z represents a substituent other than hydrogen, usually a hydrocarbon radical, in particular an alkyl radical, and wherein A and B can be the same or different from one another and together can form a ring; A and / or B contain a neutral or charged water-solubilizing group, such as ₀ like an ammonium salt group, a hydroxyl group (sugar), an acid group and.

In most! Cases, the e-nitroxy free radical has the Formula given below:

wherein the groups R can be the same or different from each other, and usually, a lower alkyl group with 1 to 3 carbon atoms, preferably the methyl group

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represent; X "means a 2 to 6 divalent radical Carbon atoms and 0 to 2 heteroatoms, namely oxygen, Sulfur or nitrogen, and 0 to 1 ethylenically unsaturated site, with a five- or six-membered ring with the Carbon atoms to which X is bonded is formed; Y means a water-solubilizing group, e.g. Tetraalkylammonium salt of 4 to 8 carbon atoms, usually with a halide, ζ.Bo a chloride G-egenion, it is also possible for O to 3 oxy groups, for example hydroxyl groups, in particular O to 1 groups, to be present; a phosphate or Phosphonate salt, especially an alkali salt (e.g. a sodium salt a nucleotide or a sugar with 5 to 24 Carbon atoms, especially mono- or trisaccharides, glucosamines or the like.

Examples of suitable connections are:

O - F

) ₂ CH ₂ CH ₂ OH

ET, F-dimethyl-U- (2 ', 2', 6 ', O' -tetramethyl-1 -oxyl-piperidyl-4 ') 2-hydroxyethylammonium ion, wherein X - represents a negative ion such as chloride, fluoride, phosphate, or the like; Tempophosphate salts with the general formula:

2Ζ ^Ί

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1-oxyl-2,2,6,6-tetramethylpiperidinyl-4-phosphate, in which Z ^{+ is} a metal or ammonium ion, for example sodium, potassium or the like.

P P P

O-P-O-P-O-P-O-CH,

ii

OH OH

NH

4Z

I - (1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) adenosine triphosphate.

Examples of useful polar but nonionic organic free radical molecules are:

CH ₂ OH

OH

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-H-

1-oxyl-2,2,6 / 6-tetramethylpiperidinyl-4-β-D-galactoside

CH ₂ OH

KO

/1

\ 0H

"Q _S _

Q H

ίι ι.

, - C-N

OH

K- (I-oxyl-2 , 2,6, 6-tetrainethylpiperidinyl-4) - ^ carbamoylmethyl - $ - D-thiogalactoside

CH "OH

CH ₂ OH JO _r 0-CH ₂

N _ /

- 0

OH

1- 0xyl-2,2,5,5-tetrainethyl-3-pyrrolidinyl> methyl-3-D-lactoside

CH ₂ OH

- ο

OH OK

(1- Oxy 1-2 , 2.5, D-thlccaiactosic.

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OK

(1-Oxyl-2,2,5,5-tetrameth.yl-3-pyrrolidinyl) -methyl-β-D-glucopyranose

CH ₃ C = O

NH

i-O

C = O

H ₂ OH

(1-Oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) -methyl-β-N-acetyl-D-glucosamine

—0., - 0 - CH,

OH

N O

HO IiH

-f

= 0

(1-033Γ1-2,2,5,5-tetramethyl-3-pyrrolidinyl) -methyl -ßciiitobiose

38/0850

CH ₂ OH

(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) -methyl- (4-0-ß-2-D-glucopyranose)

As stated earlier, the sensitizing compounds have a determinant site that is recognized by an antibody. These compounds include haptens and antigens. Antigenic substances include a wide variety of proteins, lipopolysaccharides, polysaccharides, and the like. Almost any organic compound can be a hapten. Haptenic compounds include hypnotics and sedatives, e.g., barbiturates; narcotic analgesics such as morphine _o; Central nervous system stimulants such as strychnine and xanthines; Local anesthetics such as cocaine and procaine; Anticholinesterase agents, parasympathomimetic agents, atropine-like drugs, histamines and anthistamines, polypeptides, steroids, glycosides, saponins, sapogenins, diuretics, antibiotics, thyroid and antithyroid drugs, vitamins, etc.

Depending on the hapten in question, various methods can be used to link the hapten to the "sack" can usually be the same methods that are available for linking antigens with the "sack", and have also been used successfully with haptens will. On the other hand, the hapten can also be modified so that it has an active functional group available represents, e.g. a mixed anhydride or imidate, which is capable of having functional groups in the membrane wall to react.

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There are two principal ways of performing the test: In the first variant, the complement is present, which causes the lysis of the "sacs" as a function of the quantity of the material to be analyzed. In the second variant, agglutination is used, with the Degree of agglutination a puncture the amount of too material to be analyzed and wherein the agglutination is carried out in the absence of the complement.

After separating the agglutinated "bags" from the non-agglutinated "sacks" can then be "sacks" can be lysed, and the amount of unknown material can be due to the distribution of the "sacs" between the agglutinated and non-agglutinated norms can be determined ©

In the first variant, the complement is used for the lysis. An aqueous buffered medium is prepared, which is usually buffered in the pH range from about 6 to 9, in particular from about 7 to 8. The buffered medium is isotonic with the osmolarity of the "bags ¹ O A certain deviation of the osmolarity of the medium from isotonic conditions is permissible. Various buffer substances can be used, for example barbiturates, iris, phosphates, carbonates, borates and the like. Usually inorganic salts are used The concentration of the salts is usually from about 0.01 mmol to about 0.25 mol, especially from about 0.1 mmol to about 0.15 mol. The salts provide the desired osmolarity and further stabilize the various' proteins present in the solution.

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Although the order of addition can be varied widely, a certain order of addition is preferred in certain cases. If a hapten or antigen is to be determined, the hapten or antigen will normally be combined with the appropriate antibody under conditions in which the antibody becomes bound to the antigen or hapten. If necessary, an incubation can also be carried out, which depends on whether or not the antigen or hapten in the unknown substance is to be allowed to compete with the "sacks" that have the appropriate bpitop site Substance with the antibody are then combined in the appropriate "bags" with the antibody. It can then again be carried out by incubation, _o or the complement can be added immediately, whereupon the whole mixture is incubated.

The incubations are generally carried out at about 15 to 40 ° C, usually carried out at about 20 to 37 ° C. The incubation period is generally about 5 minutes to 1 hour, usually about 10 to 45 minutes.

The concentration of the antibody used varies within wide limits, which depends on the binding constant of the antibody and the range of concentration of the unknown substance of interest, usually the concentration of the antibody is about 0.1 to 1000 times (based on the binding sites) the number of the molecules at the top of the range of interest. The ratio of antibodies is in many cases empirically, and can be determined using standards with known concentrations of antigen or hapten “The essential factor is that you have a detectable and distinguishable signal about the person of interest

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Area wants to get.

After incubation, the degree of lysis can be determined by introducing of the sample or an aliquot of the sample can be determined in an EPR spectrometer cavity, the amplitude of Is determined peak to peak. If standards with known concentrations of the unknown substance are used, see one can plot the amplitude from peak to peak in relation to the concentration of the unknown substance. The received Signal can then be directly related to a specific concentration of the unknown substance will".

The relative amounts of each reactant will depend on the purpose of the particular test. In general, the lower the concentration of the "sacks" that can be selected, the greater the sensitivity. The amount of spin-marking substance that can be released from each sack, the speed of the spontaneous "seeping out" and the sensitivity of the electrometer determine these lowest concentration of "bags", which can be applied with success to determine _o If the antibody or complement, the complement or the antibody may be added in a functional excess and the "bags" are added in a concentration that their - Lysis is a puncture of the concentration of the unknown substance. If an antigen or hapten is to be determined by lytic inhibition, a limited amount of antibody and excess complement are added together with a suitable concentration of "bags".

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In the agglutination process, the sacs and antibodies are combined to produce a suitable concentration at which agglutination occurs; that is, a substantial proportion of the bags present in the solution, usually at least 10 fo, can be separated from the bags that are not associated with an antibody, for example by centrifugation in gradients. By combining the unknown substance with the bags and the antibodies in a buffered aqueous solution of the type described above, the amount of the antigen or hapten present in the unknown substance affects the proportion of the agglutinating bags.

The agglutinated sacs can then be easily lysed in a number of ways, for example by enzymatic analysis, use of detergents, osmolysis or the like. The determination of the number of free radicals present can be related to tests performed with known amounts of antigen or hapten »

Some agglutination methods are for example in Kabat et al, "Experimental Immunochemistry" (1961) and Carpenter "Immunology and Serology", Saunders, Philadelphia (1965)

Experimental part

The following examples are given for illustrative purposes only. All temperatures are given in C, and all Parts or percentages relate to weight, unless stated otherwise.

The following were given for the individual experiments Reagents used.

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¹¹ SRBC "

Saline solution buffered with veronal

pH = 7.30 + 0.05; 0. Η molar; 4.95 mmoles of 5,5-diethylbarbituric acid; _{0.50 mmoles of MgCl 2;} 0.15 moles CaCl ₂

Sheep red blood cells washed twice with VBS and prepared as 5% and 50% suspension.

complement

Guinea pig blood was "coagulated at room temperature for one hour and then allowed to stand for about 2 hours" at 4 °. The serum was withdrawn, centrifuged at 0 °, absorbed twice with three times washed, packed SRBC (0.3 ml / 10 ml serum) at 0 ° for 5 minutes each time and divided into aliquots of 0.5 ml at 0 ° and in sealed ampoules stored at -20 ° C ¹ from Difco Laboratories can also be used.

Heat-inactivated guinea pig complement

Guinea pig complement was inactivated by heating at 56 ° for 30 minutes.

! formal rabbit serum

Decomplemented serum to be used on appropriate

Erythrocytes absorbed and stored ^{at -20 ° s}

became·

Eo coli OB Antiserum Serotype 026: B6 ATCC No.12795 Lyophilized antiserum with merthiolate in a ratio of 1: 1000 (Difco Laboratories), decomplemented (56 °, 1 hour) and stored at 4 °.

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"Anti-EA" rabbit anti-egg albumin serum Reconstituted lyophilized antiserum (Difco Laboratories), decomplemented (56 °, 0.5 hours), absorbed on appropriate erythrocytes and stored at -20.

Hemolysin (rabbit anti-SRBC serum) Reconstituted lyophilized hemolysin, decomplemented (56 °, 0.5 hours), adjusted to the desired concentrations with VBS, stored at -20 °.

Sealing solution for the "shadows"

pH = 7.6; 0.20 moles of tempocholine chloride; 0.050 moles of KCl; 8.65 mmoles of 5,5-diethylbarbituric acid; 0.875 mmol MgGl ₂ ; 0.263 mmol CaCl ₂ ; 1.0 mmol adenosine triphosphate disodium salt.

"Lecithin" egg lecithin standard solution

The lecithin was made from egg yolk in a non-oxidizing atmosphere by the method of Singleton et al, J. Am. Chem. Soc. £ 4.53 (1965) extracted and as a 50 / umolar / ml solution in with

■ ο

Ethanol flushed with argon and stored at -20.

"Cholesterol" standard cholesterol solution

Recrystallized cholesterol was dissolved in ethanol up to 50 / UMol / ml, flushed with argon and stored at 4 °.

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"ATLPS"

Di c e tylpho sphat

Dicetyl phosphate at a concentration of 5.5 / µmol /

ml in toluene, flushed with argon and increased at 4

Lipopolysaccharide treated with alkali

50 mg E. coli lipopolysaccharide 026: Ββ, extracted according to the Westphal, Bact method. Rev. 30 , 192 (1966), partially saponified (Kabat, loc. _{Cit .; Kataoka et al, Eur o} J.Biochem. 2A_, 80 (1971) were in 10 ml of 0.05 molar NaOH solution for 1 hour "at 56 ° suspended, weakly acidified with acetic acid and centrifuged for 0.5 hours at 4 ° at 27,000 G. The supernatant liquid was dialyzed against distilled water for 24 hours at 4 °, centrifuged again at 80,000 G for one hour at 4, whereupon the supernatant liquid obtained was lyophilized and above

P ₀ Or- was kept,
τ

Buffered tempocholine chloride solution

The compound was synthesized according to the method of Kornberg et al, Biochemistry 10, 111 (1971).

- from

A solution was prepared in VBS which differed from the above solution in that it was 0.045 mol The solution was prepared for a requirement of 2 days.

("PBS") phosphate buffer solution

(1) pH = 7.40 + 0.05, 0.11 moles of sodium phosphate

(2) pH = 7.2; 0.074 mole NaCl, 0.018 mole ICH ₂ PO ₄ : 0.039 mole Na

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2A11 88 A

example 1

Comparison of immunolyses of sheep red blood cells using optical measurements of released hemoglobin with shadows of sheep red blood cells filled with spin-labeling substance using EPR measurement .

Red cell shadows were made as follows:

A 50% SRBC suspension (0.2 ml) was slowly added to 1 ml of IBStHpO in a ratio of 1: 3 with mixing. The glasses were centrifuged for 10 minutes at 4500 G- and 0.5 ml of the supernatant liquid removed and discarded. The original volume and the isotonic state were restored by adding 0.5 ml of the sealing solution. The pellet was resuspended with the aid of a vortex mixer, whereupon the preparation was placed in a ^{water bath maintained at 37 °} C. for 30 minutes in order to seal the cells. The shadows were cooled by setting the lens in an ice bath, centrifuged, and the solution 10 minutes at 2000 G and discard the supernatant and was replaced by 1.90 ml of VBS (1 io URS). The pellet was resuspended and then centrifuged and suspended five more times to obtain a 5 # SRBC suspension (0.1 ml of packed SRBG was used to make the preparation, which had a final volume of about 2.0 ml).

The two methods of determination were carried out by mixing the cells or shadows with antiserum and complement, Incubate at 37 ° for 20 minutes and then cool by placing in an ice bath.

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In the case of hemolysis, de.r non-lysed cells and cell membranes dilute the supernatant fluids to a known volume, and the optical density was determined at 41 nm using a Gary spectrophotometer. At the lysis In the shadow, the EPR signal was measured using a Varian E4 EPR spectrometer. The trials were performed to demonstrate the sensitivity of the two tests to variations in antiserum concentration. The antiserum was adjusted to various dilutions with VBS, whereupon the test mixture for hemolysis by combining 0.80 ml of a 5 cell suspension, 0.10 ml of hemolysin solution and 0.10 ml Complement was established. As a control, 0.10 ml HIC with the 1: 100 dilution of the Used antiserum. In the experiment with the shadows, 0.20 ml of a 5% suspension of the shadows combined with 0.025 ml of the appropriately diluted hemolysin and 0.025 ml complement, Bs also became a Control sample performed with HIC.

The table below shows the results the results as the response of each antiserum dilution as a percentage of the 1: 200 dilution in excess of the 1: °° dilution are recorded.

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Table I.

Hemolyysin dilution

Mean ^ response amplitudes from the optical
Density at
541

nm

Peak to peak
between 3255g
and 3275 g
Gain factors ₉ -2.5x1 Q 6.2x10 ^

tion

1: 100 9.84

1: 200 9.06

1: 400 6.27

1: 800 3.15

1: 1600 2.29

1: 3200 1.54

1: 6400 1.40

1: «(VBS) 1.23

HIO · control 0.99

100 101.2 80.5 100 64.4 72.4 73.0 61.2 24.5 40.5 70.9 18.2 13.5 30.7 67.5 4.99 3.96 28.4 1.89 2.17 27.5 0.674 0 27.5 0

65.0

In both cases the HIG 'control shows a lower level Lysis as the 1: °° dilution. This can be done on a something increased stability due to the presence of more serum in the previously used G-glasses is. The shadows slowly fade and the samples were measured through in the shortest possible time. When measuring started with the samples with the highest antiserum concentration and ended with the HIC 'control, to keep the leakage effect as low as possible.

The results in Table I clearly show that antibodies are slightly below levels over a wide range of concentrations Use of shadows that contain stable free radicals can be determined.

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Example 2

Production of liposomes, sensitization with bacterial. Lipopolysaccharides !! and analysis aes Antiaerums and complements

It became a liposome in the form of 3 ml of a suspension made with 49.5 / U mole lipid per ml.

A standard Leeithin solution (1.60 ml) and 1.2 ml a standard cholesterol solution were taken under a Argon atmosphere brought into a flask and not completely evaporated to dryness with the help of a rotary evaporator. 1.6 ml of dicetyl phosphate solution were added to the residue, to dissolve the lipids, after which the solvent was removed in a rotary evaporator at 40 °. To When the solvent was completely removed, the flask was filled with argon and two glass beads were added and 3 ml tempocholine chloride solution (TC solution), the was flushed with argon, added to the lipids. The liposomes were mixed using a Variwhirl mixer manufactured.

A column with an inner diameter of 0.92 cm, which contained Sephadei G25 up to a height of 28.5 cm, was equilibrated and, after eluting with TBS at room temperature, was used to remove entrapped Spin label from the liposomes too remove. 0.5-1 ml was used for each run. It became a visibly opalescent eluate in a measuring tube collected and mixed gently by inverting to ensure homogeneity and an approximate

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To enable estimation of the αχ moles of lipid per ml. The liposomes obtained had a molar ratio between lecithin, cholesterol and dieetyl phosphate of about 2.0: 1.5: 0.22.

The sensitization of the liposomes was carried out in a passive and active manner. Passive awareness is achieved by adding ATLPS to already formed and clean liposomes. A weighed amount of ATLPS is dissolved in a small amount of VBS and added to the eluate containing the clean liposomes. The mixture is kept at room temperature for 40 minutes before it is used further.

The passive sensitization was carried out with 5, 10, 20 or / Ug ATLPS // U Mol Lipid. Maximum sensitization was obtained at 5 to 10 / Ug ATLPS // u mol lipid. The liposomes could be ^{lysed with AS and C 1} at 25 °, while unsensitized liposomes were not lysed under the same conditions. No further improvement was obtained when sensitization was carried out for more than 40 minutes.

The active sensitization was carried out by adding 20 Axg of ATLPS // U mol of lipid to the liposome preparation solution.

The test is carried out as follows. That at room temperature The liposome preparation obtained is diluted with VBS so that a concentration of about 1.0 / u mol lipid / ml is obtained.

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The temperature should be kept constant to a possible leakage of the spin marking entity as a result to prevent volume changes.

Various solutions of complement and antibody were made using TBS. aside from that several blanks were made. Should the complement be determined, then undiluted antiserum was used used} if, on the other hand, the concentration of the antiserum was to be determined, undiluted was used Complement used. The liposomes used were passively sensitized with 10 / Ug ATLPS // U mol lipid. Starting from the highest concentration of interest, the test glasses were after more than 15 minutes Measure the addition of the solution to be measured. The substances were in the order liposomes, Antiserum and complement added.

In each experiment, the amplitude of the blank was subtracted from the amplitude of all other spectra. These values were considered to be proportional to the degree of lysis in each particular Test glass were; they were used to calculate the degree of lysis as a percentage of total lysis when frozen in liquid nitrogen and thawing used. The tables below show the degree of lysis as a function the complement and antiserum concentration.

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Table II Lyscgrad al3 function of complement concentration *

/ UlC '/ 500 / Ul io

Solution lysis

50.0 28.0

25.0 28.0

12.5 28.0

6.25 25.5

3.13 18.0

1.57 13.0

0 3.3

* HIO 'control as a blank sample; AS used undiluted.

Table III Lyaegrade ale function of the antiserum concentration *

/ Ul AS / 500 / Ul i>

Solution lysis

32.0

5 31.8

2.5 '24.5

1.3 21.5

0.6 15.8

0.3 10.0

0. 1.0

♦ HIC 'control used as a blank sample j C undiluted.

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The results of Tables II and III with passive sensitized liposomes practically corresponded to the results obtained when using actively sensitized and liposomes tested in the same way were found. The results show that both the complement as well as the antibodies with LipcBomen, which contain free radicals, can be determined.

Example 3

Immunolysis of shadows sensitized with (exogenous) egg albumin

In this example, an antigen is covalently linked to a shadow. Below is the manufacture of the shadow sensitized with egg albumin. Bis-diazotized benzidine chloride (0.64g »2.5 ι 10" 'mol) was in 13 ml of 1N HOl and 77 ml of distilled water Dissolved at 4 °, whereupon 0.35 g of sodium nitrite in 10 ml of distilled water was added with stirring at 4 °. The temperature was maintained with stirring for 6 hours. A 0.5 ml aliquot in 7 ml Phosphate buffer dissolved at 0 °.

Unless otherwise stated, the further work steps were carried out at 0 °.

To 2 ml of diluted VBS (1: 3) were added 0.4 ml of 50 #, washed SRBO, whereupon the solution is mixed and the resulting lysed cell membranes become a pellet were centrifuged. It became exactly 1 ml of the supernatant

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Solution removed and replaced with 1 ml of the solution to seal the shadows. After thorough mixing it was incubated the jar at 37 for 60 minutes, chilled in an ice bath for 10 minutes and then centrifuged. To discarding the supernatant solution became the shadows Washed four times with 4 ml of VBS (1 $ NRS) and then in 4 ml a 0.1 $ solution of albumin suspended. The suspension 3 »1 ml of VBS and then 0.25 ml of the diazotized benzidine were added. After mixing gently, the The reaction was carried out for 10 minutes at room temperature, after which 15 ml of VBS (1 $ NRS) were added. After this Mixing the shadows were centrifuged off, the pellet with 15 ml VBS (1 $ NRS) and then again with

Washed 4 ml of VBS (1 $ NRS). When suspended in 4ml VBS ($ 1 NRS) gave a concentration of about

5 $> i based on the volume of the original SRBO in relation to the final volume of the shadow preparation.

The test glasses given below were prepared, incubated for 20 minutes at 37 ° in an ice bath cooled and measured the EPR spectrum. For the entire lysis, the shadow samples were given an equal volume diluted distilled water.

Table IV

Test composition, volume, ml

5 # shadow AS / NRS / VBS C »/ HIC ^f / VBS suspension

fully 0.2 sensitized 0.025 AS 0.025 C (only AS)

fully 0.2 sensitized 0.025 1: 4 AS 0.025 C ¹ (1: 4 AS)

fully 0.2 sensitized 0.025 1: 8 AS 0.025 C » (1: 8 AS)

NRS control 0.2 sensitized 0.025 NRS 0.025 C

HIC · control 0.2 sensitized 0.025 AS 0.025 HIO ¹ sensitized

Blank sample 0.2 sensitized 0.025 VBS 0.025 VBS

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non-sensitized, 0.2 non-sensitized 0.025 AS 0.025 O ¹ completely bilized

non-sensitized 0.2 non-sensitive 0.025 VBS 0.025 VBS Blank sample bilized

So the immunolysis of spin-labeled shadows is one Function of the specific antibody concentration, the complement activity and the presence of the appropriate one Antigen or the shadow membrane.

Example 4

Determination of egg albumin using egg albumin senabilized shadows containing spin labeling substances

The egg albumin sensitized shadows were prepared using the above prepared shadows in one quarter of the amount. Instead of SRBO, however, human red blood cells of type A or O were used, which were further washed with PBS (2). To the pellet obtained after the last wash, 120 mg of recrystallized egg albumin dissolved in 1.5 ml of the above-mentioned phosphate buffer was added. After mixing thoroughly, 20 mg of N-ethyl ·, Ή '- (3- dimethylaminopropyl) -carbodiimide hydrochloride in 0.2 ml of distilled water was slowly added with mixing. After the mixture was allowed to stand at 20 ° for 30 minutes, it was centrifuged at 2000 Gr for 10 minutes, and the supernatant was discarded. The shadows

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were washed four times with 3 ml of VBS (1 # NES) and finally suspended in 10 ml of fresh VBS (1 # NRS) to a Obtain preparation from an approximately 0.5 # cell suspension.

The reading was carried out as follows: A solution of 10 / Ug / ml egg albumin in VBS (1 $ NRS) has been raised several times diluted ten times. 0.1 ml of each antigen solution was added to a duplicate set of test tubes, and four test tubes in each row were given 0.1 ml VBS ($ 1 NRS) without egg albumin added. A solution of 1 part rabbit marrow anti-egg albumin serum (anti-EA, obtained from Difco Laboratories) diluted with 19 parts VBS (1 $ NRS) was added and the contents were taken under Shake well mixed. Sufficient anti-serum was present to allow 50 # lysis in the absence of egg albumin to create. In some experiments, the test glasses were placed on ice for a period of time before cooling incubated for 15 minutes 30 minutes at 37 ° while no incubation was carried out with others *

0.1 ml of 0.5 # shadow suspension were then added to each test tube, then 25 / μl G 'or, in the case of two test glasses without egg albumin, 25 / μl HIC, to give the HIO' control samples. The test glasses were mixed well with shaking and placed in a 37 ° water bath for 4-5 minutes, after which they were removed and cooled with ice. Samples were taken and placed in cells made from 50 yul micropipettes; this was done just prior to gauging at about 4 ° using a

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temperature controlled Varian-E ^ spectrometer. The cells were placed horizontally in the cavity to get through To avoid settling caused errors, the samples with the lowest concentrations of antigen were routinely used first prepared and measured. In one experiment, however, the order was reversed, with but practically the same results were obtained. The parameter used to report the results presses the difference in the amplitude of the EPR spectrum from the mean value of the two control samples without antigen as Percentage of the difference between this mean and the mean of the HIC 'control samples. The results are given in the table below.

Table V premixed at 0 ° Egg albumin
-log M -50 Hemolytic Enhancement Effect
Reaction $> +50 7th Incubation at 37 ° ¹ +40 8th -80 +20 9 +20 0 10 +58 11 +50 +30

Antigen and antibody previously incubated at 37 °; Antigen and antibody premixed at 0; in comparison with the lysis obtained in the absence of antigen in the aqueous phase.

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The above results show the extremely high sensitivity of the present method to the Determination of the antigen egg albumin. It should also be pointed out that in the concentration range from 10 to

—9
10 in the determination with the previous incubation, the iysis increases with decreasing antigen concentration,

_q where a maximum gain occurs at 10M.

_q _i -j In the concentration range from 10 to 10, the Lysis increases with increasing concentration (see Humphries and McConnell, PNAS, in press).

The invention provides "bags ^M with stable free radicals in a concentration sufficient to ensure a substantial broadening of the peaks of the EPR spectrum (enlarged peak width) and a considerable reduction in peak amplitude. The bags can be used for immunological tests for the determination of antibodies , Complements or in competitive systems with haptens and antigens. The haptens and the antigen influence the availability of the antibody for combination with the sack for lysis or agglutination of the complement. Because of the large number of stable free radicals that result from the lysis When released from the sack, a large multiplication factor is obtained, and thus extremely sensitive tests are obtained, from which a wide variety of compounds can easily be determined.

Although the invention has been described in some details for purposes of illustration, certain changes are possible and modifications can be made within the scope of the claims.

- patent claims -

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Claims (20)

Claims Method for the analysis of an organic substance or composition of matter which contains a compound with at least an epitope site, an antibody that is able to specifically bind to a specific epitope site, or represents a complement in an unknown material in which one of the organic substances mentioned is suspected is, characterized in that in an aqueous, buffered medium under conditions under which a complement lysis takes place with each other combined: (1) the unknown material; (2) Sacks containing an aqueous solution of substantially isotonic with the aqueous buffered medium stable free radicals in a concentration at which the EPR spectrum of the free eadikals occurs, include and the at least one epitope site on their surface exhibit; (3) an antibody capable of binding to the epitope sites of the sac; and (4-) a complement; with the proviso that the amount of Antibody or complement is known in the medium in the absence of the unknown material if the Antibodies or the complement are detected in the unknown material, and that at least one of the Epitope sites of the sacs and the substance is the same when the substance is determined. 409838/0850 the lysis of the sack upon binding of the antibody and the complement is made to the sack; and that at least one point of the EPR spectrum of the Medium compared to the EPR spectrum of the medium for a the concentration of the organic matter covered by the bruises. 2. The method according to claim 1, characterized in that the sacks represent shadows. 3. The method according to claim 2, characterized in that there is used a compound in which at least one Epitope site koTalent is tied to the shadow. 4 ·. Method according to claim 1, characterized in that the bags represent liposomes or vesicles. 5. The method according to claim 4 # characterized in that the bags are passively sensitized with a compound which contains at least one epitope site. 6. The method according to claim 4 »characterized in that" the sacs are actively sensitized with a compound that contains at least one epitope site. 7. Method for the analysis of an unknown material, of which is suspected of containing a compound with at least one bpitope site, characterized in that one of the unknown material with antibodies that are able to bind to the Epj-top site to create a to form aqueous solution with the unknown material and the antibody combined, that the solution of the unknown material and the antibody can be sacked to obtain an aqueous, 409838/0850 to produce buffered medium in which the bags contain an aqueous solution substantially isotonic with the medium of stable free radicals in a concentration at which a peak broadening of the EPR spectrum of the free Radicals occurs, include, and which have at least one epitope site on their surface that is dated Antibody is recognized., And the complement combined under lysis conditions, with a lysis of the sacs during Binding of the antibody and complement to the sacs occurs, and that at least one point of the EPR spectrum of the Medium compared to a medium analyzed under the same conditions with a known amount of the substance certainly. 8. The method according to claim 7, characterized in that the compound is an antigen. 9. A method of analyzing an unknown material suspected of containing an antibody thereby characterized in that one in an aqueous, buffered medium under conditions under which a complement lysis takes place, combined with each other: (1) the unknown material; (2) Sacks containing an aqueous solution substantially isotonic with the aqueous buffered medium of stable free radicals in a concentration at which a peak broadening of the BPR spectrum of the free radicals occurring, include, and the at least one epitope site on their surface which is recognized by the antibody; and (3) a complement; wherein the lysis of the sac upon binding of the antibody and the complement takes place on the sack; 409838/0850 and that one compares the EPR spectrum of the medium to the Determined EPR spectrum of the medium analyzed in the same way with a known amount of the antibody. 10. A method of analyzing an unknown material suspected of containing a complement thereby characterized in that in an aqueous buffered medium under conditions under which a complement lysis takes place, combined with each other: (1) the unknown material; (2) Sacks containing an aqueous solution of substantially isotonic with the aqueous buffered medium stable free radicals at a concentration at which a peak broadening of the EPR spectrum of the free radical occurs, include and the at least one epitope site on their surface have} and (3) an antibody that is able to bind to the epitope site of the sac, and wherein the Lysis of the sac occurs upon binding of the antibody and complement to the sac; and that one compares at least one point of the EPR spectrum of the medium to one in the same way analyzed medium with a known amount of complement. 11. Method for the analysis of an unknown material suspected of having a compound with at least contains an epitope site, characterized in that the unknown in an aqueous buffered medium Material combined with 409838/0850 Sacks that are essentially isotonic with the medium Aqueous solution of stable free radicals in a concentration at which a · peak broadening of the EPR spectrum of free radicals occurs, and which include a multitude of epitope sites on their surface which are capable of associating with the same antibody as said compound; un <y antibody for the epitope site, where due the ratio between the bags and the antibody, agglutination of a substantial proportion of the bags and the antibody is done in the absence of the unknown material j «Met that the agglitated bags are separated from the medium; lysing the bags in the agglutinated bags and / or in the separated medium; and that at least one point of the EPR spectrum of the lysed agglutinated bags or the lysed bags in the medium compared to the EPR spectrum of the lysed sacs with a known concentration of the compound were obtained. 12. Stoffzuaamaensetzung, containing a sack that is a Volume encloses and the at least one interior compartment contains, the wall of the compartment having a permeability pipe to the outside, characterized in that the sack encloses a volume of about 0.1 to 150 / u ^, which contains an aqueous medium with water-soluble stable free radicals in a concentration at which a substantial broadening of the half width of an EPR line occurs in the EPR spectrum of free radicals. 409838 ^ 0850 13. Substance composition according to claim 12, characterized in that that the free radicals represent free organic liitroiid radicals. 14. The composition of matter according to claim 13, characterized in that that the wall is a double-layer membrane. 15. The composition of matter according to claim 13, characterized in that that the hydroxide radical is a salt. 16. The composition of matter according to claim 13, characterized in that that the nitroxide radical is a neutral molecule represents. 17. Composition of matter in itora of a shadow that passes through Removal of the cell contents of a naturally occurring cell and introduction of a liquid into the cell has been, characterized in that the liquid introduced with renewed closure of the cell is an aqueous Medium with water-soluble stable free radicals in a concentration at which a substantial broadening the half width of the BPR line of the EPR spectrum of the free radicals occurs. 18. The composition of matter according to claim 17, characterized in that that the free radical is an organic nitroxide. . , 19.Material composition in the form of τοη tea particles or Liposoaen, by. Dispersing at least one amphiphilic material in an aqueous medium and enclosing an aqueous one 409838/0850 Medium have been formed, characterized in that the aqueous medium free organic radicals in a Contains concentration at which a substantial broadening of the half width of the EPR line of the EPR spectrum the free radical occurs. 20. A composition of matter according to claim 19, characterized in that that the free radical is an organic nitroxide. 409838/0850