LT4121B - Kit for the cholesterol vizualization on the skin and process for preparing conjugate for use in cholesterol determination - Google Patents

Kit for the cholesterol vizualization on the skin and process for preparing conjugate for use in cholesterol determination Download PDF

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LT4121B
LT4121B LT95-057A LT95057A LT4121B LT 4121 B LT4121 B LT 4121B LT 95057 A LT95057 A LT 95057A LT 4121 B LT4121 B LT 4121B
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Lithuania
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cholesterol
conjugate
kit
solution
skin
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LT95-057A
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Lithuanian (lt)
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LT95057A (en
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Vladas-Algirdas Bumelis
Algimantas Antanas Pauliukonis
Henrikas Kestutis Sebeka
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Akcine Bendrove Biofa
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Abstract

The offered kit for visual detection of cholesterol on skin surface consists of a conjugate related to epidermis cholesterol, and a developer solution, which contains of the following ingredients (mg/ml):citric acid: 1,000 to 2,000;3.3',5.5'- tetra-methyl-4.4'-diamynodiphenile: 70 to 83;hydrogen peroxide: 250 to 300;disodium ethylene- diamine tetraacetate: 300 to 400;disodium dihydrogen pyrophosphate: 15 to 30;acetanilide: 5 to 30;water remaining part up to 1 litre.It is recommended to produce the conjugate, which is related to epidermis cholesterol, having initially treated N- vinylpyrrolidone and maleine acid anhydride copolymer with digitonine, and then having combined the ferment peroxidase modified by treating it with glutaraldehyde and 1.6 hexamethylene diamino.

Description

Išradimas priklauso biologinės ir bioorganinės chemijos sričiai, tiksliau diagnostinėms priemonėms, skirtoms nustatymui susirgimų, kuriems būdingas cholesterino koncentracijos odos paviršiuje padidėjimas pvz., aterosklerozės.BACKGROUND OF THE INVENTION The present invention relates to the field of biological and bioorganic chemistry, more particularly to diagnostic tools for the detection of disorders characterized by elevated cholesterol levels in the skin, such as atherosclerosis.

Rinkiniai, skirti vizualiniam cholesterino nustatymui odos paviršiuje susideda iš 2 pagrindinių komponentų: konjugato, turinčio giminingumą epidermio cholesterinui ir ryškalo tirpalo. Be to, rinkinio sudėtyje būna pagalbiniai komponentai: buferinis tirpalas konjugato ištirpinimui, kitos pagalbinės medžiagos, pipetės ir pan.Kits for visual determination of cholesterol on the surface of the skin consist of 2 main components: a conjugate having affinity for epidermal cholesterol and a developing solution. The kit also contains auxiliary components: conjugate buffer, other excipients, pipettes and the like.

Rinkiniai, skirti vizualiniam nustatymui yra labai populiarūs pastaraisiais metais. Jų privalumas greitas, paprastas ir akivaizdus tam tikros medžiagos nustatymas.Kits for visual setting have become very popular in recent years. They have the advantage of being quick, easy and obvious to identify a particular material.

Paprastai vizualiniam cholesterino nustatymui konjugatas yra ištirpinamas buferiniame tirpale ir gauto tirpalo lašas užlašinamas ant delno odos. Po tam tikro fiksuoto laiko (pvz., 1 min) konjugato perteklius nuplaunamas vandeniu, o surišto su delno cholesterinu konjugato kiekis įvertinamas pridėjus lašą ryškalo tirpalo pagal spalvotos dėmės susidarymą bei jos intensyvumą.Typically, for visual determination of cholesterol, the conjugate is dissolved in buffer and a drop of the resulting solution is applied to the skin of the palm of the hand. After a fixed period of time (e.g., 1 min), the excess conjugate is washed with water and the amount of conjugate bound to palm cholesterol is evaluated by adding a drop of developer solution according to the color spot formation and its intensity.

Pagrindinio rinkinio komponento. - konjugato, turinčio giminingumą epidermio cholesterinui artimiausias gavimo būdas aprašytas TSRS autoriniame liudijime SU 1675769. Šiame išradime aprašytas iš esmės panašus konjugato, turinčio giminingumą epidermio cholesterinui, gavimo būdas prijungiant prie N-vinilpirolidono ir maleino rūgšties anhidrido kopolimero pradžioje digitoniną, po to fermentą peroksidazę. Deja, taip gautas konjugatas yra nelabai stabilus ilgai laikant.The main component of the set. - The closest method for preparing a conjugate having epidermal cholesterol affinity is described in USSR SU 1675769. The present invention describes a substantially similar method for preparing a conjugate having epidermal cholesterol affinity by attaching to the N-vinylpyrrolidone and maleic anhydride copolymer at the beginning of the enzyme digonoxide. Unfortunately, the conjugate thus obtained is not very stable over long periods of time.

Kito esminio rinkinio komponento - ryškalo tirpalo sudėtis žinoma, pvz., iš apžvalgos [Yu. M. Lopukhin, The Skin and Atherosclerosis,The composition of the other essential component of the kit, the developer solution, is known, for example, from the review [Yu. M. Lopukhin, The Skin and Atherosclerosis,

Physicochemical Aspects of Medicine, Reviews, 1992, vol. 3, part 4, psl. 46-501] . Tai yra vandeninis tirpalas, kurio sudėtyje yra N,N-dietil-p-fenillendiamino sulfatas, vandenilio peroksidas, natrio tiosulfatas, stabilizuojantis tirpalas ir kalio bromidas.Physicochemical Aspects of Medicine, 1992, vol. 3, part 4, p. 46-501]. It is an aqueous solution containing N, N-diethyl-p-phenylenediamine sulfate, hydrogen peroxide, sodium thiosulfate, stabilizing solution and potassium bromide.

Toks ryškalo tirpalas yra nestabilus ir gali būti panaudotas (išlieka bespalvis) tik 6-8 valandų laikotarpyje.This developer solution is unstable and can only be used (remains colorless) for 6-8 hours.

Minėti trūkumai žymiu mastu riboja viso rinkinio panaudojimo klinikinėje praktikoje galimybes.These shortcomings significantly limit the clinical use of the entire kit.

Pramoninei rinkinių gamybai būtina, kad rinkiniai išlaikytų savo funkcionalines savybes pakankamai ilgą laiką ( pvz., 6 mėn. ), reikalingą rinkinio transportavimui, saugojimui iki panaudojimo bei panaudojimo metu. Naudojant šiame išradime pasiūlytos sudėties ryškalo tirpalą ši problema išsprendžiama.For industrial kits, it is essential that the kits retain their functional properties for a sufficient period of time (eg 6 months) to transport, store, and use the kit. The present invention solves this problem by using the formulation of the present invention.

Tikslu pagerinti komponentų ir viso rinkinio stabilumą, ryškalo tirpalu siūloma naudoti tirpalą, vandenyje šiuos komponentus gaunamą 'tirpinant nurodytais kiekiais (mg/1): citrinos rūgštisIn order to improve the stability of the components and of the kit as a whole, it is proposed that a solution of the following components be prepared in water in the following amounts (mg / l): citric acid

3,3’, 5,5’-tetrametil-4,4'-diaminodifenilas vandenilio peroksidas dinatrio etilendiaminotetraacetatas dinatrio divandenilio pirofosfatas acetanilidas3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl hydrogen peroxide disodium ethylenediaminetetraacetate disodium dihydrogen pyrophosphate acetanilide

1000 - 2000 70 - 831000 - 2000 70 - 83

250 - 300250-300

300 - 400300-400

- 30- 30

- 30- 30

Konjugatą, turintį giminingumą epidermio cholesterinui siūloma gauti, veikiant N-vinilpirolidono ir maleino rūgšties anhidrido kopolimerą pradžioje digitoninu, po to prijungiant fermentą peroksidazę, modifikuotą glutaraldehido ir 1,6-heksametilendiamino poveikiu.It is proposed to produce a conjugate having affinity for epidermal cholesterol by reacting a copolymer of N-vinylpyrrolidone and maleic anhydride initially with digitonin followed by coupling with the enzyme peroxidase modified by glutaraldehyde and 1,6-hexamethylenediamine.

Pavyzdys 1.Example 1.

Rinkinys paruošiamas iš tokių komponentų:The kit consists of the following components:

- konjugatas t.y. N-vinilpirolidino ir maleino rūgšties anhidrido kopolimeras su prijungtais digitoninu ir peroksidaze arba modifikuota glutaraldehido ir 1,6-heksametilendiamino poveikiu peroks-ida-ze;- Conjugate i.e. A copolymer of N-vinylpyrrolidine and maleic anhydride with digitonin and peroxidase attached or modified by the action of glutaraldehyde and 1,6-hexamethylenediamine on peroxidase;

- ryškalo tirpalas;- developer solution;

- buferinis tirpalas konjugato ištirpiniraui.- buffer solution for conjugate solution.

Ryškalo tirpalas paruošiamas taip:Prepare the solution as follows:

Citrinos rūgštis ištirpinama distiliuotame vandenyje, pridedama koncentruotos druskos rūgšties iki pH 2,5, ištirpinamas 3,3', 5,5'-tetrametil-4,4'diaminodifenilas, pridedama 10%-nio natrio šarmo tirpalo iki pH 3,5 ir gautame mišinyje ištirpinama 90% viso dinatrio etilendiaminotetraacetato kiekio. Atskirai paruošiamas tirpalas, turintis vandenilio peroksidą, 10% viso dinatrio etilendiaminotetraacetato kiekio, dinatrio divandenilio pirofosfatą ir acetanilidą. Abu tirpalai gerai sumaišomi. Ryškalo tirpalo sudėties įtaką jo stabilumui iliustruoja lentelė.Citric acid is dissolved in distilled water, concentrated hydrochloric acid is added to pH 2.5, dissolved in 3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl, 10% sodium hydroxide solution is added to pH 3.5 and the 90% of the total disodium ethylenediaminetetraacetate is dissolved in the mixture. Separately prepare a solution containing hydrogen peroxide, 10% total disodium ethylenediaminetetraacetate, disodium dihydrogen pyrophosphate and acetanilide. Mix both solutions well. The influence of the composition of the solution on its stability is illustrated in the following table.

Konjugato su modifikuota peroksidaze peroksidazinis aktyvumas stabilizuojasi.Tai yra labai svarbu rinkinio naudojimo laikui, kadangi abiejų komponentų stabilumo terminas praktiškai susilygina.The peroxidase activity of the conjugate with modified peroxidase stabilizes the peroxidase activity. This is very important for the lifetime of the kit since the stability term for both components is virtually equal.

LentelėTable

Ryškalo Komponentų koncentracijos, mg/1 Stabilumas* tirpalo_ mėn.Component concentrations, mg / 1 Stability * solution_month.

Nr.No.

A B C D E FA B C D E F

1 1 1600 1600 83 83 283 283 344 344 22 22nd 13 13th > > 6 6th 2 2 2000 2000 83 83 283 283 344 344 22 22nd 13 13th 6 6th 3 3 1000 1000 83 83 283 283 344 344 22 22nd 13 13th 6 6th 4 4 1600 1600 70 70 283 283 344 344 22 22nd 13 13th > > 6 6th 5 5 1600 1600 83 83 300 300 344 344 22 22nd 13 13th 6 6th 6 6th 1600 1600 83 83 250 250 344 344 22 22nd 13 13th > > 6 6th 7 7th 1600 1600 83 83 283 283 400 400 22 22nd 13 13th > > 6 6th 8 8th 1600 1600 83 83 283 283 300 300 22 22nd 13 13th 6 6th 9 9th 1600 1600 83 83 283 283 344 344 30 30th 13 13th > > 6 6th 10 10th 1600 1600 83 83 283 283 344 344 15 15th 13 13th 6 6th 11 11th 1600 1600 83 83 283 283 344 344 22 22nd 30 30th > > 6 6th 12 12th 1600 1600 83 83 283 283 344 344 22 22nd 5 5 6 6th

kur A - citrinos rūgštiswhere A is citric acid

B - 3,3’, 5, 5'-tetrametil-4,4'-diaminodifenilas C - vandenilio peroksidasB - 3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl C - hydrogen peroxide

D - dinatrio etilendiaminotetraacetatas E - dinatrio-divandenilio pirofosfatas F - acetanilidas * Laikas, per kurį tirpalo spalvos intensyvumas, matuojant spektrofotometru prie 652 nm, esant kontroliniame pavyzdyje vandeniui, neviršija 0.05.D - Disodium ethylenediaminetetraacetate E - Disodium dihydrogen pyrophosphate F - Acetanilide * The time during which the color intensity of the solution, measured with a spectrophotometer at 652 nm in the control sample, does not exceed 0.05.

Pavyzdys 2: Konjugato gavimo būdasExample 2: Method for Preparation of Conjugate

200 mg peroksidazės ištirpinami 15 ml 0,2 M fosfatinio buferio, pH 7,0. Į tirpalą lašais pridedami 9,0 ml 25% glutaraldehido tirpalo. Mišinys maišomas 24 vai, po to praplaunamas 6 kartus 0,2 M fosfatiniu buferiu pH 7,0 ultrafiltracijos aparate. Tada į tirpalą pridedamas 1 ml 30% - nio vandeninio 1,6-heksametilendiamino tirpalo ir mišinys maišomas 24 vai. Toliau reakcijos mišinys praplaunamas 8 kartus 0,2 M fosfatiniu buferiu ultrafiltracijos aparate. Gautas modifikuotos peroksidazės tirpalas inkubuojamas su N-vinilpirolidono ir maleino rūgšties anhidrido kopolimeru, prie kurio prijungtas digitoninas. Gautas tokiu būdu konjugatas išlaiko daugiau 80% peroksidazinio aktyvumo saugojant 6 mėn.Dissolve 200 mg of peroxidase in 15 ml of 0.2 M phosphate buffer, pH 7.0. To the solution are added dropwise 9.0 ml of 25% glutaraldehyde solution. The mixture is stirred for 24 hours and then washed 6 times with 0.2 M phosphate buffer pH 7.0 in an ultrafiltration apparatus. Then 1 ml of a 30% aqueous solution of 1,6-hexamethylenediamine is added to the solution and the mixture is stirred for 24 hours. The reaction mixture is then washed 8 times with 0.2 M phosphate buffer in an ultrafiltration apparatus. The resulting solution of modified peroxidase is incubated with N-vinylpyrrolidone and maleic anhydride copolymer to which digitonin is attached. The resulting conjugate retains more 80% of the peroxidase activity for 6 months.

Šio išradimo panaudojimas suteikia sekančius pranašumus:The use of the present invention provides the following advantages:

1. Siūlomo ryškalo stabilumas (laikas per kurį tirpalas išlieka bespalvis) padidėja nuo 6-8 vai iki 6 mėnesių, t.y. 540-720 kartų, o konjugato stabilumas (laikas per kurį išsilaiko ne mažiau 80 % peroksidazinio aktyvumo) padidėja nuo 2-3 mėn. iki 6 mėn., t.y. 2-3 kartus.1. The stability of the proposed developer (the time during which the solution remains colorless) increases from 6-8 hours to 6 months, i.e. 540-720 times, and the stability of the conjugate (the time to maintain at least 80% of the peroxidase activity) increases from 2-3 months. up to 6 months, i.e. 2-3 times.

2. Konjugato peroksidazinio aktyvumo ir ryškalo tirpalo stabilizavimas ekspluatacines savybes.2. Operational properties of conjugate peroxidase activity and stabilizer solution stabilization.

pagerina rinkinioimproves the set

Claims (2)

IŠRADIMO APIBRĖŽTISDEFINITION OF INVENTION 1. Rinkinys cholesterino vizualiniam nustatymui odos paviršiuje, susidedantis iš konjugato, turinčio giminingumą epidermio cholesterinui ir ryškalo tirpalo, besiskiriantis tuo, kad ryškalas susideda iš (mg/1):1. Kit for visual determination of cholesterol on the surface of the skin, consisting of a conjugate having affinity for epidermal cholesterol and a developer solution, characterized in that the developer consists of (mg / l): citrinos rūgšties 1000 - 2000 3,3', 5,5'-tetrametil-4,4'-diaminodifenilo 70 - 83 vandenilio peroksido 250 - 300 dinatrio etilendiaminotetraacetato 300 - 400 dinatrio divandenilio pirofosfato 15 - 30 acetanilido 5 - 30 vandens likusi dalis iki 1 1citric acid 1000-2000 3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl 70-83 hydrogen peroxide 250-300 disodium ethylenediaminetetraacetate 300-400 disodium dihydrogen pyrophosphate 15-30 acetanilide 5-30 water up to 1 1 2. Konjugato, turinčio giminingumą epidermio cholesterinui gavimo būdas prijungiant prie N-vinilpirolidono ir maleino rūgšties anhidrido kopolimero digitoniną ir po to peroksidazę, besiskiriantis tuo, kad peroksidazę modifikuoja, veikiant ją glutaraldehidu ir 1,6-heksametilendiaminu.2. A process for the preparation of a conjugate having an affinity for epidermal cholesterol by attaching digitonin to a copolymer of N-vinylpyrrolidone and maleic anhydride followed by peroxidase modification by the action of glutaraldehyde and 1,6-hexamethylenediamine.
LT95-057A 1995-06-01 1995-06-01 Kit for the cholesterol vizualization on the skin and process for preparing conjugate for use in cholesterol determination LT4121B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1675769A1 (en) 1988-01-19 1991-09-07 Научно-исследовательский институт физико-химической медицины Method for preparation of agent, used for visual detection of cholesterol on the skin surface

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1675769A1 (en) 1988-01-19 1991-09-07 Научно-исследовательский институт физико-химической медицины Method for preparation of agent, used for visual detection of cholesterol on the skin surface

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LOPUKHIN YM: "The skin and atherosclerosis (a three-drop test)", PHYSIOCHEMICAL ASPECTS OF MEDICINE REVIEWS, 1992, pages 1 - 124

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