DK160446B - PHOTOMETRIC PROCEDURE FOR CONCENTRATION DETERMINED BY REACTIONS PROCESSED IN THE ESTABLISHMENT OR CONSUMPTION OF LIGHT DISTRIBUTION CENTERS - Google Patents
PHOTOMETRIC PROCEDURE FOR CONCENTRATION DETERMINED BY REACTIONS PROCESSED IN THE ESTABLISHMENT OR CONSUMPTION OF LIGHT DISTRIBUTION CENTERS Download PDFInfo
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Abstract
Description
iin
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Opfindelsen angår en fremgangsmåde til bestemmelse af koncentrationen af en reaktionspartner ved hjælp af en lysspredningsmåling.The invention relates to a method for determining the concentration of a reaction partner by means of a light scattering measurement.
Fænomenet med lysspredning ved partikler, der 5 befinder sig i et homogent medium, benyttes til koncentrationsbestemmelse ved både måling af intensiteten af spredt lys (nephelometri) og ved måling af intensitetstabet af lysstrålen, der går gennem mediet, (turbi-dimetri).The phenomenon of light scattering on particles contained in a homogeneous medium is used for concentration determination both in measuring the intensity of scattered light (nephelometry) and in measuring the intensity loss of the light beam passing through the medium (turbi-dimetry).
10 Mange kemiske reaktioner fører i flere trin til molekylaggregater eller makromolekyler, der med hensyn til deres lysspredningsopførsel adskiller sig meget stærkt fra udgangsstofferne, og hvis koncentration kan bestemmes ved hjælp af denne egenskab.Many chemical reactions, in several steps, lead to molecular aggregates or macromolecules that differ greatly in their light scattering behavior from the starting materials and whose concentration can be determined by this property.
15 For eksempel går mange ioner ved overskridelse af opløselighedsproduktet sammen til krystaller, der først viser sig som uklarhed og til slut bliver så store, at de sedimenteres fra den flydende fase.15 For example, when the solubility product is exceeded, many ions coalesce into crystals that first appear as cloudiness and eventually become so large that they are sedimented from the liquid phase.
Et andet eksempel er den immunkemiske reaktion 20 mellem et opløseligt antigen og et bivalent antistof, der kan føre til store og stærkt lysspredende molekylaggregater .Another example is the immunochemical reaction 20 between a soluble antigen and a bivalent antibody which can lead to large and highly light scattering molecular aggregates.
Det tidsmæssige forløb af sådanne reaktioner svarer meget hyppigt til det almene kinetiske forløb af 25 på hinanden følgende reaktioner af 1. orden, dvs. koncentrationskurven for dannelsen af slutproduktet udviser et vendepunkt, og den maksimale reaktionshastighed optræder således først i løbet af reaktionen.The temporal course of such reactions very frequently corresponds to the general kinetic course of 25 consecutive 1st order reactions, ie. the concentration curve for the formation of the final product exhibits a turning point, and thus the maximum reaction rate does not occur until the reaction.
Med en immunkemisk reaktion som eksempel er 30 dette forløb vist for to antigenkoncentrationer i fig.With an immunochemical reaction as an example, this process is shown for two antigen concentrations in FIG.
1 på tegningen.1 of the drawing.
Ud fra signal/tid-kurverne i fig. 1 kan der på forskellig måde fås et koncentrationsafhængigt målesignal (tabel I).From the signal / time curves of FIG. 1, a concentration dependent measurement signal can be obtained in different ways (Table I).
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22
Tabel I.Table I.
Tidsmæssige randbetingelser ved tilvejebringelse af koncentrationsafhængige målesignaler (S “ signal, t = tid).Temporary boundary conditions in providing concentration-dependent measurement signals (S “signal, t = time).
Navn Princip 5 1) Endepunkts- Målesignalet bestemmes på et så metode sent tidspunkt, at det erfarings- j mæssigt ikke mere forandres, men j at der endnu ikke finder nogen iName Principle 5 1) The endpoint measurement signal is determined at such a late stage that it is no longer experientially changed, but that no
udfældning sted. Iprecipitation place. IN
10 2) Kinetisk Det egentlige målesignal er dif- metode ferensen mellem to signaler, der | "fixed-time"- bestemmes på to forskellige, men -kinetik forud givne tidspunkter.10 2) Kinetic The actual measurement signal is the dif- ference between two signals that | "fixed-time" - determined at two different but-kinetics predetermined times.
3) Kinetisk metode Den maksimale reaktionshastighed, 15 "rate determina- dvs. den maksimale ændring af tion" "peak rate signalet pr. tidsenhed, måles: method" a) ved måling af &S ved til strækkelig korte intervaller af Lt og bestemmelse af den stør-20 s te kvotient AS/&t, b) ved elektronisk differentiering dS/dt og bestemmelse af maksimummet, c) ved konstruktion af tangen- 25 ten til signal/tid-kurven og be stemmelse af den maksimale stigning.3) Kinetic method The maximum reaction rate, 15 "rate determina- ie the maximum change of tion" "peak rate signal per unit of time, is measured: method" a) by measuring & S at sufficiently short intervals of Lt and determining it b) by electronically differentiating dS / dt and determining the maximum; c) by constructing the tangent to the signal / time curve and determining the maximum increase.
Et stort antal analytter kan i dag kvantificeres ifølge de i tabel I anførte metoder ved direkte eller 30 indirekte måling af spredt lys.A large number of analytes can today be quantified according to the methods listed in Table I by direct or indirect measurement of scattered light.
Hvis man betragter afhængigheden af et egnet målesignal (tabel I) af koncentrationen af én reaktionspartner, f.eks. antigenet, medens den anden reaktions-partner anvendes med konstant koncentration, kan man 35 f.eks. ved iramunkemiske reaktioner observere den i fig.Considering the dependence of a suitable measurement signal (Table I) on the concentration of one reaction partner, e.g. the antigen, while the second reaction partner is used at constant concentration, e.g. by iramochemical reactions observe the one in fig.
2 viste sammenhæng. Den overraskende kendsgerning, at2. The surprising fact that
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3 det samme målesignal både kan tilbageføres til en lav og en høj koncentration af analytten, fører til en dobbelttydighed af signal/koncentration-relationen, der er kendt af fagmanden som antigenoverskudsfænomenet eller 5 Heidelberg-kurven.3, the same measurement signal can be returned to both a low and a high concentration of the analyte, leading to an ambiguity of the signal / concentration relationship known to those skilled in the art as the antigen excess phenomenon or the Heidelberg curve.
Denne dobbelttydighed kan principielt observeres overalt, hvor komplekser med forskellig støkiometri er mulige, alt efter overskuddet af den ene eller anden reaktionspartner, og disse komplekser ikke adskil-10 ler sig med hensyn til signalegenskaber, f.eks. lys spredning, f.eks.This ambiguity can, in principle, be observed where complexes of different stoichiometry are possible, depending on the excess of one or other reaction partner, and these complexes do not differ in signal properties, e.g. light scattering, e.g.
MonxCoMonx+m og Monx+mCoMonx hvori Mon = monomerMonxCoMonx + m and Monx + mCoMonx wherein Mon = monomer
CoMon = Co-monomer 15 Ag2 Ak og AgAk2 hvori Ag = antigenCoMon = Co-monomer Ag2 Ak and AgAk2 wherein Ag = antigen
Ak - homologt antistofAc - homologous antibody
Lee2 GP og Lee GP2 hvori Lee = lectin GP = glycoprotein 20 (AgxClx+1)- og ^Ågx+lC1x*+ hvori A9 = sølvionLee2 GP and Lee GP2 wherein Lee = lectin GP = glycoprotein 20 (AgxClx + 1) - and ^ Ågx + 1C1x * + wherein A9 = silver ion
Cl = chloridionCl = chloride ion
Hvis man gennemfører sådanne reaktioner med det formål at bestemme koncentration, definerer og op-25 timerer man metoden til en bestemt overskudssituation.If such reactions are carried out for the purpose of determining concentration, one defines and optimizes the method for a particular surplus situation.
I praksis fører dette til vanskeligheder, hvor analytten kan foreligge i et meget stort koncentrationsområde, og koncentrationsoverskuddet af en reaktionsdeltager ikke kan sikres.In practice, this leads to difficulties where the analyte can be present in a very large concentration range and the excess concentration of a reaction participant cannot be assured.
30 Denne situation spiller især en rolle ved im munkemisk bestemmelse af immunoglobuliner, hvis koncentration kan variere med en faktor 1000. Testbetingelserne og den økonomisk optimale antistofkoncentration til bestemmelse af den normale og subnormale koncen-35 tration tillader ikke indbefatning af ;en koncentration, der er forhøjet med en faktor 1000.30 This situation plays a particular role in immunochemical determination of immunoglobulins, the concentration of which may vary by a factor of 1000. The test conditions and the economically optimal antibody concentration to determine the normal and subnormal concentration do not allow the inclusion of; increased by a factor of 1000.
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Det i det følgende anførte vedrører derfor kun immunkemiske reaktioner, selv om fremgangsmåden principielt er anvendelig på alle reaktioner, der har de ovenfor diskuterede egenskaber og udviser en analog 5 kinetisk opførsel.Therefore, what follows is only for immunochemical reactions, although the method is in principle applicable to all reactions having the properties discussed above and exhibiting an analogous kinetic behavior.
Med hensyn til immunkemiske reaktioner er der blevet foreslået et stort antal fremgangsmåder og udførelsesformer til at erkende, på hvilken del af Hei-delbergkurven man befinder sig med en given analyse- 10 blanding.With regard to immunochemical reactions, a large number of methods and embodiments have been proposed to recognize which part of the Hei-delberg curve is present with a given assay mixture.
I det følgende nævnes nogle eksempler: 1. Den ældste og sikreste metode til erkendelse af et antigenoverskud er gennemførelse af en dobbeltbestemmelse med to forskellige prøvefortyndinger. Når 15 der foreligger et antigenoverskud, får man et højere signal ved den største fortynding og finder tilsyne- . ladende et højere proteinindhold end i den koncentrerede prøve (H.E. Schultze og G. Schwick, Prot. Eiol. Fluids 5, 15-25 (1958)).The following are some examples: 1. The oldest and safest method for recognizing an antigenic excess is the implementation of a dual assay with two different sample dilutions. When an antigen excess is present, a higher signal is obtained at the largest dilution and detection is seen. loading a higher protein content than in the concentrated sample (H.E. Schultze and G. Schwick, Prot. Eiol. Fluids 5, 15-25 (1958)).
20 Ifølge forbedrede udførelsesformer for denne fremgangsmåde sker der en yderligere tilsætning af antistoffer til prøveblandingen, efter at reaktionen har nået en vis ligevægt. Når der foreligger et antigenoverskud, optræder der en signalforøgelse (T.O. Tiffany et 25 al., Clin. Chem. 20^, 1055-1061 (1974)). Ligeledes kan et antigenoverskud erkendes ved efterdosering af antigenmateriale med kendt koncentration (J.C. Sternberg,According to improved embodiments of this method, further addition of antibodies to the sample mixture occurs after the reaction has reached a certain equilibrium. When an antigen excess is present, a signal increase occurs (T. O. Tiffany et al., Clin. Chem. 20, 1055-1061 (1974)). Likewise, an antigen excess can be recognized by post-dose antigenic material of known concentration (J.C. Sternberg,
Clin. Chem. 23, 1456-1464 (1977)).Clin. Chem. 23, 1456-1464 (1977)).
2. I det specielle tilfælde, hvor der er tale 30 om "continuous flow"-teknik observeres det, at der foreligger et antigenoverskud, ved at der optræder en dobbelttop (R.F. Ritchie, Protides Biol. Fluids 21, 569 (1974)).2. In the special case of continuous flow technique 30, an antigen excess is observed by a double peak (R.F. Ritchie, Protides Biol. Fluids 21, 569 (1974)).
3. Ved betragtning af reaktionskinetikken ef-35 ter grafisk afbildning af reaktionsforløbet kan man ved turbidimetrisk måling skelne mellem antistofoverskud og3. In view of the reaction kinetics after graphically depicting the reaction course, by turbidimetric measurement one can distinguish between antibody excess and
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5 "mildt" antigenoverskud (I. Deverill, Protides Biol.5 "mild" antigen excess (I. Deverill, Protides Biol.
Fluids 2£, 697 (1979); P.J.J. van Munster et al., Clin.Fluids 2, 697 (1979); P.J.J. of Munster et al., Clin.
Chim. Acta 76, 377-388 (1977)).Chim. Acta 76, 377-388 (1977)).
4. Ved bestemmelse af varigheden af reaktionen, 5 indtil der optræder den maksimale reaktionshastighed, ved nephelometrisk måling kan der beregnes en størrelse, der tillader diskriminering mellem antigen- og antistofoverskud (DE-A 2.724.722 C2).4. In determining the duration of the reaction, 5 until the maximum reaction rate occurs, by nephelometric measurement, a size that allows discrimination between antigen and antibody excess (DE-A 2,724,722 C2) can be calculated.
De under punkt 2 og 4 nævnte metoder adskiller 10 sig fordelagtigt fra de andre metoder ved, at ikke alle prøver skal undersøges for, om der foreligger et antigenoverskud, men at der under målingen fås information om, hvorvidt der skal gennemføres en prøvning for antigenoverskud .The methods mentioned in paragraphs 2 and 4 10 differ advantageously from the other methods in that not all tests need to be tested for antigen excess, but during the measurement information on whether an antigen excess test should be performed.
15 Metode 2 er bundet til en bestemt teknik og er ikke alment anvendelig til nephelometriske eller turbi-dimetriske proteinbestemmelser.Method 2 is bound to a particular technique and is not generally applicable to nephelometric or turbimetric protein determinations.
Ifølge DE-A 2.724.722 C2 gås der som kendt teknik ud fra, at der eksisterer en funktion af den maksimale reak-20 tionshastighed, der ved hjælp af en tærskelværdi af denne funktion gør det muligt at skelne mellem antigenoverskud og antistofoverskud (krav l). I spalte 12, linie 8-21, i dette patentskrift anføres det, at der som diskriminatorfunktion kan anvendes den tid (T), der forløber fra reaktionens start 25 og indtil der optræder en maksimalværdi (H) af reaktionshastigheden. Det her anførte begrænses imidlertid stærkt i spalte 21, linie 55-68, hvor det anføres, at der ikke eksisterer nogen enkelt tidsværdi, ved hvilken antistofoverskudskurvedelen ligger på den ene side af denne værdi, og antigen-30 overskuds-kurvedelen ligger på den anden side. Ifølge patentskriftet løses problemet ved koordinattransformation og indføring af nye variable. Endvidere er metoden karakteriseret ved, at man, når der konstateres en antigenoverskudstilstand, gentager målingen efter tilsvarende fortynding af 35 prøven til opnåelse af en antistofoverskudstilstand. Denne yderligere måling på en prøve, der findes at være i antigen-According to DE-A 2,724,722 C2, as known in the art, it is assumed that a function of the maximum reaction rate exists which, by means of a threshold value of this function, makes it possible to distinguish between antigen excess and antibody excess (claim 1 ). Column 12, lines 8-21 of this patent states that as a discriminatory function, the time (T) elapsing from the start of the reaction 25 until a maximum value (H) of the reaction rate can be used. However, the foregoing is greatly limited in column 21, lines 55-68, where it is stated that no single time value exists at which the antibody excess curve portion is on one side of this value and the antigen excess curve portion is on the other. page. According to the patent, the problem is solved by coordinate transformation and introduction of new variables. Furthermore, the method is characterized in that when an antigen excess state is found, the measurement is repeated after corresponding dilution of the sample to obtain an antibody excess state. This additional measurement on a sample found to be in antigen
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6 overskuds-tilstand, er materiale- og tidskrævende.6 surplus state is material and time consuming.
Det har derfor været formålet med den foreliggende opfindelse at tilvejebringe en fremgangsmåde, der gør det muligt at bestemme en prøves indhold af et an- j 5 tigen eller antistof uden yderligere måling, også når j denne befinder sig i den foreliggende prøveblandings antigenoverskudstilstand .It has therefore been the object of the present invention to provide a method which allows the determination of a sample's content of an antigen or antibody without further measurement, even when it is in the antigen excess state of the present sample mixture.
Det har nu overraskende vist sig, at der ved måling af tiden, der forløber fra reaktionens„start og 10 indtil der optræder en maksimalværdi af reaktionshastigheden, ved en kinetisk metode, dvs. ved bestemmelse af den maksimale reaktionshastighed som funktion af koncentrationen, kan måles kvantitativt på begge sider af Heidelbergkurven. Det er ikke mere nødvendigt at skel-15 ne mellem antistofoverskud og antigenoverskud.It has now surprisingly been found that by measuring the time elapsed from the onset of the reaction and 10 until a maximum value of the reaction rate occurs, by a kinetic method, i.e. by determining the maximum reaction rate as a function of concentration, can be quantitatively measured on both sides of the Heidelberg curve. It is no longer necessary to distinguish between antibody excess and antigen excess.
Opfindelsen angår således en fremgangsmåde til fotometrisk bestemmelse af koncentrationen af en reaktant i en reaktion, der forløber under dannelse eller forbrug af lysspredningscentre i retning af en o-20 vergang fra Rayleigh- til Mie-spredning, hvorved den maksimale reaktionshastighed (V ax) og tidsrummet fra reaktionens begyndelse og indtil der optræder den maksimale reaktionshastighed (tmax) bestemmes, man til éntydig bestemmelse af koncentrationen empirisk bestemmer den funk-25 tionelle sammenhæng mellem koncentration, Vmax og tmax ved hjælp af et standardpræparat, og Vmax og tmax måles på prøven, hvilken fremgangsmåde er ejendommelig ved, at der blandt to mulige koncentrationer udvælges den rigtige koncentration ved hjælp af den 2. reaktionsparameter, altså Vmax eller 30 ^max*The invention thus relates to a method for photometric determination of the concentration of a reactant in a reaction which proceeds during the formation or consumption of light scattering centers towards an o-transition from Rayleigh to Mie scattering, whereby the maximum reaction rate (V ax) and the time from the beginning of the reaction until the maximum reaction rate (tmax) is determined, the unambiguous determination of concentration determines the functional relationship between concentration, Vmax and tmax using a standard preparation, and Vmax and tmax are measured on the sample, which method is peculiar in that, from two possible concentrations, the correct concentration is selected by means of the second reaction parameter, ie Vmax or 30
Relationen mellem koncentration og V og t * max ^ max kan f.eks. foreligge i en tabel, en matriks eller en eller flere matematiske funktioner.The relationship between concentration and V and t * max ^ max can e.g. are present in a table, a matrix, or one or more mathematical functions.
Det er kendt, at reaktionshastigheden og sig-35 nalintensiteten af antigen-antistofreaktioner kan påvirkes inden for et vidt område alt efter reaktions-It is known that the rate of reaction and the signal intensity of antigen-antibody reactions can be affected in a wide range depending on the reaction time.
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7 mediets ionstyrke, ionart og polymertilsætning. Den følgende redegørelse er derfor ikke begrænset til de i eksemplerne anførte betingelser, men gælder for udfældningsreaktioner, der kan påvirkes på den ovennævnte måde.7 the ionic strength, ionic nature and polymer addition of the medium. The following disclosure is therefore not limited to the conditions set forth in the Examples, but applies to precipitation reactions which may be affected in the above manner.
5 Hvis man bestemmer den maksimale reaktionshas tighed (V ) i afhængighed af koncentrationen, får man den i fig. 2 viste sammenhæng. Hvis man samtidig med Vmax t,esteiraner det tidsrum, der forløber fra begyndelsen af reaktionen og indtil nås (t„,_), fås den i fig.5 If the maximum reaction rate (V) is determined depending on the concentration, the one obtained in FIG. 2. If, at the same time as Vmax t, esteirans, the time elapsing from the beginning of the reaction until reaching (t „, _) is obtained, it is obtained in fig.
10 3 viste sammenhæng. Hver af de to signal-koncentrations kurver udviser antigenoverskuds-fænomenet. Bestemte signaler kan tilordnes to koncentrationer.10 3. Each of the two signal concentration curves exhibits the antigen excess phenomenon. Certain signals can be assigned to two concentrations.
Ved projicering af de to funktioner i én afbildning (fig. 4) ser man, at der ikke eksisterer et værdi- 15 par (Vm!iv, t aw) , der kan tilordnes to forskellige max max koncentrationer.Projection of the two functions in one image (Fig. 4) shows that there is no value pair (Vm! Iv, t aw) that can be assigned two different max max concentrations.
Den maksimale reaktionshastighed (Vmax) og tidsrummet til opnåelse af den maksimale reaktionshastighed (t x) er åbenbart af hinanden uafhængige variable 20 af koncentrationen, og Heidelberg-kurven er en tredimensional kurve (fig. 5), der ved passende reaktionsstyring ikke udviser nogen dobbelttydighed med hensyn til koncentrationen.The maximum reaction rate (Vmax) and the time to obtain the maximum reaction rate (tx) are obviously independent variables 20 of the concentration, and the Heidelberg curve is a three-dimensional curve (Fig. 5) which, with appropriate reaction control, exhibits no ambiguity with taking into account the concentration.
Målemetoder, hvorved der kun arbejdes med en 25 todimensional projektion af denne kurve, f.eks. måling af V som funktion af koncentrationen (fig. 6), ud-viser altid en dobbelttydighed med hensyn til koncentrationen .Measurement methods that only work with a 25 two-dimensional projection of this curve, e.g. measuring V as a function of concentration (Fig. 6), always shows an ambiguity with respect to concentration.
Ved den praktiske vurdering af de fundne sam-30 menhænge skal der tages hensyn til, at koncentrationen i nærheden af en signal-koncentrationskurves minimum eller maksimum kun kan bestemmes med mindre præcision, da relativt ringe signaldifferenser svarer til en stor koncentrationsdifferens. Ved den her omhandlede metode til 35 bestemmelse af to uafhængige målesignaler forbedres præcisionen især, når minimummet af funktionen kone. =In the practical assessment of the correlations found, it must be taken into account that the concentration in the vicinity of a minimum or maximum signal concentration curve can only be determined with less precision, since relatively small signal differences correspond to a large concentration difference. In this method of determining two independent measurement signals, the accuracy is improved especially when the minimum of the function wife. =
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f(tmax) ligger så langt fra maksimummet af funktionen kone. = f(V ) som muligt.f (tmax) is so far from the maximum of the function wife. = f (V) as possible.
Arbejdsmåden og reaktionsbetingelserne ved den her omhandlede fremgangsmåde vises i de følgende eksemp-5 ler.The operation and reaction conditions of the present process are illustrated in the following examples.
Eksempel 1Example 1
Bestemmelse af maksimumsområdet af en kalibreringskurve for IgG.Determination of the maximum range of a calibration curve for IgG.
t0 Fotometer: DU-8 med tilsatsdel til nephelometriske målin ger (Beckman Instruments).t0 Photometer: DU-8 with additive part for nephelometric measurements (Beckman Instruments).
Reaktionsmediumr 0f02 mol/liter kaliumphosphatpuffer, pH-værdi 7,3, med 40 g/liter polyethy-lenglycol 6000 (Serva), Heidelberg, 15 Vesttyskland, og 64 g/liter natriumbro- mid.Reaction medium rf02 mol / liter potassium phosphate buffer, pH 7.3, with 40 g / liter polyethylene glycol 6000 (Serva), Heidelberg, West Germany, and 64 g / liter sodium bromide.
Prøvefortyndingsmedium: fysiologisk natriumchloridopløsning .Sample dilution medium: physiological sodium chloride solution.
Antiserum: LN-antiserum mod humant IgG/kappa-kæde (fra 20 kaniner) (Behringwerke AG, OSAS 14), fortyn det 1:31 med reaktionsmedium.Antiserum: LN antiserum against human IgG / kappa chain (from 20 rabbits) (Behringwerke AG, OSAS 14), dilute it 1:31 with reaction medium.
Standard: T-protein-standard-serum (humant), (Behringwerke AG, OSKT 06).Standard: T protein standard serum (human), (Behringwerke AG, OSKT 06).
Prøveblanding: til 20/Uliter prøve-/standardfortynding AE ' sættes 500yUliter antiserumfortynding, og der måles straks i fotometeret under anvendelse af kinetik-II-programmet i maksimalt 3 minutter ved en bølgelængde på 334 nm.Sample Mix: At 20 µL sample / standard dilution AE, 500 µL liter antiserum dilution is added and measured immediately in the photometer using the kinetics-II program for a maximum of 3 minutes at a wavelength of 334 nm.
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9 a) Turbidimetrisk måling.9 a) Turbidimetric measurement.
Tid fra reaktionensTime from the reaction
Koncentration af Maksimal reaktions- begyndelse og indtil standardfortynding, hastighed (vmax i der optræder maksimum 5 mg/dl IgG_ mE/min.)_ (t i sek.)_ 110,4 425 38 92 466 30 78.9 542 24 73.6 569 20 10 69 583 18 64.9 576 16 61,3 567 16 55.2 548 14 22.1 219 20 15 11,0 75 30 b) Nephelometrisk måling. Tid fra reaktionensConcentration of Maximum Reaction onset and up to standard dilution, rate (vmax in which maximum 5 mg / dl IgG_ mE / min occurs) _ (ten sec) _ 110.4 425 38 92 466 30 78.9 542 24 73.6 569 20 10 69 583 18 64.9 576 16 61.3 567 16 55.2 548 14 22.1 219 20 15 11.0 75 30 b) Nephelometric measurement. Time from the reaction
Koncentration af Maksimal reaktions- begyndelse og indtil standardfortynding, hastighed (Vmax i der optræder maksimum mg/dl IgG mE/min.) (t i sek.) —*--------- * ' ' 1 — * 1 -—max........ 1 1 20 110,4 1410 36 92 1490 30 78.9 1710 22 73.6 1730 22 69 1780 20 25 64,9 1800 20 61.3 1810 20 55.2 1720 18 22,1 706 24 11,0 325 42 30Concentration of Maximum Reaction Beginning and Up to Standard Dilution Speed (Vmax in Maximum mg / dl IgG mE / min Occurred) (Ten Secs) - * --------- * '' 1 - * 1 - —Max ........ 1 1 20 110.4 1410 36 92 1490 30 78.9 1710 22 73.6 1730 22 69 1780 20 25 64.9 1800 20 61.3 1810 20 55.2 1720 18 22.1 706 24 11.0 325 42 30
Eksempel 1 viser, at der både ved turbidimetrisk og ved nephelometrisk måling éntydigt kan bestemmes koncentrationer i og på begge sider a£ Heidelberg-kurvens maksimum. Til bestemmelse af en ukendt prøvekoncentra-35 tion interpoleres der efter måling af V og t mel-lem standardens tabellerede værdier. Måleområdet fast-Example 1 shows that both turbidimetric and nephelometric measurements can unambiguously determine concentrations in and on both sides of the maximum Heidelberg curve. To determine an unknown sample concentration, after measuring V and t between the tabulated values of the standard, interpolation is interpolated. Measuring range fixed
DK 160446 BDK 160446 B
10 o lægges ved passende fortynding af prøverne - i eksempel 1 ved en 1:51-fortynding af prøven på 560 til 5600 mg/dl IgG - og kan vælges således, at delområdet med den ringere præcision ikke falder inden for det klinisk 5 relevante område for bestemmelse af de tilsvarende parametre .10 o is added by appropriate dilution of the samples - in Example 1, at a 1: 51 dilution of the sample of 560 to 5600 mg / dl IgG - and can be selected so that the sub-area of the inferior precision does not fall within the clinically relevant range for determining the corresponding parameters.
Eksempel 2.Example 2.
Optagelse af en kalibreringskurve for IgM 10 (turbidimetrisk).Recording a calibration curve for IgM 10 (turbidimetric).
Fotometer: DU-8 (Beckman Instruments).Photometer: DU-8 (Beckman Instruments).
Reaktionsmedium: 0,15M natriumchlorid-phosphatpuffer, pH-værdi 7,2, med 39 g/liter PEG 6000 (Serva) og 8 g/liter NaCl.Reaction medium: 0.15M sodium chloride phosphate buffer, pH 7.2, with 39 g / liter PEG 6000 (Serva) and 8 g / liter NaCl.
15 Prøvefortyndingsmedium: fysiologisk natriumchloridop løsning.Sample dilution medium: physiological sodium chloride solution.
Antiserum: LN-antiserum mod humant IgM^u-kæde (fra kaniner) (Behringwerke AG, OSAT 14), fortyndet 1:11 med reaktionsmedium.Antiserum: LN antiserum against human IgMβ chain (from rabbits) (Behringwerke AG, OSAT 14), diluted 1:11 with reaction medium.
20 Standard: T-protein-standard-serum (humant) (Behring werke AG, OSKT 06).Standard: T protein standard serum (human) (Behring werke AG, OSKT 06).
Prøveblanding: til 200^,uliter prøve-/standardfortynding sættes 500^uliter antiserumfortynding, og der måles derefter straks i fotometeret 25 under anvendelse af kinetik-II-programmet i maksimalt 3 minutter ved en bølgelængde på 312 nm.Sample Mix: To 200 µL of sample / standard dilution, 500 µL of antiserum dilution is added and then immediately measured in Photometer 25 using the Kinetics II program for a maximum of 3 minutes at a wavelength of 312 nm.
30 3530 35
11 DK 160446 B11 DK 160446 B
oisland
Tid fra reak-Time from reaction
Konc. af stan- Kone. af prøve Maks. reakti- tionsbegyndelse dardfortynding, fortyndet 1:21, onshastighed til maksimum m9/dl IgM mg/dl IgM (V i mE/min.) (t i sek.) -----i-max--- —max- — 5 110 2310 371 34 74 1540 508 28 55 1155 591 18 44 924 639 14 10 37 770 639 12 31 660 620 12 28 578 611 10 24 513 598 10 22 462 556 10 15 20 420 508 10 18 385 488 10 17 355 467 10 16 330 460 10 15 308 419 10 20 14 290 405 10 13 272 378 10 12 257 368 10 11 231 330 10 5,5 116 158 10 25 2,8 58 69 10 1,4 29 27 12Conc. of stan- Wife. of sample Max. reaction commencement dard dilution, diluted 1:21, on-speed to maximum m9 / dl IgM mg / dl IgM (V in mE / min) (ten seconds) ----- i-max --- —max- - 5 110 2310 371 34 74 1540 508 28 55 1155 591 18 44 924 639 14 10 37 770 639 12 31 660 620 12 28 578 611 10 24 513 598 10 22 462 556 10 15 20 420 508 10 18 385 488 10 17 355 467 10 16 330 460 10 15 308 419 10 20 14 290 405 10 13 272 378 10 12 257 368 10 11 231 330 10 5.5 116 158 10 25 2.8 58 69 10 1.4 29 27 12
Det fremgår af eksempel 2, at der ved den valgte prøvefortynding kan måles en koncentration mellem 30 og 30 600 mg/dl IgM med en bedre præcision end mellem 600 og 900 mg/dl. Ved en normalområde mellem 70 og 300 mg/dl IgM i serum ligger værdier over 600 mg/dl imidlertid altid nær en mistanke om monoklonal gammopati og kræver uafhængigt af værdiens præcision yderligere undersøgel-35 ser.It is apparent from Example 2 that at the selected sample dilution, a concentration between 30 and 30 600 mg / dl IgM can be measured with a better precision than between 600 and 900 mg / dl. However, at a normal range between 70 and 300 mg / dl IgM in serum, values above 600 mg / dl always lie near a suspected monoclonal gammopathy and, regardless of the value of the value, require further investigations.
Claims (5)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19833347162 DE3347162A1 (en) | 1983-12-27 | 1983-12-27 | PHOTOMETRIC METHOD FOR DETERMINING CONCENTRATION IN REACTIONS WHICH ARE GIVING OR USING SPREAD CENTERS |
| DE3347162 | 1983-12-27 |
Publications (4)
| Publication Number | Publication Date |
|---|---|
| DK626584D0 DK626584D0 (en) | 1984-12-21 |
| DK626584A DK626584A (en) | 1985-06-28 |
| DK160446B true DK160446B (en) | 1991-03-11 |
| DK160446C DK160446C (en) | 1991-09-16 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK626584A DK160446C (en) | 1983-12-27 | 1984-12-21 | PHOTOMETRIC PROCEDURE FOR CONCENTRATION DETERMINED BY REACTIONS PROCESSED IN THE ESTABLISHMENT OR CONSUMPTION OF LIGHT DISTRIBUTION CENTERS |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0148463B1 (en) |
| JP (1) | JPS60158337A (en) |
| AT (1) | ATE37749T1 (en) |
| AU (1) | AU587368B2 (en) |
| CA (1) | CA1236707A (en) |
| DE (2) | DE3347162A1 (en) |
| DK (1) | DK160446C (en) |
| ES (1) | ES8605639A1 (en) |
| FI (1) | FI81448C (en) |
| NO (1) | NO166381C (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5019999A (en) * | 1988-09-30 | 1991-05-28 | Technicon Instruments Corporation | Immunoanalysis method for discriminating between antigen excess and antibody excess conditions |
| JPH0820355B2 (en) * | 1988-11-24 | 1996-03-04 | 元二 神保 | Particle size measurement method |
| DE4034509A1 (en) * | 1990-10-30 | 1992-05-07 | Boehringer Mannheim Gmbh | IMMUNOLOGICAL PRECIPITATION METHOD FOR DETERMINING A BINDABLE ANALYTIC AND REAGENT FOR CARRYING OUT THIS METHOD |
| DE4221807C2 (en) * | 1992-07-03 | 1994-07-14 | Boehringer Mannheim Gmbh | Method for the analytical determination of the concentration of a component of a medical sample |
| DE19524414A1 (en) * | 1995-07-05 | 1997-01-09 | Behringwerke Ag | Antigen excess free nephelometric and turbidimetric protein determinations |
| DE19602491A1 (en) | 1996-01-25 | 1997-07-31 | Bayer Ag | Methods for increasing the assay area and avoiding the hook effect in simultaneous sandwich immunoassays and corresponding test kits |
| DE19640121B4 (en) * | 1996-09-28 | 2007-04-26 | Dade Behring Marburg Gmbh | Method for determining a time-dependent variable to be measured |
| CN104991056B (en) * | 2015-08-05 | 2017-01-04 | 武汉林勉生物技术有限公司 | A kind of Serologic detection and the method for quantitative analysis |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4157871A (en) * | 1976-06-02 | 1979-06-12 | Beckman Instruments, Inc. | System for rate immunonephelometric analysis |
| US4268171A (en) * | 1977-07-18 | 1981-05-19 | Beckman Instruments, Inc. | Method determining concentration in rate nephelometric immunochemical analysis |
| US4174952A (en) * | 1978-01-23 | 1979-11-20 | Massachusetts Institute Of Technology | Immunoassay by light scattering intensity anisotropy measurements |
| US4204837A (en) * | 1978-03-14 | 1980-05-27 | Beckman Instruments, Inc. | Method of rate immunonephelometric analysis |
-
1983
- 1983-12-27 DE DE19833347162 patent/DE3347162A1/en not_active Withdrawn
-
1984
- 1984-12-17 DE DE8484115626T patent/DE3474478D1/en not_active Expired
- 1984-12-17 AT AT84115626T patent/ATE37749T1/en not_active IP Right Cessation
- 1984-12-17 EP EP84115626A patent/EP0148463B1/en not_active Expired
- 1984-12-21 NO NO845212A patent/NO166381C/en not_active IP Right Cessation
- 1984-12-21 DK DK626584A patent/DK160446C/en active
- 1984-12-21 FI FI845103A patent/FI81448C/en active IP Right Grant
- 1984-12-24 AU AU37164/84A patent/AU587368B2/en not_active Ceased
- 1984-12-24 ES ES84539023A patent/ES8605639A1/en not_active Expired
- 1984-12-26 JP JP59273534A patent/JPS60158337A/en active Granted
- 1984-12-27 CA CA000471071A patent/CA1236707A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| DK160446C (en) | 1991-09-16 |
| ES8605639A1 (en) | 1986-03-16 |
| JPH0231339B2 (en) | 1990-07-12 |
| DE3474478D1 (en) | 1988-11-10 |
| EP0148463A3 (en) | 1986-04-30 |
| EP0148463B1 (en) | 1988-10-05 |
| JPS60158337A (en) | 1985-08-19 |
| AU3716484A (en) | 1985-07-04 |
| ES539023A0 (en) | 1986-03-16 |
| NO845212L (en) | 1985-06-28 |
| FI845103L (en) | 1985-06-28 |
| NO166381B (en) | 1991-04-02 |
| AU587368B2 (en) | 1989-08-17 |
| FI81448C (en) | 1990-10-10 |
| FI81448B (en) | 1990-06-29 |
| CA1236707A (en) | 1988-05-17 |
| EP0148463A2 (en) | 1985-07-17 |
| FI845103A0 (en) | 1984-12-21 |
| NO166381C (en) | 1991-07-10 |
| ATE37749T1 (en) | 1988-10-15 |
| DE3347162A1 (en) | 1985-07-04 |
| DK626584A (en) | 1985-06-28 |
| DK626584D0 (en) | 1984-12-21 |
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