DD152424A1 - METHOD FOR SEPARING THE FREE AND ANTIBODY-LINKED LIGANDS FOR LABELED LIGAND IMMUNOASSAYS - Google Patents

Abstract

According to the invention, a novel, universally rationally applicable method for the separation of the free and antibody-bound ligand in isabelled ligand immunoassay systems is described using an immuno-immobilized Precipitationreagens' (IIPR). This method combines the advantages of dual antibody technology and solid-phase methods, while circumventing their disadvantages. The low unspecific binding of the labeled ligand provides an important prerequisite for sensitive, precise assay systems. By employing the novel separation procedure, the labeled ligand immunoassays are to be standardized both in the preparation as complete kits and in the performance itself, thus resulting in a substantial cost reduction e.g. the ridioimmunologischen determinations is possible.

Description

Field of application of the invention

The invention includes a universal method for the separation of the free and antibody-bound labeled antigen. Haptens for all Mgand-Iiniaunoassay systems for quantitative determination, predominantly biologically active substances such as hormones, enzymes or pharmaceuticals up to concentrations of 10 ~ 10 mmol / l, mostly in body fluids. The most extensive field of application is Eadioimmunoassay (ElA) and Enzyme Immunoassay (EIA) _β

Charak g terisierun the known technical solutions

After the introduction of the Eadiotune Assay (EIA) by YALOW and BEESOi? (J, "elin" Invest (1960) 1157) for insulin determination in serum have been published to date for over 200 substances ligand immunoassays They are based on the measurement of the percentage binding of the labeled ligand ^X L by the ligand-specific antibody AS-, in Dependence on the concentration of the unlabelled ligand L ₀ to be determined For all methods, the following balance is established for the ligand-specific immune reaction

marked

LIGAKD ^(X L)

+ ANTIBODIES (AK ₁ ) ^; ^ L-iE ^ 4- ²¹ L

LIGAIID (L)

unknown concentration

Concentration ² L and AK ₁ are kept constant in an incubation procedure. "After equilibration, separation of free (F) and antibody-bound (B) labeled ligands is required." Although the immune complex itself is soluble, various separation methods have been developed So far, no universally advantageous applicable method result * Thus, oils are the largest differences

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published radioimmunoassays and commercially available RIA kits in carrying out the separation of B and F.

The separation should be as quantitative as possible, thereby significantly affecting the lower detection limit of an EIA and also its precision. The separation process must be suitable for a high sample throughput. The balance of the ligand-specific immune response should not be demonstrably altered by the separation. Since these requirements are often difficult to meet, many separation methods have been developed.

Thus, electrophoresis, chromatography and ultracentrifugation were used because of the different mobility of B and F for separation. Disadvantages of these methods are the strong balance disturbances, the high expenditure of time and a low sample throughput.

Separation can also be achieved when B is precipitated by ethanol, dioxane, polyethylene glycol at 125 g / L or ammonium sulfate at 215 g / L. The first two, otherwise elegant methods have the disadvantage that with increasing protein content of the labeled free ligand (F) increasingly coprecipitated. In addition, the immune complexes of low affinity antibodies are cleaved here.

Another group of separation methods is based on the adsorption of the free ligand (F) on activated carbon, ion exchange resins, cellulose or silicates. The disadvantage of these methods is that B is also partially adsorbed, with the extent of binding being dependent on the medium.

Another possibility is the chemical fixation of the IgG-specific antibody before the reaction,. e.g. on cellulose or sephaxose (solid phase method) or on the inner Yiand of the incubation tube (tube-nethod), whereby a common. Centrifuge Ration or a simple decanting to "separate"

These methods have the disadvantages that the immobilized antibodies are mostly impaired in their affinity and thus the EIA in their sensitivity. The consumption of the usually expensive specific antibodies (AK-) is increased many times over by the fixation the incubation tubes have to because of the heterogeneous reaction phases.

, be moved constantly during the incubation.

All methods mentioned so far are physico-chemical. A further group of separation methods are to be summarized under the term double-antibody method. Here, the separation of B and F takes place in that by means of a second antibody (AKP) j which is directed against the immunoglobulin of the donor species for AK-, the soluble immune complex " ² L-AK _{1 is} precipitated" Since the AK ₁ in very low

3 6

Concentration (dilution of the antiserum 1: 1Cr to 1:10) is used, a normal serum (ITS) of the species of the AK -, - dispenser is added to the incubation at a dilution of about 1 i 100 »The concentration ratio of NS to AKp is tuned to achieve maximum immunoprecipitation. After the precipitation has ended, B is centrifuged off. The double antibody technique gives the best separation effect between B and F, the non-specific precipitation (Bu) is very low. The disadvantage of this method is that the binding of the. ^X L ~ AK- to AK ₂ by immunoglobulins of individual patients (BRUNFELDT, K ₆ and KeR ₆ JÖRGENSEN, Acta Endocrin. (1967) 34-7) is competitively inhibited, and thus false high values of L in the plasma are determined. In addition, the precipitation is quantitatively influenced by the complement.

In order to avoid the aforementioned disadvantages, was the so-called binding reagent, the specific vorpräzipitiertem AK- with AII ₂ with the addition of NS, worked (Hales ₅ CN. And PJ _e RANIjLS ₅ Biochen. _E £ J 8 (1963) 137), In this case, the advantage of the low Bu was retained, and the cross-reaction of the inununglobulin with the AE ₂ and the co-claviation factor is largely suppressed, but at the same time the advantages are obtained

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the homogeneous reaction phase for the ligand-specific immune reaction abandoned.

Another variant has been described by HOLLANDER et al., J. of Immunol., Methods Λ (1972) 2 * 4-7). "After this, AK _{2 is} fixed to a solid support and the soluble immune complex -, is obtained by immunoadsorption of the free ligand ^X. L separately. Since the fixed AKo can be added in excess, the cross-reactivity of the immunoglobulin with _AK? and the complement reaction ,. the disadvantage of this method is without influence on the quantitative binding of the ^X L-AK- due to the fact that chemical in the carrier fixing a significant portion of AK is inactivated _2, this increases the AKp consumption. Secondly, the Solid phase AKp is not stable, can be found after a few days of storage free AKp, making false high concentration of the ligand to be determined L are then found In addition, the unspecific binding can be increased by the solid phase, in addition to which the carrier fixation, after still partly unclear non-controllable reactions a and thus the coupling yield and the degree of inactivation of AKp and thus the binding properties of the solid phase AC for AK- can vary widely. However, the biggest disadvantage is the performance itself, because during the entire incubation (several hours) of the ^X L ~ AK ₁ binding, the samples must be shaken.

Object of the invention

The aim of the invention is the introduction of a universal method for the separation of the free (-F) and antibody-bound (B) ligands in the advantageous ligand immunoassay systems for determining, for example, hormones, enzymes, pharmaceuticals, specific proteins and others. Substances, 'mainly in biological but also other media, regardless of the type of labeling of the ligands (radioimmunoassay, enzyme immunoassay and other immunoassays) _β For the knowledge gain in biochemical and clinical research, the highly specific, sensitive analysis is an essential prerequisite, thus the

Need for very expensive test sets very high. With the introduction of the universal separation process according to the invention, a standardization in production and test execution would be given and, at the same time, a considerable cost saving would be associated.

Explanation of the essence of the invention-

The object of the invention is to introduce a novel, universal separation method for the separation of the free (F) and antibody-bound (B) ligands for the radio, enzyme, spin or generally labeled ligand-imx-monoassays.

The novel separation process is characterized in that the double antibody method with its implementation advantages is modified in such a way that additionally the advantages of the solid phase method are utilized, while maintaining the homogeneous reaction phase for the ligand-specific immune reaction, without the chemical fixation of the ligand-specific antibody ( AK-.) Binding second antibody (AKp) and the associated partial inactivation of the AK,

The combination of the mentioned advantages of the different separation methods has succeeded in that after the ligand-specific immune reaction in a homogeneous liquid phase, the soluble immune complex L-AK-v by adding a finely dispersed immunoprecipitate IIPR (immuno-immobilized precipitation reagent), which by immuno-priming of the AKp is bound with an ITS of the species of the AK -, - donor, is bound:

³¹ L-AK ₃ (B) + ^X L (F) + HFR ^ ²¹ L-AK ₁ -IIER j (B) + ^X L t. (F)

After incubation for only 30 minutes with the HPR, each sample is mixed with 2 ml of the nonionic-containing assay buffer, the final concentration of which (ITPLp ₂ SO being 1.16 mol / l Samples are centrifuged at 2000 g for 15 min, then additional wash is done.

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usually not necessary. "Instead of (M ^) ₂ SO. Polyethylene glycol 6000 (Merck, Darmstadt) may also be used at the final concentration of 30 g / l.

The IIPR is conveniently produced in a large batch (several thousand ampoules). The optimal concentration ratio of the AIL, species-like normals around (NS) and the second antiserum (AKo) directed against AK.sup.-species-like immunoglobulin is tested in advance. "

For this purpose, for example

 0.1 ml NS dilution (1: 4-00) with

0.1 ml of the AS ₂ dilution series (1: 2 to 1: 1000) for 1 h "incubated at room temperature (RT)

0.2 ml of an ^x Ir-AK-, incubation added after 30 min incubation at RT.

the incubate is mixed with 2.0 ml of 1.4 mol / l (HH ^ gSO ^ assay buffer)

and for more

 Centrifuged for 30 min at 2000 g for 15 min.

The supernatant is discarded, the activity measured in the precipitate. From the AK ₂ dilution, which guarantees the maximum binding of the ²¹ Ir-AK ₂ , the optimal NS / AK ₂ ratio is determined for the large-scale approach for the production of the HPR.

In order to guarantee consistently good binding and Prazipitationssescnaiten the HPR, the two components are filled according to the invention in an ampoule and lyophilized that the reaction is excluded with each other until the application of HPR: per ampoule about 0.5 ml AK _{2 are} frozen, -then, 0.05 ml of the CE graduation are pipetted into each ampoule. The KS freezes immediately without the AIi ₂ thaws. Subsequently, lyophilized ₀

In laboratory terms, AK ₀ and KS

processing the optimal concentration ratio so _s metered 'that each ampoule IIPR for 100 test tube contains · For the purposes of HPR the lyophilizate a vial is dissolved with 10 nil assay buffer for 1 h preincubation at ET of IIPPu per test tube, 100 / ul pipetted *

Since it is almost exclusively the custom-specific antisera obtained by immunization of guinea pigs (GP) and rabbits (Rab), it is a preparation of only two IIPR (GP-HPE and Rab-IIPR) for B / F separation in the said immunoassays Thus, the standardization of B / F separation according to the method of the invention results in a high efficiency in the production of the test kits, in addition it comes to simplifying the implementation of EIA, EIA and other immunoassay for the user, z * T * are very under Different laboratory equipment is required for the various radi oimmunoassays.

The following examples are intended to illustrate the principal application of the described separation process with the help of the HPR

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example 1

Radioimmunoassay for pancreatic glucagon in plasma:

0.5 nil of the piasm map will be used

0.1 ml of a diluted rabbit anti-glucagon serum for 2 days at 4 ⁰ C and after addition of

0.05 ml of 125 I-glucagon solution.

Incubated at 4 ^° C. for 20 hours

Thereafter, for B / F separation, 0.1 ml of Rab-IIPR is added and

Incubated for 30 min at RT, mixed with 2.0 ml of 1.6 mol / l (KEL) p ^{S (} \ ^{in the} assay buffer and

again .

Incubated at RT for 30 min.

After subsequent centrifugation for 15 minutes at 2000 g, the supernatant is poured off with the free 125 I glucagon (F) and the radioactivity in the precipitate is measured.

The RIA for pancreatic glucagon performed in this way shows an unspecific binding of the 125J glucagon of 3.4- + 0.4 % and a precision index of 3 »4 + 0.3% (5 + SEM, η = 14) through 100 pg glucagon In the incubation, the bound 125J-glucagon (B) dropped to 38 % compared to B in the absence of glucose.

Example 2

Radioimmunoassay for insulin in plasma:

0.05 ml of the plasma samples are with ^;

0.1 ml of a diluted guinea-pig anti-insulin serum

20 h at RT and after addition of 0.1 ml 125J-insulin

Incubated for 90 min at RT.

Thereafter, 0.1 ml of GP-IIPR are added for B / F separation

Incubated at RT for 30 min, with

98

2.0 ml 1.36 mol / l (Jffi ₄ ) ₂ S0 ^ in the assay pouch ex

mixed and incubated again for 30 min

After subsequent centrifugation for 15 "at 2000 g, the supernatant is decanted with the free 125 I-insulin (F) and the radioactivity (B) measured in the precipitate,

The RIA for insulin thus performed shows an unspecific binding of the 125 I ~ insulin of 2.4-5 ± 0.05% and a precipitation index of 2.6 + 0.3% (x ± SEM, η = 7) 300 pg (Oj3 / UE) insulin in the incubate, a decrease of the bound i25 <T-insulin (B) to 12 _S 6% compared to B in the absence of unlabelled insulins

Claims (5)

Invention claim: 1β Method for the separation of the free and antibody-bound ligand in carrying out labeled ligand immunoassays, characterized in that the separation is carried out with the aid of an iioiauno-immobilized precipitation reagent ¹ (IIPR). 2. The method according to item 1, characterized in that the IIPR only after the ligand-specific immune reaction in the homogeneous phase, as a finely dispersed immunoprecipitate obtained after Kurζzeitinkubation of normal serum (KS) of the species of the donor animals of the ligand-specific antisera with an antiserum (AlL ₃ ) against the immunoglobulins of this species at optimized antigen / antibody ratio, is mixed with the specific incubation. The B / F separation is achieved by a subsequent simple centrifugation and pouring of the supernatant. 3. The method according to item 1 and 2, characterized in that the two components (NS and AILO IIPR, although both "in a liquid state in an ampoule pipetted, but not mixed by a freezing intermediate step, lyophilized, can be stored for years, where it comes only after absorption with buffer, shortly before application of the IIPR, to form a finely dispersed, highly reactive IIPR. 4. Method according to items 1 to 3 »characterized in that with only two Proouktionsansätzen, each for GP-IIPR and Rab-IIPR in almost all labelled ligand immunoassays, the B / F separation can be durchgefüiirt. 5. Method according to items 1 to 4, characterized dadurcii, öaS by addition of e.g. Ammoniumsulf at or Polyätiiylenglykol aer time for the B / F separation to only 1 h Kai ^ "r ^ i_Lj u f & Gi ίχί * <Sto ^ ni ^ t &? <Xo * the concentration of said additives and the precipitation time is selected such that the ² L-AK ₁ -AKo complexes which are soluble without (M ₂ ) ₂ SCV or PEG addition, but not the labeled free (F ) Ligand ("L) and the immunoglobulins themselves precipitate" 6 _e method according to item 1 to 5, characterized in that the ligand to be determined may be any substance against which directly or after conjugation to a support material specific antibodies can be produced and which reacts in the ligand immunoassay as antigen or hapten, wherein the Determination of ligands in the plasma of the species of the donor animal for AK- is excluded « 7c method of item i to 6, characterized in that for the ligand immunoassay, the ligand itself or an antigenic bz?, Labeled i _ö haptenic ligandspezifische- structural unit with a delicate to be measured markers, while maintaining the reactivity to the specific antibody, is used, preferably the mark is made with a radioactive isotope (Radioimniunoassay) or with an enzyme (enzyme immunoassay)