DD153240A1 - METHOD FOR PRODUCING STANDARD MEDIA FOR IMMUNOASSAYS - Google Patents

Abstract

The invention relates to a process for the preparation of standard media for immunoassays, which operate on the principle of analysis Saetigungsanalyse, whereby the task is solved to match animal sera in terms of their properties hormone-free or hormone poor human sera. The method consists in either digesting the animal serum with assay buffer or concentrating it or adding to the animal serum proteins, in particular albumin, until identical or nearly identical antiserum-specific binding is achieved in the animal and human serum. The choice of animal serum must be such as to avoid a cross-reaction between the hormone it contains and the hormone used or to be determined as a standard, which requires prior testing. For the determination of the hormone TSH herdeserum is particularly suitable.

Description

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Process for the preparation of standard immunoassays

Field of application of the invention

The invention relates to methods for the preparation of standard media for immunoassays which operate on the principle of saturation analysis (radioimmunoassay RIA, Pluoreszenzimmunoassay - PIA, enzyme immunoassay - EIA, etc.), with a specific composition of these standard media, a precise determination of biologically active substances, in particular of hormones , can be reached·

Characteristic of the known technical solutions

In the determination of biologically active substances, mainly of hormones, according to the principle of saturation analysis (radioimmunoassay, Pluoreszenzimmunoassay, enzyme immunoassay, etc.), it has been found that, in view of the accuracy of the achievable results, the standards for determining the reference curves in human sera to be set. Only in this Palle is a largely the same behavior of standards and samples guaranteed. In order to avoid congestion, only sera can be used in which the mirror is to be determined at the. Hormone practically indistinguishable from zero (deficient sera, null sera). The procurement of such human serums with extremely low. Hormone content is common

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2 Z- 9 3 5

However, the hormones used as standards react in buffers - even "with the addition of protein - unlike the hormones in the sample serum, which can be of considerable consequence for clinical diagnostics and therapy.

Because of the difficulty of obtaining so-called null sera for preparing the standards in sufficient quantity, attempts have been made to artificially deplete hormones of normal serum or to avoid animal sera. Preparative enrichment of the hormone in question from the serum then encounters great difficulties Substances with regard to their chemical and physical parameters which approximate the serum proteins · Upon extraction, the total protein content would decrease so much that no native serum would be present · Only affinity chromatography offers the possibility of selective depletion, but is too expensive for routine purposes.

The use of animal sera to prepare the standards has encountered difficulties so far. Thus, Pekary et al. Reported the use of calf serum in the radioimmunoassay for the determination of the proteohormone hTSH, thus obtaining falsely elevated levels (J. Elin. Endocr. Metab. 41 (1975), 676). Erhard has reported that standard curves in horse serum also give too high values for TSH (Der liuklearmediziner 1, 2 (1979), 24). Using the native horse serum for the standards results in low TSH values for human samples.

As already mentioned, obtaining native human serums with the required extremely low hormone levels is difficult. In the case of proteohormone TSH such sera z. For example, only in subjects who have been hypophysectomized, or in extreme hyperthyroidism of other genesis. Patients with corresponding clinical pictures are rare

In addition, the production of a test system does not suffice to obtain the quantities of serum that can be obtained in this way, in order not only to have recourse to patients, an attempt was made to win test persons their pituitary gland can be suppressed by administration of thyroid hormones (T3 and / or T4) and then donated blood Apart from the fact that absolute freedom from hormones or very low levels of TSH are rarely achieved in this preparation, rather a residual function of the pituitary gland with corresponding TSH blood level is maintained, the effort is very significant. Also, this procedure is not without dangers for the subject, which is why, the serum extraction is bound to certain conditions.

It is clear from the above that the milieu in which the standards are set is of great importance for obtaining the correct values of the jj analysis. Both buffer solutions with protein as well as native animal sera are unsuitable solvents for standard production, since too high or too low hormone levels are determined, resulting in considerable diagnostic and. can have therapeutic consequences.

Object of the invention

The aim of the invention is to obtain sera which are suitable as standard media in immunoassays »

Explanation of the nature of the invention

The object of the invention is to specify a method for the preparation of animal sera which, in terms of their properties as standard media in immunoassays, are virtually hormone-free or hormone-poor human sera.

The inventive method for obtaining animal

Sera as Stardard Media in Immunoassays "is that the animal sera is either diluted with assay buffer or concentrated to give identical or nearly identical antiserum-specific binding in the animal and human serum environment. The selection of the animal serum must be such that a cross-reaction between the in vivo hormone and "standard" or hormone to be determined is avoided (so-called species cross reactivity), which made a previous testing necessary

If the Staadardkurve in animal serum to the left of in hormone-free human serum, the animal serum must be diluted accordingly. In the other case, the curve is shifted to the right ^ the animal serum has to be concentrated or adjusted by addition of proteins © In addition to achieving identical antisummer-specific binding in animal and human serum, the so-called non-specific binding must also be included in the investigations; it must also be practically the same.

In the work-up of the assay, the so-called B / i ¹ separation, i. ii. the separation of antibody-bound and free activity, preferably carried out according to the double antibody principle. In this case, the procedure according to the invention is such that no disruption of the precipitation reaction occurs through constituents of the animal serum.

The subtle testing of the double antibody reaction must be done in both Husian and modified animal serum environments. For the most part there are kinetic differences. It is advantageous to select those working conditions in which the reactions come close to equilibrium in both environments. The non-specific binding is very low in the double antibody method and hardly differentiated between sera of different provinia.

For radioimmunoassay for determination of hormone TSH

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is particularly suitable horse serum. In this case, preferably second antibody from goats should be used. A disorder occurs when the second antibody is donkey mule-gamma-globulin (mule, mule) because there is a partial mating relationship between horse and donkey. "There is no cross reaction between horse TSH and human TSH on.

embodiment

FIG. 1 shows the adjustment of the horse serum concentration, FIG. 2 shows the standard curve η in different sera.

Pur radioimmunoassay for the determination of the hormone TSH horse serum was chosen as the standard medium. Dilute native pooled horse serum with assay buffer to prepare the solution media for the standards. The buffer has the following composition:

7.58 g Fa ₂ HPO ₄ + 2 H ₂ O

0.93 g KH ₂ PO ₄

3.72 g Chelaplex III

1.0 g HaF ₃

5.0 g human serum AlTdumin ·

These substances are made up to one liter and the pH is adjusted to 7.4.

To determine the optimal dilution, an animal serum / assay buffer dilution series is prepared. Using this dilution series, the zero point of the system is set first, which is compared to a range of THS-poor human sera. The binding rates in the radioimmunoassay for the investigated TSH poor human sera are between 24,000 and 25 × 300 pulses / 5 min. With 50 * $ horse serum, a binding rate of 25 400 pulses / 5 is kept pure. This concentration is considered optimal.

When 0.2 ml of test serum is used for the assay, the standards are in 0.2 ml of 50 $ horse serum.

In addition, the horse serum concentration adjusted to the zero point must also be secured or slightly corrected for the entire standard curve range. For this reason, standard curves were dissolved in buffer containing 0.5 fo HSA (i), in the best of our human Uullserum (II), in un ~ treated horse serum (III) and (IV) in adjusted horse serum was added.

The course of these curves clearly shows that in the present case 50% horse serum can be used as standard medium and gives the same results as best human null serum.

Claims (5)

invention claim A method for the preparation of standard antibodies for immunoassays, characterized in that animal serum is either diluted with buffer or concentrated until identical antiserum-specific binding in animal and human serum is achieved. 2. The method according to item 1, characterized in that the adjustment of the animal serum by the addition of proteins, in particular of albumin, is made ·. 3. Method according to item 1, characterized in that such animal serum is selected which does not cross-react between the hormone contained therein and the human hormone used as the standard or to be determined. 4. The method according to item 1, characterized in that "in the TSH immunoassay horse serum is used · 5. The method according to item Λ and 4, characterized in that antibody from goats is used as the second antibody. Here are the pages of the series