WO1995023972A1 - Heterogeneous immunoassay and its use for detecting proteins - Google Patents

Abstract

A heterogeneous immunoassay is characterised in that it contains a strongly binding partner irreversibly immobilised on a solid substrate. The substrate may be exposed to a liquid buffer flow so that the bonded non-marked counterpart to be detected be eluted, if required after washing with an appropriate buffer, and detected by a detector. Also disclosed is the use of this heterogeneous immunoassay.

Description

 Heterogeneous immunoassay and its use for the determination of proteins

Various immune techniques are used for the selective and sensitive determination of proteins. The RIAs (radioimmunoassays) or ELISAs (enzyme linked immunoassays) are particularly noteworthy. They are used by default for the analysis of biotechnological samples. Other assays (see, for example, Freitag, R.; Scheper, T.; Spreinat, A., Anranikian, G. (1991b) On-line monitoring of pullulanase production during continuous culture of Clostridium thermosulfurogenes. Appl. Microbiol. Biotechnol. 35, 471-476, Freitag, R.; Scheper, T.; Schügerl, K. (1991) Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes, Enzyme Microb. Technol., 13, 969-975, Matiasson, B .; Hakanson H. (1992) Immunochemically based assays for process control, in: Modem Biochemical Engineering (ed.: A. Fiechter) Advances in Biochem. Eng. / Biotechnol ., Vol, 46, Springer Verlag Berlin, pp.: 81-101, Middendorf, C.; Schulze, B .; Freitag, R.; Scheper, T .; Howaldt, M.; Hoffmann, H. (1993) On -line immunoanalysis for bioprocess control, J. Bio¬ technol., 3, 395-403, Miyabayashi, A.; Mattiasson, B. (1990) A dual Streaming potentiai device used as an affinity sensor for monitoring hybridoma cell cultivatio n, anal. Biochem., 184, 165-171 and Stöcklein, W.; Jäger, V .; Schmid, R.D. (1992) Monitoring of mouse immunoglobulin G by flow injection analytical affinity chromatography, Anal. Chim. Acta, 245 (1), 1-6) can be used.

Various steps are necessary in a conventional ELISA:

Sample dilution to the optimal working range, immune reaction, washing step and detection step. Individual steps can be automated, but the assay remains time-consuming, personnel-intensive and error-prone. This also applies to all other immunoassays. Another disadvantage of these assays with regard to bioprocess analysis is that these methods are all designed for the detection of very small amounts of protein. Since there are higher protein concentrations in bioprocess samples (above 1 mg / l), the samples must therefore be extremely diluted, which is also time, personnel and error-intensive.

In addition to these points, there are no methods for bioprocess analysis that can quickly, ie within a few minutes, measure a large number of samples and quickly convert to other questions that work fully automatically and that also work online on the bioprocess can work.

In addition to the pure off-line processes listed above, some automated processes have also been established, for example the following are mentioned:

- Methods for the detection of immune reactions with the aid of the change in flow potential (Miyabayashia and Mattiasson, 1990), methods for turbidity measurement in the formation of immune complexes (turbidimetric assay, TIA) (Freitag et al; 1991a, Middendorf, et al., 1993) ,

- Process for the automation of heterogeneous, competitive assays with subsequent UV detection (Freitag et al., 1991b; Stöcklein et al., 1992) and

- Automated flow ELISA technique (Mattiasson and Hakanson, 1992).

With the exception of the TIA system, these processes are quite complex and can only be automated with great effort. The TIA system is the only one that has already been used in real industrial processes and has also been validated. A disadvantage is the high consumption of antibodies, since new antibody solutions have to be applied for each assay. - -. -

All systems have a high consumption of analysis in common, but the downtimes (except for TIA) and the degree of automation (except for TIA) are low. The systems are susceptible to contamination and a separation of interfering substances is not possible at all or only possible through dilution steps. Only the TIA manages without a dilution step.

The present invention is therefore based on the task of developing an immunoassay which meets the following minimum requirements:

- General analysis system using affinity reactions;

- Immobilization of the affinity component for repeatable use, e.g. B. of at least 100 assays;

- no sample dilution necessary;

- easy removal of interfering substances;

- fully automated;

- easy to adjust measuring range;

- no risk of contamination;

- variable detection options;

- Can be calibrated at any time;

- Use for on-line and off-line analysis (at least 10 samples per hour);

- easy to change over;

- Possibility of analyzing different IgGs with an immune cartridge (with immobilized protein A or G).

According to the invention, the measuring system (see FIG. 1) is based on a new heterogeneous immunoassay (heterogeneous elution assay). say) according to the principles of flow injection analysis. Here, a partner of a strong binding reaction is irreversibly immobilized on a solid support, which is preferably implemented in a flow-through cartridge. After the binding partner has been immobilized on a suitable carrier material, the unlabeled sample is placed in a buffer stream which is constantly flowing through the system and passed through the immobilizate (see FIG. 2). The binding partner binds its specific counterpart from the sample, while all other substances are discharged through the carrier stream. There is a separation between analytes (counterpart) and interfering substances. The bound substances are then discharged by changing from the carrier buffer to an elution buffer. Washing and conditioning steps can be implemented depending on the analysis problem. The discharged substances are then analyzed with a detector, for example, for their concentration or biological properties. Depending on the detector signal and evaluation method, the concentration of the analyte in the sample can be proportional to the peak integral, for example. The cartridge can be cleaned, conditioned or equilibrated before performing a new sample.

The present invention thus relates to a heterogeneous immunoassay, which is characterized in that it contains a partner with a strong binding reaction, the partner being irreversibly immobilized on a solid support and the support in a flowing buffer stream It is possible to expose that the counterpart to be determined and bound, after washing, if necessary, is eluted with a suitable buffer and determined with a detector, its use for the determination of proteins and a method for the determination of proteins below Use of the immunoassay according to the invention.

The system and analysis principle described is suitable for analyzing samples in a few minutes. Proteins can be determined (the corresponding antibodies are immobilized in the cartridge), immunoglobulins (through immobilized proteins A / G) or other systems with high binding constants such as avidin / biotin or lectins / glycosylated macromolecules.

The immunassay according to the invention has the following advantages:

- The device works independently and is fully automatable;

- Both on-line and off-line samples can be analyzed;

- there are only short analysis times, e.g. B. only a few minutes necessary;

the sensitivity of the immunoassay can be increased by coupling to fluorescent dyes or other markers before or after the cartridge;

an analysis in cell-containing samples is possible;

- By optimizing the elution conditions it can be achieved that in the cartridge as well as in the flow system there is no growth with microorganisms and no further cleaning cycles are necessary;

the measuring range of the immunoassay can be adjusted by varying the injection volume (cumulative effect), which means that very small concentrations can also be measured;

- When using the measuring system, samples can be used directly (i.e. undiluted, not conditioned). Here, the concentration of the substance to be analyzed is very large, e.g. B. several milligrams per milliliter, can be adjusted upwards by an increased dispersion of the measuring range;

the system can be coupled to an autosampler, which enables simple analysis of several hundred samples in succession; the detection can be carried out in the most varied of ways, the determination of the fluorescence of the proteins or of the UV absorption being particularly suitable;

since there is a separation between interfering substances and analytes, reliable analyzes can also be carried out if the composition of the interfering substances changes; a reference measurement is not necessary here;

- With the help of this new combination, contamination-free, trouble-free, automated analysis is possible, since different rinsing steps can be switched on. The system can be used repeatedly for any system.

Suitable carriers are the usual carriers such as synthetic or natural polymers or glasses, in particular Sepharos 4B, Eupergit, Eurocell ONB and VA epoxy biosynth, the grain size preferably being between 100 and 200 μm. The binding of the partner of the desired strong binding reaction to the carrier used can be carried out adsorptively, ionically or covalently, but the latter is preferred.

To form a covalent bond, the carrier must be activated for this by customary methods. This can be done by means of hydroxy groups on the support by reaction with bifunctional substances such as glutardialdehyde and subsequent binding of the binding partner, after conversion of the hydroxy groups into amino or carboxy groups and subsequent binding of the binding partner by way of example or by way of example Hydroxide groups occur in groups with terminal epoxy functions and subsequent addition of the binding partner, the latter method being preferred.

The following example is intended to explain the invention in more detail: - 1 -

Bf-i.spiel

The system was tested for the following analytes: antithrombin III, rt-PA and antibodies of the IgG type. The first two analytes were tested with poly- and monoclonal antibodies, the IgG antibodies with proteins A and G.

Sepharose 4B, Eupergit, Eurocell ONB and VA-Epoxy Biosynth were tested as carriers for the rt-PA assay. Potassium phosphate buffer (0.1 M, pH = 7.4) was used as a carrier, washing / conditioning and equilibration buffer. Elution was possible in basic with 0.1 M potassium phosphate buffer (pH = 12.3) and in acid with glycine buffer (0.1 M, pH 2.0).

In each case 20-100 μl sample was injected (duration 10 sεc); then 170 see was washed and 150 see eluted and detected. After an equilibration time of 30 seconds, a new injection could be made.

Detection was carried out in a flow spectrophotometer (excitation at 273 nm, emission measurement at 340 nm).

Figure I shows the calibration line for rt-PA with various immobilizates.

Figure II shows the influence of the pH value of the elution solution on the protein fluorescence.

Figure III shows the long-term stability for the ATIII measurement: the correlation coefficients are very high, even if the slope of the straight line decreases. A simple two-point calibration per measuring day is sufficient to take this effect into account.

Figure IV shows the agreement of the measurement results from two different measurement days after a previous two-point calibration. Figure V shows the agreement of the measurement results of the conventional ELISA's and the measurement values of the automated heterogeneous assay.

Figure VI shows that interference from foreign proteins can be excluded by the washing step.

Claims

claims

1.) Heterogeneous immunoassay, which is characterized in that it contains a partner with a strong binding reaction, the partner being irreversibly immobilized on its solid support and the support being exposed to a flowing buffer stream in such a way that the determining and bound unmarked counterpart after washing, if necessary, eluted with a suitable buffer and determined with a detector.

2.) Heterogeneous immunoassay according to claim 1, characterized in that the binding partner is contained in a cartridge.

3.) Heterosrogenic immunoassay according to claims 1 and 2, so that the counterpart is an antibody or a protein.

4.) Heterogeneous immunoassay according to claims 1 and 2, characterized by the fact that the binding partner is a protein, an immunoglobulin or a system with high binding constants such as avidin / biotin or εin lectin / glycosylated macromolecule.

5.) Heterogeneous immunoassay according to claims 1 to 4, characterized in that a marked counterpart is used to increase the sensitivity as counterpart.

6.) Method for determining a gene part, characterized in that the counterpart is bound to an immobilized partner on a solid support, which is eluted after washing, if necessary, and then determined with a detector.