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AU701926B2 - Heterogeneous immunoassay and the use thereof for detecting proteins - Google Patents

Heterogeneous immunoassay and the use thereof for detecting proteins Download PDF

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Publication number
AU701926B2
AU701926B2 AU26074/95A AU2607495A AU701926B2 AU 701926 B2 AU701926 B2 AU 701926B2 AU 26074/95 A AU26074/95 A AU 26074/95A AU 2607495 A AU2607495 A AU 2607495A AU 701926 B2 AU701926 B2 AU 701926B2
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counterpart
partner
binding partner
immobilised
heterogeneous immunoassay
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AU2607495A (en
Inventor
Wolfgang Noe
Martin Reinecke
Thomas Scheper
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Boehringer Ingelheim Pharma GmbH and Co KG
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Dr Karl Thomae GmbH
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Description

1- S016-603.590 DR. KARL THOMAE GMBH D-88397 BIBERACH Case 12/165-FL
PCT
Heterogeneous Immunoassay and the use thereof for detecting proteins Various immuno techniques are used for the selective and sensitive detection of proteins. The RIAs (Radioimmunoassays) or ELISAs (Enzyme Linked Immunoassays) should be mentioned in particular. They are used as a standard procedure for analysing biotechnological samples. Other assays may also be used (cf. for example Freitag, Scheper, Spreinat, A., Antranikian, G. (1991b) On-line monitoring of pullulanase production during continuous culture of Clostridium thermosulfurogenes. Appl. Microbiol.
Biotechnol. 35, 471-476, Freitag, Scheper, T.; Schigerl, K. (1991) Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes, Enzyme Microb. Technol., 13, 969-975, Mattiasson, Hakanson H. (1992) Immanochemically based assays for process control, in: Modern Biochemical Engineering A. Fiechter) Advances in Biochem.
Eng./Biotechnol., Vol, 46, Springer Verlag Berlin, pp.: 81-101, Middendorf, Schulze, Freitag, R.; Scheper, Howaldt, Hoffmann, H. (1993) On-line immunoanalysis for bioprocess control, J. Biotechnol., 3, 395-403, Miyabayashi, Mattiasson, B. (1990) A dual streaming potential device used as an affinity sensor for monitoring hybridoma cell cultivation, Anal.
Biochem., 184, 165-171 and St6cklein, Jager, V.; Schmid, R.D. (1992) Monitoring of mouse immunoglobulin G by flow injection analytical affinity chromatography, \F
I
2 Anal. Chim. Acta, 245(1), 1-6).
In a conventional ELISA, various steps are necessary: Sample dilution to the optimum working range, immune reaction, washing step and detection step.
Admittedly, individual steps can be automated but the assay still remains time-consuming, labour intensive and prone to errors. This also applies to all other immunoassays. Another disadvantage of these assays with regard to bioprocess analysis iF that these methods are all designed to detect tiny amounts of protein. Since higher protein concentrations (above 1 mg/l) are present in bioprocess samples, the samples have to be diluted substantially, which is also time-consuming, labour intensive and prone to error.
In addition to the points mentioned above, there are no processes available for bioprocess analysis which operate fast, ie. within a few minutes, measure a large number of samples and can quickly be adapted to other kinds of problems, operate fully automatically and can also operate on-line in the bioprocess.
In addition to the pure off-line processes mentioned above, some automated processes have been established of which the following are mentioned by way of example: Process involving detecting immune reactions with the aid of the change in flow potential (Miyabayashia and Mattiasson, 1990), process for measuring turbidity in the formation of immune complexes (turbidimetric assay, TIA) (Freitag et al; 1991a, Middendorf, et al. 1993), Process for automating heterogeneous competitive assays with subsequent UV detection (Freitag et al., 3 1991b; St6cklein et al., 1992) and Process using an automated throughflow-ELISA technique (Mattiason and Hakanson, 1992).
Apart from the TIA system these processes are highly laborious and can only be automated at great cost. The TIA system is the only one which has been used and proved its worth in real industrial processes. A disadvantage is the high consumption of antibodies since new antibody solution has to be used for each assay.
All the systems share a high consumption of material for analysis, but the service life (apart from TIA) and the degree of automation (apart from TIA) are low. The systems are susceptible to contamination and separation of unwanted substances is impossible or can only be achieved by dilution. Only TIA does not require dilution.
The aim of the present invention is therefore to develop an immunoassay which satisfies the following minimum requirements: generally applicable system of analysis using affinity reactions; immobilisation of the affinity component for repeatable use, eg. at least 100 assays; no need for dilution of sample; easy removal of unwanted substances; fully automated; easily adapted measuring range;
I
4 no risk of contamination; variable detection possibilities; can be calibrated at any time; use for on-line and off-line analysis (at least samples per hour); easy to adjust; possibility of analysing different IgGs with an immune cartridge (with immobilised protein A or G).
According to the invention, the measuring apparatus (see Figure 1) is based on a new heterogenous immunoassay (heterogeneous elution assay) based on the principles of flow injection analysis. Here, one partner of a strong binding reaction is irreversibly immobilised on a solid carrier, the latter preferably being inserted in a throughflow cartridge. After the immobilisation of the binding partner on a suitable carrier material the unlabelled sample is placed in a buffer current flowing constantly through the system and is passed through the immobilised material (see Figure The binding partner binds its specific counterpart from the sample, whilst all other substances are removed by the carrier current. Separation of the analyte (counterpart) and unwanted substances takes place. The bound substances are then extracted by changing from the carrier buffer to an elution buffer. Washing and treatment steps may be carried out depending on the particular analysis problem. The extracted substances are then analysed with a detector in order to discover, for example, the concentration thereof or their biological properties.
Depending on the detector signal and method of evaluation, the concentration of the analyte in the Mi' P I'DOCIJ( 10 WS1& CIfl I N VJ sample may, for example, be proportional to the peak integral. Before a fresh sample is put in the cartridge may be purified, treated or equilibrated.
The present invention thus relates to a heterogeneous immunoassay which is characterised in that it contains one binding partner with a strong binding reaction, said binding partner being a binding partner for a protein, an immunoglobulin, avidin, a lectin or a glycosylated macromolecule and being irreversibly immobilised on a solid carrier, said carrier being exposed to a flowing buffer current containing the unlabelled counterpart which is to be detected, such that said unlabelled counterpart binds to said binding partner, and after optional washing with a suitable buffer, said unlabelled counterpart is eluted and measured with a detector.
In another aspect, the present invention relates to a process for detectin3 a counterpart, characterized in that said counterpart is bound to an immobilised partner on a solid carrier, said immobilised partner being a binding partner for a protein, an immunoglobulin, 20 avidin, a lectin or a glycosylated macromolecule, and after optional washing said counterpart is eluted and measured with a detector.
o The system and principle of analysis described are suitable for analysing samples in a few minutes. It is possible to detect proteins (in which case the corresponding antibodies are immobilised in the cartridge), immunoglobulins (by means of immobilised 1 protein A/G) or other systems with high binding constants such as avidin/biotin or lectins/glycosylated macromolecules. The immunoassay according to the invention has the following advantages: the apparatus works independently and can be fully automated; samples may be analysed both on-line and off-line; only short analysis times, eg. just a few minutes, are required; the sensitivity of the immunoassay can be increased by 6 coupling to fluorescent dyes or other markers ahead of or after the cartridge; analysis may be carried out in cell-containing samples; by optimising the elution conditions it is possible to ensure that no growth of microorganisms occurs either in the cartridge or in the flow system and no further purification cycles are required; the measuring range of the immunoassay can be adapted by varying the injection volume (cumulative effect), and in this way very small concentrations can also be measured; by using the measuring apparatus, samples can be put in directly (ie. undiluted, untreated). If the concentration of the substance to be analysed is very great, eg. several milligrams per millilitre, the measuring range can be adjusted upwardly by increasing the degree of dispersion; the apparatus can be coupled to an autosampler which enables simple analysis of several hundred samples one after the other; detection may be carried out in numerous ways, measurement of the fluorescence of the proteins or UV absorption are particularly suitable; since a separation occurs between the unwanted substances and the analyte, reliable analyses can also be achieved when the composition of the unwanted substances changes; reference measurements are not required; i
I
7 using this new combination it is possible to carry out analysis automatically, free from contamination and free from disruption, as various rinsing steps may be included. The system may be used repeatedly for any desired systems.
The carriers used may be the conventional carriers such as synthetic or natural polymers or glass, particularly Sepharose 4B, Eupergit, Eurocell ONB and VA-Epoxy- Biosynth, the particle size preferably being between 100 and 200 pm. The partner of the desired strong binding reaction may be bound to the carrier by adsorption, ionically or covalently, the latter being preferred.
In order to form a covalent bond the carrier must be activated by conventional methods. This can be done by means of hydroxy groups on the carrier by reacting 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, eg. using the carbodiimide method, or after converting the hydroxy groups into groups having terminal epoxy functions and subsequent addition of the binding partner, this latter method being preferred.
The example which follows is intended to illustrate the invention: Example 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 and the IgG antibodies with protein A and G.
WI,-
8 For the rt-PA assay, Sepharose 4B, Eupergit, Eurocell ONB and VA-Epoxy Biosynth were tested as carriers.
Potassium phosphate buffer (0.1 M, pH 7.4) was ased as the carrier, washing/treating and equilibrating buffer.
Elution was possible in the alkaline range with 0.1M potassium phosphate buffer (pH 12.3) and in the acid range with glycine buffer (0.1 M, pH 20-100 pl of sample were injected in each case (duration seconds), then washing was carried out for 170 seconds followed by elution for 150 seconds and detection. After an equilibration time of 30 seconds a new injection could be effected.
Detection was carried out in a throughflow spectrophotometer (excitation at 273 nm, measurement of emission at 340 nm).
Figure I shows the calibrating line for rt-PA with different immobilised materials.
Figure II shows the influence of the pH of the eluting solution on the protein fluorescence.
Figure III shows the long term stability for ATIII measuremenc the correlation coefficients are very high, even when the pitch of the straight line decreases. One simple two-point calibration per measuring day is sufficient to take this effect into consideration.
Figure IV shows the similarity between the measurements on two different measuring days after previous two-point calibration.
Figure V shows the similarity between the measurements from conventional ELISAs and the measurements from the I 9automated heterogeneous assay.
Figure VI shows that interference caused by foreign proteins can be excluded by means of the washing step.

Claims (5)

1. A heterogeneous immunoassay which is characterised in that it contains one binding partner with a strong binaing reaction, said binding partner being a binding partner for a protein, an immunoglobulin, avidin, a lectin or a glycosylated macromolecule and being irreversibly immobilised on a solid carrier, said carrier being exposed to a flowing buffer current containing the unlabelled counterpart which is to be detected, such that said unlabelled counterpart binds to said binding partner, and after option±i washing with a suitable buffer, said unlabelled counterpart is eluted and measured with a detector. V:
2. A heterogeneous immunoassay according to claim 1, characterised in 15 that the binding partner is contained in a cartridge.
S3. A heterogeneous immunoassay according to either claim 1 or claim 2, characterised in that the counterpart is an antibody or a protein. 20
4. A heterogeneous immunoassay according to anyone of claims 1 to 3, characterised in that the counterpart used is labelled in order to Sincrease the sensitivity.
5. A process for detecting a counterpart, characterised in that said counterpart is bound to an immobilised partner on a solid carrier, said immobilised partner being a binding partner for a protein, an immLnoglobulin, avidin, a lectin or a glycosylated macromolecule, and after optional washing said counterpart is eluted and measured with a detector. I 11 Abstract The present invention relates to a heterogeneous immunoassay which is characterised in that it contains a partner with a strong binding reaction, :he partner being irreversibly immobilised on a solid carrier and the carrier being adapted to be exposed to a flowing buffer current in such a way that the unlabelled, bound counterpart which is to be determined, after being washed with a suitable buffer if necessary, is eluted and measured with a detector; and the use thereof. 'N i I
AU26074/95A 1994-03-04 1995-02-28 Heterogeneous immunoassay and the use thereof for detecting proteins Ceased AU701926B2 (en)

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Application Number Priority Date Filing Date Title
DE19944407142 DE4407142A1 (en) 1994-03-04 1994-03-04 Heterogeneous immunoassay and its use for the determination of proteins
DE4407142 1994-03-04
PCT/EP1995/000727 WO1995023972A1 (en) 1994-03-04 1995-02-28 Heterogeneous immunoassay and its use for detecting proteins

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AU701926B2 true AU701926B2 (en) 1999-02-11

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Publication number Priority date Publication date Assignee Title
DE10015448A1 (en) * 2000-03-29 2001-10-11 November Ag Molekulare Medizin Detection and quantification of binding between affinity molecules useful in screening for drugs, does not require removal of reaction solution prior to measurement

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0796787A1 (en) * 1996-03-18 1997-09-24 HE HOLDINGS, INC. dba HUGHES ELECTRONICS Satellite spin vector control with spun sensor

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* Cited by examiner, † Cited by third party
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FR2459479B1 (en) * 1979-06-21 1983-07-08 Inst Nat Sante Rech Med PROCESS FOR SEPARATING A PROTEIN SUBSTANCE FROM A SOLUTION CONTAINING IT BY AFFINITY FILTRATION AND APPLICATION OF SAID METHOD FOR ENZYMATIC ASSAYS
US4818687A (en) * 1985-02-28 1989-04-04 Massachusetts Institute Of Technology Affinity column and process for detection of low molecular weight toxic substances
FR2585367B1 (en) * 1985-07-26 1987-10-02 Lyonnaise Eaux METHOD FOR THE CONTINUOUS IMMUNOLOGICAL ASSAY OF ORGANIC COMPOUNDS IN A CIRCULATING FLUID, AND DEVICE FOR IMPLEMENTING SAME
WO1991016116A1 (en) * 1990-04-23 1991-10-31 Cellpro Incorporated Immunoselection device and method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0796787A1 (en) * 1996-03-18 1997-09-24 HE HOLDINGS, INC. dba HUGHES ELECTRONICS Satellite spin vector control with spun sensor

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AU2607495A (en) 1995-09-18
DE4407142A1 (en) 1995-09-07
EP0748448A1 (en) 1996-12-18
JPH09509745A (en) 1997-09-30
CA2184714A1 (en) 1995-09-08

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