WO1998009172A1

WO1998009172A1 - Method for analysing proteins and system for its implementation

Info

Publication number: WO1998009172A1
Application number: PCT/FR1996/001334
Authority: WO
Inventors: André BURCKEL
Original assignee: Optimum Research
Priority date: 1996-08-30
Filing date: 1996-08-30
Publication date: 1998-03-05
Also published as: AU6933196A

Abstract

The method for analysing proteins in a plasma or human serum comprises, for each analysed protein (Pi), the following steps: taking, from a set of sample containers, a predetermined volume of one sample to be analysed; taking, from a set of reagent containers, a predetermined volume of one reagent (Ri) associated with the analysed protein; reacting said sampled volume and said reagent volume (Ri); photometric measuring for determining an amount of precipitate compound, and processing this measurement for extracting therefrom a measurement for analysing protein. Each reagent (Ri) associated with each protein analysis (Pi) is diluted beforehand according to a dilution ratio (Ti) determined after an optimising step (SO). The invention is useful in analytical laboratories.

Description

"Protein dosing method and system for its implementation"

DESCRIPTION The present invention relates to a method for assaying proteins in human plasma or serum. It also relates to a system for its implementation.

We recall below some definitions of terms and concepts which will be used in the following:

Proteins are nitrogenous organic macromolecules, of high molecular weight, which give amino acids by hydrolysis. We treat here the case of plasma proteins, i.e. contained in a blood plasma, and serum proteins contained in a human serum. Nephelometry and turbidimetry are methods that involve the formation of a precipitate.

By measuring the fraction of the transmitted light intensity (turbidimetry) or by measuring the scattered fraction (nephelometry), the quantity of precipitated compound can be determined.

An antibody is an immunoglobulin whose synthesis is triggered in the body by injection of an antigen, with which they combine to neutralize the possible toxic effect. An antigen is a substance, the injection of which causes the body to synthesize specific antibodies.

An antiserum is the serum of an animal immunized by injection of an antigen, containing the corresponding antibody.

Current methods for assaying proteins in human plasma by turbidimetry or nephelometry are implemented in automated analysis machines. We can for example cite the BM / HITACHI 911 analysis machine from the company HITACHI. This machine implements a method for determining concentrations of antigens or antibodies by turbidimetry such as that described in the document. J60100764. This method implements a measurement of the increase in absorbance caused by the formation of a complex i provided with a macromolecule formed by an antigen-antibody reaction. In this method, an antigen with a variable concentration is diluted or not in a first reagent (volume given for each type of antigen). This is then brought into contact with an antibody at a given concentration specific to the type of assay. This antibody is prediluted extemporaneously in a second reagent, but it could be by the analyzer. The antigen-antibody mixture results in the formation of a haze measured by absorbance.

This process notably allowed the automation of nephelometry and turbidimetry using an antigen-antibody reaction, by performing a calibration curve of linear relationship between the concentration of antigen or antibody and the absorbance, for a zero concentration and for a known concentration. These automated machines are designed to use dosing products supplied by industrial laboratories. These assay products generally comprise reagent units, control units: for example a low control unit and a high control unit, and a calibrator unit. These products are supplied optimized and ready to use and are associated with predetermined protocols. Reaction buffer and dilution buffer solutions are also available. We can quote for example the reagents proposed by the company DAKO.

It turns out, for use in medical analysis laboratories, that reagents represent a very significant part in the cost of plasma protein assays. The object of the invention is to provide a protein dosage method which is more economical than current methods, while providing the same level of reliability. It is a process for assaying proteins in human plasma or serum, comprising, for each assayed protein, the following sequences:

- sampling, from a set of sample containers, of a predetermined volume of a sample to be analyzed, used as it is or diluted in a first reagent,

- sampling, from a set of reagent containers, of a predetermined volume of a reagent associated with the assayed protein, reaction of said volume withdrawn from sample and said volume of reagent,

- photometric measurement to determine an amount of precipitated compound, and - processing of this measurement to extract a measurement of protein dosage.

According to the invention, each reagent associated with each protein assay is diluted beforehand according to a dilution rate determined at the end of an optimization sequence.

The predilution of the reagents thus allows a substantial saving to be made, insofar as the dilution rates used can reach high levels. Furthermore, the dilution operations are carried out in situ at instants close to the instants of measurement. For example, the diluted reagents can be used reasonably during the day. Indeed, it would be impossible to provide for a distribution of prediluted reagents at the manufacturing stage, for reasons of stability and preservation of the products.

It goes without saying that the dilution of the reagents must necessarily be accompanied by an appropriate dilution of the plasma, serum, saliva or urine samples to be analyzed, in order to respect the stoichiometric ratios normally used in the reaction of dosage. Each sequence for optimizing a dilution rate of a reagent comprises a photometric measurement of the change in absorbance as a function of the concentration of reagent and a determination of a limiting dilution rate corresponding to a detection of excess antigen.

In a particular embodiment of the method according to the invention, the operations of prior dilution of the reagents are carried out directly within the containers of reagents.

In another embodiment, operations for prior dilution of the reagents are carried out when the reagents are withdrawn before reaction.

Optimized dilution rates differ depending on each protein. To determine these optimized dilution rates, optimization sequences were previously performed on the analysis machine, for each protein to be assayed.

A remarkable reliability of the assays has been observed experimentally, which is absolutely not altered by the dilutions - even high - employed. Furthermore, the assay method according to the invention anyway includes the control steps provided for in the assay protocols. According to another aspect of the invention, there is provided a system for assaying proteins in human plasma, implementing the assay method according to the invention, this system comprising: means for taking from a set of containers of 'samples a given volume of a sample to be analyzed and deposit it in a reaction receptacle, means for taking from a set of reagent containers a given volume of a reagent associated with a protein to be assayed and depositing it in said reaction receptacle, photometric means for measuring a fraction of light scattered or transmitted through said reaction receptacle and determining an amount of precipitated compound,

- And processing means to extract from said measurements, protein dosage information. According to the invention, this system further comprises means for diluting each reagent beforehand in an aqueous diluent according to a dilution rate specific to each protein to be assayed.

The system according to the invention is advantageously provided with means for determining, for each protein to be assayed, an optimum dilution rate of the associated reagent.

In a particular embodiment of a system according to the invention, the means for prior dilution cooperate with the means for withdrawing the reagents in order to dilute said reagents before each reaction.

However, provision may also be made for the prior dilution means to be arranged for carrying out the prior dilutions of each reagent within each reagent container.

In practice, the metering system according to the invention consists of an adaptation of an existing analysis and metering machine making it possible to implement the metering method according to the invention under the best possible conditions.

Other features and advantages of the invention will appear in the description below. In the accompanying drawings given by way of nonlimiting examples: - Figure 1 schematically illustrates an automatic analysis machine implementing the method according to the invention;

- Figure 2 illustrates the essential steps of the method according to the invention; FIG. 3 illustrates a typical example of a curve of evolution of the absorbance index as a function of the concentration of antigens, used for determining the dilution rates; - Table I provides optimized dilution rates for common monoreagents; and

- Table II provides optimized dilution rates for common bi-reagents. We will now describe a preferred form of implementation of the method according to the invention, together with a metering system for its implementation, with reference to FIGS. 1 to 3.

An automatic machine S comprises for example an analysis device 1, a control and processing device 2 and a control and acquisition station 3. The analysis device 1 comprises for example:

- a disk or tray for supporting plasma or serum samples to be analyzed; - two disks or trays DR1, DR2 of reagents, supporting a set of bottles CR containing reagents R'i,

- a DA reaction disk or plate consisting for example of 120 CV vessels grouped into 6 racks of 20 cuvettes, driven by a reaction rotor placed in an incubation bath;

- a PE sample pipette, to take a given quantity of plasma or serum from a cup of the DE sample support disc, and transfer it to a CV tank of the DA reaction disc;

- reagent pipettes PR1, PR2, to take from the bottles of the reagent support disks DR1, DR2, a given quantity of reagent and transfer it to a tank CV of the reaction disk DA, - and a photo caliper device PH to measure the fraction of the light intensity transmitted (turbidimetry) or scattered (nephelometry) through the reaction vessels.

The sample pipettes PE and reagents PR1, PR2 are normally subjected to washing cycles in which washing devices LE, L1, L2 are activated. The sample tray DE comprises for example an external crown CE, an intermediate crown CI and an internal refrigerated crown CF, and is designed to receive, in addition to the samples to be measured, high HC and low LC control units and calibration or CS standards. The samples can be contained in wells or tubes.

Each reagent tray DR1, DR2 comprises, in addition to the reagent bottles CR, bottles DL of diluent for samples and standards, and bottles of detergent DT. The dilution operations for samples and standards can be carried out in CV reaction vessels.

An automatic machine of this type allows several modes of analysis well known in the field of medical analyzes, in particular static at one point (prozone control) or at two points, and kinetic at two points

(prozone control), t in two final points, twin in one point and kinetic, kinetic A and kinetic B. The drive of the plates of reagents DR1, DR2, of samples DE and reactional DA and the device of photometry PH are fully automated. A barcode identification system manages samples, reagent bottles, diluents and other system components.

In a DA rotating reaction tray, the following operations are carried out:

- washing of reaction vessels,

- injection by pipette of a sample unit into a reaction tank,

- possible dilution of the sample unit,

- reception of a reagent unit,

- agitation of reaction vessels

- photometric measurements and "cell blank" measurements.

In an analysis machine according to the invention, the dilution of the reagents can be carried out, either on the reagent tray in the reagent bottles, either directly in each reaction tank.

We will now describe the essential steps of the metering method according to the invention, with reference to FIG. 2.

For the determination of a particular protein Pi, industrial suppliers design and market a reagent Ri, a Cal ^ calibrator, a low LCi control unit and a high HCi control unit. These different components can thus constitute a set or kit Ki dedicated to a given protein. With these components, a prior sequence SO is carried out for determining an optimal dilution rate Ti of the reagent Ri. It is then possible, within the framework of the operation of the analysis machine, to carry out the dilution of the reagent according to the dilution rate thus determined. This dilution can be carried out extemporaneously or even within the machine. The reagent thus diluted is inserted or placed in a tray or disc of reagent DR1, DR2. Furthermore, the samples subjected to analysis are also subjected to a dilution so as to maintain the normal stoichiometric proportions of the antigen-antibody reaction. Samples are then taken by pipette respectively of a volume of diluted reagent and a volume of diluted sample which are reacted in a tank CV of the reaction disk DA. The antibody-antigen reaction causes the formation of a precipitated compound which is quantified by photometric measurement. This measurement is then processed to deliver an assay result for the Pi protein concerned.

We will now describe an example of determining an optimum rate Ti of dilution of a reagent Ri. For several increasing dilution rates

TI, T2, .., Tn of the reagent, the ABS absorbance index is measured by photometry as a function of the antigen concentration and a zone of linearity of measurement ZL, as illustrated in FIG. 3. This determination is carried out using a standard supplied by the laboratory marketing the reagent. By varying the concentration of antigen, it is noted, in practice automatically, the evolution of the absorbance index. This gives a set of absorbance curves Dl, D2, .. Dn corresponds to the different dilution rates TI, T2, .. Tn. The optimum dilution rate Ti corresponds to the maintenance, with a sufficient safety margin, of a determined level of precision and reproducibility of the measurements which can be controlled by using the calibrators and the controls associated with the reagents.

The assay method according to the invention has been successfully implemented for the assay of numerous proteins, in particular the following proteins, with reference to Tables I and II corresponding respectively to the monoreagents and to the bi-reagents used for the assay of the following antibodies :

IgG: Immunoglobulin G

IgA: Immunoglobulin A

ApoAl: Apolipoprotein Al

TRF: Transferrin HpT: Haptoglobin

ORO: Orosomucoid or alpha-1-glycoprotein

Alb: Albumin αlAT: Alpha- 1-Antitrypsin

IgM: Immunoglobulin M

C3: C3c complement

C4: C4c complement

Fib: Fibronectin

ApoB: Apolipoprotein B α2M: Alpha-2-Macroglobulin

Prealb: Prealbumin

CRP: Ceruloplasmin

Lp (a): Lipoprotein a

The monoreagents are preferably diluted in a diluent such as the reference diluent S2006 sold by DAKO. The bi-reagents are of preferably diluted in a diluent such as 5% PEG, with the exception of the reagents α2M and Préalb which are diluted in a reference diluent S2006 (DAKO) and CRP and Lp reagents (a) diluted in a reference diluent S2011 (DAKO).

The sample sera are diluted in diluent S2005 for the assays with monoreagents. For dosages with bi-reagents, 5% PEG or diluent S2005 is used as the serum diluent, depending on the proteins to be assayed. The diluents used in the case of checks on reduced samples are indicated in italics. When a prozone control is planned, an R4 reagent is used which is a pool of serum diluted to 1/60 in physiological water. The dilution operations implemented in the process can obviously be fully automated and integrated into the operating protocols of automatic analysis machines.

Of course, the invention is not limited to the examples which have just been described and numerous modifications can be made to these examples without departing from the scope of the invention. Thus, the process according to the invention can be implemented with all the dosing machines currently available. Furthermore, it is not limited to a particular family of proteins and may integrate other proteins whose dosage in plasmas, serums, saliva or urine of human origin could be subsequently recommended. In addition, the method and the system according to the invention can be applied to protein assays using several reagents which are then each subjected to a specific predilution.

Claims

1. Method for assaying proteins in human plasma or serum, comprising, for each assayed protein (Pi), the following sequences: - sampling, from a set of sample containers (CE), of a volume predetermined of a sample to be analyzed,

- sampling, from a set of reagent containers (CR), of a predetermined volume of a reagent (Ri) associated with the assayed protein (Pi), reaction of said volume withdrawn from sample and said volume reagent,

- photometric measurement to determine an amount of precipitated compound, and - processing of this measurement to extract a protein dosage measurement, characterized in that each reagent (Ri) associated with each protein dosage (Pi) is previously diluted according to a dilution rate (Ti) determined at the end of an optimization sequence (SO), and in that the sample to be analyzed is also diluted so as to maintain the normal stoichiometric proportions.

2. Method according to claim 1, characterized in that the sample to be analyzed is previously diluted in a first reagent.

3. Method according to one of claims 1 or 2, characterized in that each optimization sequence (SO) of a dilution rate (Ti) of a reagent (Ri) comprises an acquisition of a set of curves (Dl, D2, .., Dn) evolution of the absorbance (ABS) as a function of the antigen concentration (CA), for a series of dilution rates (TI, T2, .., Tn) of said reagent (Ri).

4. Method according to one of the preceding claims, characterized in that operations of prior dilution of the reagents (Ri) are carried out directly in the reagent containers (CR).

5. Method according to one of the preceding claims, characterized in that operations of prior dilution of the reagents (Ri) are carried out at the time of sampling of said reagents (Ri) before reaction.

6. System (S) for assaying proteins (Pi) in human plasma, implementing the method according to one of the preceding claims, comprising:

- means (PE) for taking from a set of sample containers (CE) a given volume of a sample to be analyzed and depositing it in a reaction receptacle (CV),

- Means (PR1, PR2) for withdrawing from a set of reagent containers (CR) a given volume of a reagent (R'i) associated with a protein (Pi) to be assayed and depositing it in said reaction receptacle (CV),

- photometric means (PH) for measuring a fraction of light scattered or transmitted through said reaction receptacle (CV) and determining an amount of precipitated compound, - and acquisition and processing means (2,

3) to extract from said measurements, protein dosage information, characterized in that it further comprises means for previously diluting each reagent (Ri) in an aqueous diluent according to a dilution rate (Ti) specific to each protein ( Pi) to be dosed.

7. System (S) according to claim 6, characterized in that it further comprises means for determining, for each protein (Pi) to be assayed, an optimum dilution rate (Ti) of the associated reagent (Ri).

8. System (S) according to one of claims 6 or 7, characterized in that the means of prior dilution cooperate with the means of sampling (PR1, PR2) of the reagents to achieve a dilution (Ti) of said reagents (Ri) before every reaction.

9. System (S) according to one of claims 6 or 7, characterized in that the prior dilution means are arranged to perform the prior dilutions of each reagent (Ri) within each reagent container (CR).