FR2481318A1 - IMMUNOLOGICAL ANALYSIS METHOD - Google Patents

Abstract

PROCESS FOR THE QUALITATIVE OR QUANTITATIVE ANALYSIS OF A SUBSTANCE USING IMMUNO-ACTIVE REAGENTS, ONE OF WHICH IS MARKED BY AN ENZYME AND THE OTHER FIXED ON A SUBSTANCE INSOLUBLE IN WATER, BY INCUBATION OF THE SUBSTANCE SUBMITTED ON ANALYSIS WITH THESE REAGENTS, SUBSEQUENT SEPARATION OF THE SOLID PHASE AND THE LIQUID PHASE AND MEASUREMENT OF THE DEGREE OF ENZYME LABELING IN THE SOLID PHASE OR IN THE LIQUID PHASE. FOR THE IMMUNOLOGICAL REACTION, THE SUBSTANCE UNDER ANALYSIS IS SUBMITTED FROM THE BEGINNING TO INCUBATION WITH THE IMMUNO-ACTIVE REAGENTS TOGETHER, AND IN A SINGLE INCUBATION.

Description

The present invention relates to an immunological method for

  qualitative or quantitative analysis

of a substance.

  The process according to the invention is an immunoassay

  enzymatic based on what is called the "sandwich principle". According to this principle, the analyte, which may be an antigen, an antibody or a hapten, is reacted with two reagents having the immunological activity. In

  As a general rule, one of these reagents

  The immunoassay is fixed on a water-insoluble support and the other is associated with a suitable enzyme. In practice, the analyte is first

  react with the reagent fixed on a support; after separation

  The substance, which has been fixed in the meantime on the support by immunological means, is reacted with the second reagent, labeled with an enzyme. After a new phase separation, the enzymatic reaction used for the assay of the substance under analysis is

  performed in the solid phase or in the liquid phase.

  Until now it was thought that in

  enzyme immunoassay, the immunological reaction of the test substance with the two reagents

  be performed in two successive reaction stages.

  According to the invention, we now have

  that contrary to this earlier conception, we can

  would operate almost in a "one-off" technique.

  "in which the substance under analysis

  is reacted simultaneously with the two reagents pos-

  sedating immunological activity during a single and

unique incubation.

  The invention thus relates to an immunoassay

  zymatic for the qualitative or quantitative analysis of a substance using a reagent with activity

  immunological and enzyme-labeled and of a reagent pos-

  seducing an immunological activity which is fixed or will be

  on a water-insoluble support, by incubation

  of the substance submitted for analysis with the reagents

  immunological activity, subsequent separation of the solid phase and the liquid phase and measurement of the degree of labeling with the enzyme, either in the solid phase or in the liquid phase, as a measure of the quantity

  of the substance under analysis, this process is

  in that, for the immunological reaction, the analyte and the reagents possessing the immunological activity are incubated as soon as possible.

  start and in common and only once.

  Among the substances subjected to analysis, mention will be made of all the constituents of physiological liquids, cell or tissue extracts for which there are immunological reagents or for which immunological reagents can be formed, and which possess two or more sites possessing immunological activity. It can be peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipids,

  nucleic acids, enzymes, hormones, polysaccharides

  wrinkles, alkaloids. Preferred immunoactive substances

  are the natural antigens such as hormones,

  enzymes, organ-specific antigens,

  connective tissue, the antigens of blood cells

  guines, plasma proteins, pathogenic globulins

  c. Particularly valued substances are

  carcinoembryonic antigen (CEA) and gonado-

human chorionic tropin (HCG).

  In the method according to the invention, the enzyme-labeled immunoactive reagent serves as an indicator for the immunological reaction. The preferred enzymes are the

  alkaline phosphates, malate dehydrogenase, glucose

  se-6-phosphate dehydrogenase, glucose oxidase, glucose

  coamylase, galactosidase and acetylcholine esterase. A preferred enzymatic marker is horseradish peroxidase.

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  The enzyme serving as an indicator of the immunological reaction is assayed according to known methods in the liquid phase or, preferably, in the solid phase, and is a measure of the amount of the substance under analysis. When the label is horseradish peroxidase, the amount of enzyme is preferably assayed by the existing catalytic activity, which is determined using H 2 O 2 and o-phenylenediamine.

  which serves as a redox indicator. After a catalytic reaction

  than 30 minutes, the intensity of the

  staining of the oxidized redox indicator.

  The second immuno-active reagent serves to separate

  the substance to be assayed for the sample submitted for analysis. For this separation stage, the second immuno-active reagent can be fixed from the beginning on a support

  insoluble in water or fixed on a suitable support

  or after the immunological reaction.

  Among the insoluble carriers in water which

  for the second-highest immuno-active reagent are organic and inorganic polymers (amylase, dextran,

  natural or modified celluloses, polyacrylamide, agar

  pink, magnetite, porous glass powder, poly-fluoride

  vinylidene (Kynar) and latex), the inner wall of

  analytical vessels (small test tubes, test plates)

  glass or synthetic resin troughs or bowls) and

  if the body surface colides <glass rods and plastic, rods with terminal bulge, rods with fins or end slats). The supports which are especially suitable in the process according to the invention are the glass and synthetic resin beads. The second immuno-active reagent may be attached to the water-insoluble support by a physical (adsorption) or chemical mechanism; it can also be fixed using another reagent itself fixed on a support,

during or after the reaction.

  The essential point in the process according to the invention is that the substance subjected to the analysis has at least two immunoactive sites (epitopes), which can be recognized by the two immunoactive reagents, Y that is fixed on a support and the one that is marked by

  an enzyme, and react with them. Immunoassay reagents

  are preferably two different components which, of course, both react with the substance

  analyzed, but each directed to immunoassay sites.

  different. Reagents which are suitable

  specifically for the analysis of antigens are two anti-.

  body to this antigen, produced by two animal species

  different and which are directed towards different epitopes

  of this antigen. The combination of clot antibodies

  different or monoclonal antibodies and antibodies

  of a different animal species is especially suitable.

  For the immunological reaction, we can leave

  with the sample containing the substance subject to the

  lysis as is or diluted beforehand or subjected to

  appropriate prior art. For pre-treatment, the analyte can be isolated, the sample enriched with the substance, or riddled with

troublesome constituents.

  According to the invention, the subject substance

  the analysis is reacted simultaneously with the reaction

  an enzyme-labeled immuno-active protein and with the immuno-active reagent fixed on a support. The order of addition of reagents depends on the nature of the support system

  selected. When operating with a ball of plastic material

  sensitized tick, the enzyme labeled reagent and the analyte substance are first placed in a

  appropriate test tube and then add the sensitizer

sensitized by the other reagent.

  The immunological reaction is performed in a suitable buffer system to maintain a pH

  optimal, which can be between 4 and 9. The buffers

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  Examples are, for example, acetate buffer,

  citrate, phosphate buffer, Tris buffer,

  triethanolamine, borate buffer or glycine buffer. It is also possible to use buffer mixtures. The immunological reaction is preferably carried out at a temperature of 0 to 55C. NoTmally, the speed of the immunological reaction increases with temperature and under operating conditions elsewhere.

  identical, the equilibrium is reached more quickly.

  Incubation of the analyte with enzyme-labeled reagent and fixed reagent

  on support can be continued until the equilibrium. always

  However, the immunological reaction can also be stopped earlier, by separating the solid phase and the liquid phase after

  a determined incubation period and determining the

  by labeling with the enzyme either in the liquid phase or

in the solid phase.

  For the immunological reaction, it is possible to take

  special measures to stabilize the immunological activity

  reagents, the substance under analysis and

  the enzyme. In addition, one can add to the incubation solution

  constituents such as proteins and detergents.

  used to eliminate non-specific reactions,

  to lessen inhibitory or activating influences.

  The process according to the invention is extremely sensitive

  sible and distinguished by its simplicity in handling

  tions. The following examples illustrate the invention without limiting it; in these examples, the indications

  parts and percent are by weight unless otherwise stated

  milked. EXAMPLE Quantitative Analysis of CEA in Patient Plasma Using a CEA Monoclonal Antibody and a

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  CEA antibody (goat) In the required number of small test tubes (10 x 75 mm), 0.2 ml in each case of the test solution (0.2 mol / l NaH2PO4 / Na2HPO4, pH : 6.5, at 2 g / liter of bovine serum albumin,% of normal goat serum and 0.2 microgram / ml of anti-CEA conjugate of goat-peroxidase),

  add by mixing 0.050 ml of the patient's plasma to

  lysate or CEA standard (0 ng / ml CEA, 2.5 ng / ml

  CEA, 10 ng / ml CEA and 20 ng / ml CEA) and serum

  less than CEA (5.0 ng / ml CEA + 1.0 ng / ml), a polystyrene bead (diameter 6.5 mm) sensitized with anti-mouse monoclonal anti-CEA was added to each tube and we

  incubates for 16 hours at 37 ° C.

  The mouse monoclonal anti-CEA is prepared by a procedure analogous to that described in Journal of Immunological Methods, 32 (1980) 297-304, using as the initial cell line for fusion the line

  Sp 2/01-AG myeloma deposited under CRL 8006 at ATCC.

  Fusion is performed with mouse spleen cells immunized with CEA. The immunization of the mice was carried out as described in Table I of the publication

  above, the first two immunizations with 50 micro-

  grams of CEA each time, immunizations 3 and 4 were deleted, immunization 5 with 50 micrograms of CEA and immunizations 6 to 8 with 200 micrograms

each of CEA.

  The polystyrene beads are then washed three times with 2 to 5 ml of distilled water each time,

  transferred in 0.5 ml of buffer-substrate for deter-

  peroxidase activity (0.1 mole / liter of potassium citrate buffer, pH 5.0, with 6 millimoles / liter of H2O2 and 20 millimoles / liter of o-phenylenediamine) and incubated for 30 minutes at room temperature

  (22 C). To stop the peroxidase activity and intensify

  the color depth, 2.0 ml of N HCl are mixed and measured by photometry, within 30 min.

  maximul, extinction at the wavelength of 492 nm.

  Table 1 below shows the values obtained from

  in a CEA determination, compared to values obtained by Roche's radioimmunoassay.

Table 1

  Sample submitted for analysis LE492 n / TA / 30 Min.

E ha = u I m L E492 nm / TA / 30 Min.

  CEA standard O ng / ml CEA 0.103 2.5 ng / ml CEA 0.330, 0 ng / ml CEA 0.978, 0 ng / ml CEA 1.850 CEA control serum, 0 ng / ml CEA 0.540 RIT plasmas Roche Patient procedure No. 7212 0.6 ng / ml 1.2 ng / ml No. 7188 2.2 ng / ml 1.0 ng / ml No. 7220 1.2 ng / ml 1.4 ng / ml No. 7218 1.2 ng / ml 1.3 ng / ml No. 7234 2.5 ng / ml 2.7 ng / ml No. 7249 2.4 ng / ml 2.0 ng / ml No. 7203 2.3 ng / ml ml 2.0 ng / ml No. 7223 3.0 ng / ml 3.3 ng / ml No. 7247 3.1 ng / ml 2.8 ng / ml No. 7258 4.6 ng / ml 4.3 ng / ml No. 7215 4.9 ng / ml 5.5 ng / ml No. 7219 5.0 ng / ml 5.9 ng / ml No. 8180 8.6 ng / ml 8.5 ng / ml No. 7248 11.2 ng / ml 10.7 ng / ml No. 7262 14.2 ng / ml 14.3 ng / ml No. 7201 15.6 ng / ml 15.3 ng / ml Values less than 2.5 ng / ml of CEA are

  normal; values greater than 2,5 ng / ml correspond to

dent to a pathological state.

Example 2

  Assay of CEA in patient plasmas at

  using CEA antibodies from two animal species

  the various (goat and guinea pig) In the necessary number of small test tubes (10 x 75 mm), 0.2 ml of the test solution (0.2 mol / l NaH 2 PO 4 / Na 2 HPO 4, pH 6) are pipetted in each case. , 5, at 2 g / liter of bovine serum albumin, 20% of

  normal goat serum and 0.2 microgram / liter of

  goat-peroxidase anti-CEA conjugate), 0.050 ml of analyzed patient plasma or CEA standard (0 ng / ml CEA, 2.5 ng / ml CEA, ng ml / ml, and 20 ng / ml CEA) and control serum at CEA (5.0 ng / ml CEA + 1.0 ng / ml), a polystyrene bead (diameter: 6.5 mm) sensitized to guinea pig anti-CEA and incubated during

  16 hours at 37 C. The polystyrene beads are then washed

  on three occasions with 2 to 5 ml of distilled water

  each time each is transferred into 0.5 ml of

  pon-substrate for determining the activity of the

  peroxidase (0.1 mole / liter of potassium citrate buffer)

  sium, pH 5.0 with 6 millimoles / liter of H2O2 and 20 milligrams

  the / liter of o-phenylenediamine) and subjected to incubation

  30 minutes at room temperature (22 C). To cut off

  peroxidase and increase the depth of

  2.0 ml of HCII are mixed and, within a maximum

  30-minute photometry is used to measure the extinction at

  492 nm. Table II can be found

  below, the values obtained in a CEA assay,

  in relation to the values obtained in the radioimmunoassay

Rock.

Table II

  Sample submitted for analysis eE492 m / TA / 30 Min.

  CEA standard 0 ng / ml CEA 0.098 2.5 ng / ml CEA 0.270, ng / ml CEA 0.830 0, ng / ml CEA 1.515 CEA control serum, 0 ng / ml CEA 0.470 RIT plasmas Roche Procedure according to parts 1 l No. 7220 1.2 ng / ml 1.3 ng / ml No. 7218 1.2 ng / ml 1.3 ng / ml No. 7234 2.5 ng / ml 2.4 ng / ml No. 7249 2.4 ng / ml 2.2 ng / ml No. 7203 2.3 ng / ml 2.2 ng / ml No. 7223 3.0 ng / ml 3.0 ng / ml No. 7247 3.1 ng / ml ml 3.2 ng / ml No. 7258 4.6 ng / ml 4.5 ng / ml No. 7215 4.9 ng / ml 5.3 ng / ml No. 7219 5.0 ng / ml 5.6 ng / ml / ml No. 8180 8.6 ng / ml 8.5 ng / ml No. 7248 11.2 ng / ml 10.9 ng / ml No. 7262 14.2 ng / ml 14.2 ng / ml No. 7201 15.6 ng / ml 15.0 ng / ml The values below 2.5 ng / ml of CEA are

  normal; values greater than 2,5 ng / ml correspond to

dent to pathological states.

Example 3

  Assay of CEA in Patient Plasma Using CEA Monoclonal Antibodies from Two

different clones.

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  In the required number of small test tubes (10 x 75 mm), 0.2 ml of the test solution (0.2 mol / l NaH 2 PO 4 / Na 2 HPO 4 pH 6.5, with bovine serum albumin, 20% normal goat serum and 0.15 microgram / ml

  monoclonal anti-CEA mice-peroxy-conjugation conjugate

  dase), 0.050 ml of the plasma of

  Analyzes or CEA standard (O ng / ml CEA, 2.5 ng / ml CEA, 10 ng / ml CEA and 20 ng / ml CEA) and control serum at CEA (5, 0 ng / ml CEA + 1.0 ng / ml). In each case is added a polystyrene ball (diameter: 6.5 mm) sensitized to the anti-mouse monoclonal anti-CEA and

  incubated for 16 hours at 37 ° C.

  The mouse monoclonal anti-CEA was prepared by a procedure analogous to that described in Journal of Immunological Methods, 32 (1980) 297-304, using as the initial cell line for fusion the Sp 2/01 myeloma line. -Ag deposited under CRL 8006 at the ATCC. The

  fusion was performed with spleen cells from

  immunized at the CEA. The mice were immunized by a procedure analogous to that shown in Table 1 of the above publication, the first two immunizations with 50 micrograms of CEA in each case,

  immunizations 3 and 4 have been suppressed, immunization

  5 was carried out with 50 micrograms of CEA and immunizations 6 to 8 with 200 micrograms each of CEA. Two different monoclonal antibodies were used

  and appropriate, directed to different epitopes of the

tigene of the CEA.

  The polystyrene beads were then washed three times with 2-5 ml of distilled water each, each transferred into 0.5 ml of buffer-substrate for determination of peroxidase activity (0.1 mole / ml). 1 liter of potassium citrate buffer, pH 5.0, with

  6 millimoles / liter of H2O2 and 20 millimoles / liter of o-phe

  nylenediamine) and incubated for 30 minutes at room temperature (22 C). To stop the peroxidase activity and intensify the color depth,

  2.0 ml of HCl was mixed and then photomolarized.

  within a maximum delay of 30 minutes, quenching at 492 nm wavelength. The values obtained in a CEA determination are shown in Table III below, compared with the values obtained in the table below.

radio immunoassay.

Table III

  Lower than normal values; values greater than

to pathological states.

  2.5 ng / ml of CEA are 2.5 ng / ml correspond

  Sample submitted for analysis E492 nm / TA / 30 min.

  CEA standard O ng / ml CEA 0.390 2.5 ng / ml CEA 0.440, 0 ng / ml CEA 0.620, 0 ng / ml CEA 0.860 CEA control serum, 0 ng / ml CEA 0.510 RIT plasmas Roche Procedure according to patients 1 No. 7220 1.2 ng / ml 0.8 ng / ml No. 7234 2.5 ng / ml 3.0 ng / ml No. 7223 3.0 ng / ml 3.5 ng / ml No. 7247 3.1 ng / ml 3.2 ng / ml No. 7258 4.6 ng / ml 4.4 ng / ml No. 7215 4.9 ng / ml 5.0 ng / ml No. 7219 5.0 ng / ml ml 5.4 ng / ml No. 8180 8.6 ng / ml 8.6 ng / ml No. 7248 11.2 ng / ml 11.0 ng / ml No. 7262 14.2 ng / ml 14.0 ng / ml No. 7201 15.6 ng / ml 16.0 ng / ml

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Example 4

  Determination of HCG in serum / plasma / urine

  In the necessary number of small tubes to

  (0.2 x 75 mm) 0.2 ml of the solution for analysis (0.1 mol / liter of NaH 2 PO 4 / Na 2 HPO 4, pH 7.0, 2 g / liter of bovine serum albumin) were pipetted each time. 0

  microgram / ml of monoclonal anti-HCG conjugate

  mouse-peroxidase clone) (the monoclonal anti-HCG can be prepared by one of the procedures described in Journal of Immunological Methods 32 (1980) 297-304),

  adds in each case a polystyrene ball (diameter

  6.5 mm) sensitized with rabbit anti-HCG and incubated for 16 hours at room temperature in an atmosphere saturated with moisture. The polystyrene beads are then washed three times with 2-5 ml of distilled water each time, each is transferred to

  0.5 ml buffer-substrate for determination of the activity

  peroxidase (0.1 mole / liter of potassium citrate buffer, pH 5.0, with 6 millimoles / liter of H2O2 and 20 millimoles / liter of o-phenylenediamine) and is subject to

  incubation for 30 minutes at room temperature (16 to 30 C).

  To stop the peroxidase activity and intensify the staining depth, 2.0 ml of HC1N are mixed and measured by photometry within a maximum of 30 minutes.

  extinction at the wavelength of 492 nm. We find

  in the table below the values obtained during

  a determination of HCG in serum and urine.

  Determination of HCG in serum and urine 1. Standard curves A E 492 nm / 30 min / TA mUI HCG / ml urine serum

O 0.185 0.385

- 0.385 0.525

0.530 0.720

- 0.830 1 1.05

250 1.185 1 1.59

  2. Samples from patients Calculation: 1 ng of pure HCG corresponds to approximately 10 mIU dHCG

Example 5

  Determination of HCG in serum with anti-serum

  monoclonal bodies of HCG from two different clones

  In the required number of small test tubes (10 x 75 mm), 0.2 ml of the test solution (0.1 mol / l NaH 2 PO 4 / Na 2 HPO 4,

  pH 7.0 at 2 g / liter bovine serum albumin and 1.0 micro-

  gram / ml mouse monoclonal anti-HCG conjugate) (the mouse HCG monoclonal antibody is prepared by the procedures described in Journal of Immunological Methods, 32 (1980) 297-304. appropriate monoclonal different directed

  to different epitopes of the HCG antigen),

  0.050 ml of the patient's plasma to be analyzed or

  1 g of HCG (0.25, 50, 100 and 200 mIU / ml of HCG), a diameter polystyrene ball is added to each tube.

  6.5 mm sensitized with monoclonal anti-HCG

  and incubated at 37 ° C for example for 2 hours. The polystyrene beads are then washed three times with 2 to 5 ml of distilled water each, each transferred to 0.5 ml of buffer-substrate for determination of peroxidase activity (0.1 mol). / liter of potassium citrate buffer, pH 5.0, with

  6 millimoles / liter of H2O2 and 20 millimoles / liter of o-phe

  nylenediamine) and incubated for 30 minutes at room temperature.

  ambient temperature (16 to 30 C). To stop the activity

  oxidizing and intensifying the color depth,

  1.0 ml of 2N HCl and measured by photometry in

  a maximum delay of 30 minutes the extinction to the length of

  from 492 nm. The following table shows the values obtained in serum HCG determinations.

  Determination of HCG in human serum (mean values

  yennes in duplicate measurements) 1. Standard curves mUI / ml HCG 1) serum AE 492 inm / 30 min / TA 2) plasma 3) urine 4) buffer

0 0.038 0.054 0.158 0.180

0.226 - - -

0.475 0.544 0.557 0.716

0.905 0.955 0.970 1.40

1.75 1.55 1.90 2.44

  The HCG values found in the sera of

  listed below have been recorded on the curve.

  heel 1). The calibration curves 2), 3) and 4) have been

  erected later with new HCG standards.

  2. Patient sera 6E 492/30 min / TA mIU / ml HCG Measured serum taken on curve 1, Mixture 091080. 0.041 0 No. 2801 0.025 0 No. 3881 0.450 47 No. 2673 1.13 127 No. 1167 2,12 (1: 2) 470 No. 4891 0,975 (1:50) 51450 No. 3240 1,06 (1:20) 2,280 No. 4,418 1,475 (1: 1000) 167,000

248, 2

Claims (12)

  1) Immunological process for qualitative analysis   ve or quantitative of a substance using a reagent   enzyme-labeled immuno-active agent and an immunological reagent   - active fixed on a support insoluble in water or which will be fixed on a support-insoluble in water, by incubation   of the substance submitted for analysis with the immunological reagents   no-active, subsequent separation of the solid phase and the liquid phase and measurement of the degree of enzyme labeling   either in the solid phase or in the liquid phase,   what constitutes a measure of the amount of substance   analysis, characterized in that for the reaction   the substance under analysis and the reactions   Immunoactive cells are incubated from the beginning together and only once.   2) Process according to claim 1, characterized in   the substance under analysis is an antigen.   3) Process according to claim 1 or 2, characterized   in that the immuno-active reagents are   different bodies directed towards the same antigen but towards   different epitopes of this antigen.   4) Method according to claim 3, characterized in that the two reagents are two different monoclonal antibodies.   5) Process according to claim 1 or 2, characterized   in that the substance subject to analysis is the carcinoembryonic tigena (CEA).   6) Process according to claim 5, characterized in   that the immuno-active reagent fixed on an insolu-   ble in water is a mouse monoclonal anti-CEA.   7. Process according to claim 5 or 6, characterized   rised in that the labeled immunoactive reagent is another   Monoclonal anti-CEA mouse labeled with an enzyme.   8) Process according to claim 5 or 6, characterized   in that the immuno-active reagent is an anti-   CEA goat brand with an enzyme. 17 2481318   9) Method according to claim 6 or 7, characterized   in that the enzyme is peroxidase.   Method according to claim 1 or 2, characterized   in that the antigen is human chorionic gonadotropin (HCG). 11) Method according to claim 10, characterized in that the immuno-active reagent fixed on a support   insoluble is rabbit anti-HCG.   12) A process according to claim 10 or 11, characterized   in that the labeled immunoactive reagent is a   Monoclonal anti-HCG of mouse labeled with an enzyme.   13) Method according to claim 12, characterized   in that the enzyme is peroxidase.   14) Eimmunological process for qualitative analysis   of a substance with a labeled immuno-active reagent and an immuno-active reagent fixed on a support insoluble to water or which will be fixed on a   water-insoluble support, by incubation of the subspace.   analyzed by the immo-active reagents, subsequent separation of the solid phase and the liquid phase and measurement of the degree of labeling either in the solid phase or in the liquid phase, which is a measure of the quantity of substance being analyzed, characterized in that, for the immunological reaction, the analyte and the immunoactive reagents are incubated from the beginning together and once, and that analyte is an antigen and that the immunoactive reagents are antibodies of different clones, directed towards epitopes different from this antigen.