CH669265A5 - Multi indicator test strips for immunological analysis, their production and use. - Google Patents

Abstract

Various antigens are adsorbed each on a sheet of water-insoluble, adsorptive material, each sheet is cut into parallel tracks, the latter are individually affixed next to one another on a support foil and the track support foil is cut into parallel strips, across the longitudinal direction of the tracks. The test strips obtained are used for immuno-assays, by being allowed to incubate with the biological liquid to be examined and subsequently with the solution of a conjugate of a complementary antibody and covalent-bonded enzyme, radioisotope, a fluorescent agent or dye.

Description

DESCRIPTION

The methods and methods of both the radioimmunoassay (RIA) and the enzyme immunoassay (EIA) have been developed from methodological aspects of basic research [E. Engvall and P. Perlmann, Immunochemistry 8 (1971), 1971]; they are well known.

In general, three steps are characteristic of these processes and methods: 1. A relevant substance is bound covalently or by adsorption forces to a solid support. The relevant substance has a stable, three-dimensional structure and is referred to below as an antigen.

2. The sample, which may contain the corresponding antibody and is intended for determination, is brought into contact with the antigen-bound carrier; in this way binding of a complementary antibody molecule (if antibodies are present in the sample) to the antigen is effected.

3. The binding of the antibody contained in the sample to the corresponding antigen is determined by adding a further or second complementary structure.

This complementary structure is usually an antibody that is conjugated to either an enzyme or a radioactive isotope.

In most cases, the solid support consists of the actual test tube, latex particles, Sephadex beads, glass beads, nitrocellulose sheets or even intact cells and tissues [K. Catt and G. W. Tregear, Science 158 (1967), 1570; H. Towbin and co-workers, Proc. Natl.

Acad. Sci. (USA) 76, (1979), 4350]. The antigen to be bound to this carrier can be any molecule, a subcellular structure, an organelle, an enzyme, a hormone, lymphokines, etc., but also an endogenous substance. Even antibodies can sometimes act as antigens. The antibody contained in the sample is a structure that needs to be determined.

This molecule can be present in human or animal serum or another body fluid as well as in the cell culture supernatant.

The second complementary structure is in most cases an antibody specific for a structure of the antibody contained in the sample. An enzyme or radioactive isotope is bound to this second complementary structure; the connection of the antibody and the enzyme or the radioactive isotope is called conjugate in the following. The color changes are measured after adding a suitable substrate or radioactive decay.

In recent years, a method has been developed from this which is known in the literature as Gordon's dot immunoassay [European patent application 63 810; J. Gordon and M. Rosenthal, J. Rheumatol. 12: 257-264 (1985)]. According to this method, the antigen substances are applied in the form of small droplets (0.5) 11) to a strip of nitrocellulose; the evaluation of the results is advantageously carried out by densitometry.

Surprisingly, a method has now been found by means of which novel multi-indicator test strips are obtained, which are free from the restrictions inherent in Gordon's strips and which enable immunological analyzes to be carried out in series in a particularly elegant and safe manner. In particular, all analytical steps can be carried out simultaneously under the same conditions for all antibody-antigen bonds.

The process according to the invention for producing the multi-indicator test strips is characterized in that one sheet of a water-insoluble, porous, homogeneous, adsorptive, polymeric material is in each case in a solution or suspension of a different antigen (or a different one in a living organism as the antigen) active substance) (au), (A2), (A3) etc., leaves to incubate the leaves to block unoccupied binding sites in a solution of proteins not specific for the antigen or the substance and then dry, each leaf in parallel Cut webs and each one web of each of the sheets on a carrier film made of solid, coherent, non-brittle, neutral for the biological fluid to be analyzed, non-adsorbent material so that the webs are parallel to each other, and the resulting web-carrier film composite in parallel Stripes across Cut the longitudinal direction of the previously fastened webs, so that each strip carries the antigens (Al), (Az), (A3) etc. in adjacent, separate square test areas.

The result is a multi-indicator test strip consisting of (a) neutral, non-adsorbing carrier strips made of solid, coherent, non-brittle material for the biological fluid to be analyzed, on which (b) square test surfaces made of water-insoluble, porous, homogeneous, adsorbable, polymeric material , of the width of the carrier strips, are fastened next to one another in a separate arrangement, in such a way that a gripping surface is left free at one end of the strips, said test surfaces in each case (c) a different antigen (or a different antigen acting in a living organism Substance) (Al), (Al), (A3) etc. in a uniform distribution and the binding sites of the test areas not occupied by the antigen (Al), (Az), (A3) etc. by (d) for the antigen or the substance is not blocking specific proteins.

The new test strips (with the adsorbed antigens or substances (Al), (A2), (A3) etc. which act as antigens in a living organism) are used to identify and quantify a specific antibody (B) in a biological fluid to be examined, by allowing said or said test strips to be incubated in the liquid to be examined, possibly containing the antibody (B), and then freed from the unbound components of the liquid by washing, the test strip or strips in a solution of a conjugate (CD) which consists of a for the antibody to be determined in the liquid (B) complementary antibody (C) and an enzyme, radioactive isotope, fluorescent agent or coloring agent (D) covalently bound to the latter, and then incubated by the unbound portion of the conjugate (CD) freed by washing, and the presence and amount of the antibody (B) using the enzymatisc hen reaction, the radioactivity, the fluorescence or the color of component (D) of the substance formed on the test strip (s) (ABCD) is determined.

The invention is explained in more detail below.

The sheet that is used for the uptake and binding of the antigens can generally be extracted from any suitable, i.e. H.

water-insoluble, porous, homogeneous, adsorbable, polymeric material. It is suitable for this, among others, without this list being intended to be conclusive: natural and chemically modified carbohydrates, e.g. As cellulose, regenerated cellulose (chemical cellulose fibers), cellulose esters such as cellulose acetate, nitrocellulose etc., cellulose ethers such as highly etherified methyl cellulose, ethyl cellulose etc., cross-linked dextran and starch products, then fully synthetic polymeric compounds, e.g. B. from the series of polyvinyl compounds such as polyvinyl chloride, polyvinyl acetate, polyethylene, polypropylene, polystyrene etc., from the series of polyacrylic compounds such as the polyacrylates and polymethacrylates, the polyacrylamides etc., or from the series of polymeric organic condensation products, e.g. B. polyamides, polyurethanes, polyesters, polyepoxides etc.

Sheets of nitrocellulose are preferred, the natural adsorption capacity and binding capacity for antigens of which are quite sufficient for the purposes of the invention. For example, sheets made of nitrocellulose of the HAWG type from Millipore Corporation (Bedford, MA / USA) can be used; among others, those with an average pore size of 0.01 to 10 μm, for. B. of 0.5 m.

The carrier film on which the individual antigen webs are bound can generally be made from any suitable, i.e. H. solid, coherent, non-brittle material, which should also be neutral and non-adsorbing for the biological fluid to be analyzed. Suitable for this purpose include other foils made of appropriate plastics, glass, porcelain, wood and the like. Thin foils made of non-porous polyethylene or polypropylene are preferred.

According to a preferred embodiment of the invention, the procedure is as follows (FIG. 1): 1. The solid support, preferably a nitrocellulose sheet from Schleicher and Schuell GmbH (Dassel, FRG) of 21 x 21 cm, is soaked in the solution or suspension of the antigen substance. The binding capacity of the carrier depends on the nature of the antigen substance and is in the order of 100 FLg / Cm2 and more. The solution or suspension usually contains the antigen substance in excess, so that several nitrocellulose sheets can be treated in succession in one and the same solution or suspension.

2. After an incubation period of 1-2 hours, the nitrocellulose sheet is removed from the solution or suspension of the antigen substance and immersed in a solution of bovine serum albumin or rabbit serum in order to block the binding sites which may not be occupied. The leaf is then dried.

3. The nitrocellulose sheet is cut into approximately 3 mm wide strips. You get about 60 sheets from one sheet.

4. Steps 1-3 are repeated with further antigen substances and a new nitrocellulose sheet. Binding conditions, temperature and pH can be individually adapted to the respective antigen substance.

5. A solid plastic film (for example made of polypropylene, manufacturer: Papyrus AG, Basel) of 10 x 20 cm is provided with a double-sided adhesive tape, and one sheet of each of the nitrocellulose sheets treated with the antigen substance is glued onto the plastic film. The spacing between the individual tracks can be chosen freely; However, a close arrangement with a distance of approximately 1 mm is advantageous.

6. The plastic film treated in this way is then cut vertically into individual strips. Each strip (approx. 3-4 mm wide) contains all previously used antigen substances. Calculated on the entire nitrocellulose sheet, this results in 60 plastic films stuck with appropriate sheets and 60 x 60 = 3600 test strips.

A typical test strip is 8 cm long, 3 mm wide and has 10 areas of 3 x 3 mm each with a spacing of 1 mm, the lower half of the strip accommodating all 10 test strips and the upper half serving as a grip area.

The test strip is used for immunological analysis (EIA) as follows: - A strip is immersed in the test liquid and left to incubate for one hour.

- Unbound components of the test liquid are removed by immersing the strip in containers filled with water or a buffer solution.

- The strip is immersed in an antibody-enzyme conjugate and left to incubate for one hour.

- The unbound portion of the conjugate is removed by washing the strip in a container filled with water or a buffer solution.

- The strip is immersed in a substrate solution. After the reaction, the discoloration (or other changes) of the individual areas of the strip are measured.

For example, each test strip is immersed in a tube of diluted serum (usually 1/100 to 1/1000 dilutions) and allowed to incubate for one hour with occasional agitation of the strips. The strip is then washed in tris (hydroxymethyl) aminomethane buffer in physiological saline, pH 7.5 (TBS buffer) and incubated in the solution of an enzyme-antibody conjugate (dilution 1/1000) for a further hour. After 15 minutes of washing in TBS buffer, the strip is immersed in a substrate specific for the enzyme and, after 15 minutes of reaction, washed, dried and evaluated.

Compared to the so-called dot immunoassay mentioned above or the Gordon method, the test strips according to the invention and their use are distinguished by the following advantages: 1 a) The small square test areas are as homogeneous as possible and there are therefore no side effects, as is the case with the Gordon's droplet process may be the case.

b) The test areas attached to the strips are, for example, 3 x 3 mm in size, but they can certainly be of a size of e.g. B. 0.3 x 0.3 mm can be miniaturized.

However, the Gordon method is limited by the droplet coalescence in this regard.

c) According to the Gordon method, many droplets can be applied side by side, but the method of application (adsorption) must be the same for all droplets. The Gordon method is therefore not indicated in cases in which an antigen (e.g. double-stranded DNA) requires the formation of a covalent bond.

When using high baking temperatures, all droplets have to go through the same process, while with the new process each sheet can be treated individually and as desired. An antigen with low binding capacity can achieve an improved total binding in this process by repeated soaking. This is difficult to do with the dot immunoassay.

2. The advantage of the new test strips when used for the actual analysis is that they are made of a relatively stiff material and are therefore easy to handle; at one end you can touch the strip, add a code, label it and manipulate it as you wish. The test strips can be immersed in tubes, individual strips or groups of strips removed by hand or by means of a comb from the solution (tubes or other containers), washed under the water jet or by immersing them in a washing liquid, etc. The for the dot immunoassay strips using, on the other hand, are incubated in horizontal containers; the individual strips must be handled with tweezers or another device; the washing processes are accordingly complicated and cumbersome.

3. When evaluating with the naked eye, both methods, i. H. the dot immunoassay and the present method, equivalent. When evaluating using densitometry, however, the alignment of the measurement beam in the new system is straightforward, since a uniform distribution of the antigen is guaranteed over the entire width of the strip.

With Gordon's Dot Immunoassay, the measurement beam must pass through the center of the spot or capture and integrate the entire spot, otherwise the measurement is incorrect.

In addition, it is now possible to include new antigens that are still in the research phase in addition to the already known antigens in the analysis, and thereby to achieve a higher reliability of the statement.

If one finally considers the production of the test strips and their use together with the subsequent evaluation of the results, the invention as a whole has brought about an unexpected, drastic simplification of all relevant steps in comparison with the conventional methods. For the first time, this opens up a practical possibility to produce test strips of any antigen composition for individual patient groups or special conditions (epidemics, geo-specific conditions) immediately and in a simple manner from a collection of ready-made antigen webs.

example 1 10 nitrocellulose sheets of 21 x 21 cm are immersed in tris (hydroxymethyl) aminomethane buffer in physiological saline, pH 7.5 (TBS buffer).

Simultaneously, 50 ml of the following antigen solutions are placed in 10 plastic containers measuring 23 x 23 cm with a pipette: (a) rabbit immunoglobulin, (b) lysate from HeLa cells, (c) nuclear antigen, (d) salmon DNA, ( e) mitochondria, (f) streptolysin O, (g) histone, (h) single-stranded DNA, (i) normal rabbit serum, (j) control TBS buffer.

Each of the moistened nitrocellulose sheets is placed in one of the plastic containers (a) to (j) and incubated for 2 hours at room temperature. The nitrocellulose sheets (a) to (h) are then immersed (if desired after drying) in 3% normal rabbit serum in order to block the free binding sites.

Nictrocellulose sheet (i) is left in the original container. Leaf (.1) does not come into contact with the rabbit serum and serves as an unblocked control. After 30 minutes, all leaves are removed from the solutions and air dried. Sheet (d) is dried in a hot air cabinet at 80 "C.

60 polypropylene films of 10 x 20 cm are provided with double-sided adhesive tape 665 (manufacturer: Minnesota Mining and Manufacturing Co., St. Paul, MN / USA). The pasted area corresponds to 4 x 20 cm. Each of the 10 nitrocellulose sheets is cut into 3 mm wide strips using a precision cutting machine. In this way, 60 sheets are obtained from each of the 10 nitrocellulose sheets. The individual sheets are glued to the polypropylene film in the order (a) to (j). The foil matrices created in this way are then cut vertically into 3 mm wide strips (Fig. 3). In this way, 3600 test strips are obtained.

Example 2 The procedure up to the production of the strips is identical to that of Example 1. Each 12 finished strips are combined in the form of a comb and glued to a cardboard base (Fig. 4), so that 12 samples can be analyzed in parallel and simultaneously.

Example 3 The course of the process is identical to that of Example 1. However, the antigenic substances are the following molecules: (a) antibody against mouse chain, (b) antibody against mouse yl chain, (c) antibody against mouse y2a chain, (d) antibody against mouse 2b chain, (e ) Antibody against mouse X chain, (f) Antibody against mouse K chain. All of these antibodies were produced in rats. For the determination of the immunoglobulin isotype, the test strips in hybridoma cell cultures [G. Köhler and C. Milstein, Nature (London) 56 (1975), 495] and used miniaturized strips for their determination in small volumes. The miniaturization consists, on the one hand, of strips 1.5 mm wide and, in addition, two films of different colors back to back stick on, so that both sides of the strip can be used for the analysis.

The different film color serves as a color code. 1.5 mm was selected as the strip width.

Example 4 Four tubes (inner diameter 7 mm) are filled with 2 ml TBS (Tris-buffered isotonic saline, pH 7.5) containing 3% rabbit serum. 20 pLI are added to two tubes and 2 Ill of test serum are added to the other two tubes. One test strip each is immersed in a tube and allowed to incubate for 1 hour at room temperature with occasional agitation.

The strips are then removed from the tubes and washed in excess TBS (beaker content 100 ml) for 15 minutes. Four tubes of conjugate solution (antibodies produced in rabbits with specificity against human immunoglobulins, coupled with peroxidase, commercially available from Dakopatts, Copenhagen / Denmark) are now being prepared. Two of the tubes are diluted 1/100 (in TBS + 3% rabbit serum) and the other two with 1/1000 (2 ml each). The washed strips are immersed in the conjugate solutions according to the following scheme: serum 1/100, conjugate 1/100; Serum 1/100, conjugate 1/1000; Serum 1/1000, conjugate 1/100; Serum 1/1000, conjugate 1/1000. After 45 minutes, the strips are removed from the conjugate solution and the unbound portion of the conjugate is washed in excess TBS (beaker content 100 ml). Four tubes of substrate solution are now being prepared. For 10 ml substrate solution, 0.6 ml 4-chloronaphthol stock solution (the stock solution contains 3 mg 4-chloronaphthol in 1 ml methanol) and 4 ltl H202 (30%) are added to 9.4 ml TBS. 2 ml of substrate are pipetted into each tube and a strip is immersed in the contents. After 15 minutes, the strips are removed from the tube and washed in distilled water.

The color reaction is read either with the naked eye or with the densitometer. A blue colored area indicates a positive reaction. As a rule, only some of the surfaces will change color. These are then positive for the corresponding antibody.

Example 5 A test with nine sera and a negative control was carried out. The negative control consists of 3% rabbit serum, and the possible coloring of individual areas of the strip provides information about a non-specific binding of the conjugate. Of the nine sera, three were positive controls. Positive control means sera in which antibodies have been previously detected by another method. In this case, it was one serum positive for anti-DNA antibody, another serum positive for streptolysin antibody and the third serum positive for rheumatoid factor. Six sera were previously non-analyzed patient sera, in which an independent one The antibodies were only determined after analysis with the test strip in order to determine the correlation between the new multi-indicator test strip and previous tests. The comparative tests were the Crithidia test for the anti-DNA antibodies [L. A. Aarden and R. Smeenk, Immunological Methods, editors I. Lerkovits and B. Pernis, Vol. 2, page 75 (1981)], the hemolysis inhibition test for the streptolysin antibodies [K. Kalbak, Acta Med. Scand. 130 (1984), 358] and an enzyme immunoassay in microtiter plates for the rheumatoid factor [A. Faith and co-workers, J. Immunol. Methods 55 (1982), 169].

Two serum dilutions (1/100, 1/1000) and two conjugate dilutions (1/100, 1/1000) were used for the present test. After performing the test, all four strips were considered to avoid false positives and false negatives.

The test is false positive if the coloring is produced by a non-specific binding; Under certain circumstances, this can occur either when the concentration of irrelevant serum proteins is too high or when the concentration of conjugate is too high. The test is false negative if the staining to be performed does not occur due to excessive dilution of the specific serum components or the conjugate.

The results of the test described above can be summarized as follows: a) The three positive controls were positive for corresponding reactions also in the multi-indicator test strip.

b) The following results were obtained for the six unknown sera: Serum 1: positive for streptolysin, Serum 2: positive for streptolysin, Serum 3: negative for all tested antigens, Serum 4: positive for the rheumatoid factor, Serum 5: negative for all tested Antigens, serum 6: positive for streptolysin, the rheumatoid factor and anti-DNA.

All six sera were also analyzed by the standard methods mentioned above, and identical statements were obtained as with the multi-indicator test strip. The correlation between the previous methods and the new test is therefore satisfactory.

c) The negative control (rabbit serum) gave negative results for all antigens.

Claims (8)

PATENT CLAIMS 1. Multi-indicator test strips for immunological analyzes, consisting of (a) neutral, non-adsorbing carrier strips made of solid, coherent, non-brittle material for the biological fluid to be analyzed, on which (b) square test surfaces made of water-insoluble, porous, homogeneous, adsorbable, polymeric material, of the width of the carrier strips, are fastened next to one another in a separate arrangement, in such a way that a gripping surface is left free at one end of the strip, said test surfaces each carrying (c) different antigens in a uniform distribution and not occupying those by the antigens Binding sites of the test areas are blocked by (d) proteins not specific for the antigens. 2. Test strip according to claim 1, characterized in that the square test surfaces (b) from water-insoluble, natural or chemically modified carbohydrates, in particular from cellulose, regenerated cellulose, cellulose esters, cellulose ethers or crosslinked dextran and starch products, or from fully synthetic polymeric compounds, in particular consist of the series of polyvinyl compounds, polyacrylic compounds or polymeric, organic condensation products. 3. Test strip according to claim 2, characterized in that the square test surfaces (b) consist of nitrocellulsoe. 4. Test strip according to claim 1, characterized in that the carrier strips (a) consist of a non-porous plastic or of glass, porcelain or wood. 5. Test strip according to claim 4, characterized in that the carrier strips (a) consist of polypropylene or polyethylene. 6. A method for producing the multi-indicator test strips for immunological analyzes according to one of claims 1 to 5, characterized in that one sheet of a water-insoluble, porous, homogeneous, adsorbable, polymeric material is allowed to incubate in a solution or suspension of a different antigen which leaves leaves to block unoccupied binding sites in a solution of proteins which are not specific for the antigen and then dries, cuts each sheet into parallel tracks and places a sheet of each of the sheets on a carrier film made of solid, coherent, not brittle, for the biological fluid to be analyzed neutral, non-adsorbing material is attached in such a way that the webs lie parallel next to one another, and the resulting web-carrier film composite cuts into parallel strips, transverse to the longitudinal direction of the previously attached webs, so that each strip is arranged next to one another eten, separate square test areas carries the respective antigens. 7. A method for producing a test strip set, characterized in that several test strips according to one of claims 1 to 5 are attached next to one another and parallel to one another by means of an empty end with respect to the antigens on a common base. 8. Use of a test strip according to one of claims 1 to 5 for the identification and quantification of a specific antibody (B) in a biological fluid to be examined by an immunological analysis, characterized in that said test strips carrying different antigens (A) in the examining, possibly containing the antibody (B) incubated and then freed from the unbound components of the liquid by washing, the test strip in a solution of a conjugate (CD), which consists of an antibody to be determined in the liquid (B) complementary antibody (C) and an enzyme covalently bound to the latter, radioactive isotope, fluorescent agent or colorant (D), can be incubated and then freed from the unbound portion of the conjugate (CD) by washing, and the presence and amount of the antibody (B) using the enzymatic reaction, the Radioactivity, the fluorescence or the color of component (D) of the substance formed on the test strip (ABCD) determined.