AU8037187A

AU8037187A - Test device and method of characterizing biological material

Info

Publication number: AU8037187A
Application number: AU80371/87A
Authority: AU
Inventors: Anne Bolmstrom; Magnus Ericsson
Original assignee: Biodisk AB
Current assignee: Biodisk AB
Priority date: 1986-04-04
Filing date: 1987-09-28
Publication date: 1989-04-18
Also published as: FI892489A0; NO891892L; SE8601511L; WO1989002927A1; DK252489A; SE460472B; FI892489A; SE8601511D0; JPH02501349A; NO891892D0; EP0335866A1; DK252489D0

Description

TEST DEVICE AND METHOD OF CHARACTERIZING BIOLOGICAL MATERIAL

The present invention relates to a test device for characterization of biological materials by means of microbiological, immunological and/or biochemical analyses. The invention further comprises a method of characterizing biologic materials.

Today, for example, determination of the minimum inhibitory concentration of an antimicrobial substance is i.a. carried out by means of plastic test plates in which there is a number of circular wells. A dilution series of the antimicrobial substance, of which is to be determined is introduced into these test wells. The antimicrobial substance can either be introduced in the form of a solution which is introduced into the wells when performing the analysis or in advance and dried at the bottom of the wells. In these determinations each well contains its individual concentration of the antimicrobial substance. A suspension of the microorganism, the sensitivity of which is to be tested, is applied into a culture medium. An equal and exact volume of this suspension is added to each well, after which the whole test plate is incubated. The results are thereafter read in such a way that growth or no growth of the microorganism is noted in the different wells, and in this manner the concentration to be determined is etablished.

The disadvantages of these systems are numerous. Antimicrobial substance dried at the bottom of each well this gives a short shelf-life of the thus prepared plates because a prolonged storage results in a degradation of the substance. Moreover, the choice of antibiotics and antibiotic concentrations is limited in such a plate. When the antimicrobial substance is added to the wells in conjunction with the analysis it is very time-consuming to prepare these dilution series of substance. Said methods thus are rather expensive and non-flexible.

These disadvantages are eliminated by means of the present invention comprising a test device for characterization of biological materials by means of microbiological, immunological and/or biochemical analyses, in which test device there is a plurality of. wells, each comprising two distinct zones communicating with one another, one of which is a reagent zone and the other a reading zone.

The shape of the test device can be rectangular, round or oval, and the wells in the test device can be of different designs. This invention is not restricted to any special shape of the test device or any special designs according to which the wells are made in the test device. Some embodiments of the test device according to the invention are shown in Fig. 1 consisting of the test plate A, a reagent zone B and a reading zone C of each well.

The shape of the wells can vary within the scope of the invention and according to one embodiment the wells have the shape of a key-hole, the circular portion being the reagent zone and the elongated portion the reading zone. In another embodiment each well consists of an elongated portion comprising the two zones where constricting projections are arranged between the zones. In this embodiment the ends of the wells can be rounded off or straight. In another embodiment of the invention each well consists of a square reagent zone and a squar reading zone which are of different sizes. Fig. 2 shows some different embodiments of wells according to the invention, B designating the reagent zone, C the reading zone and D the constricting projections. According to the invention other shapes of the wells can also be used and of importance is that the carrier of the active substance is retained in the reagent zone. Tapering zones for example are also possible.

As shown in Fig. 3 the wells can also have such a shape that the reagent zone and reading zone are lying above each other, i. e. in the vertical plane of the test device or consecutively, i. e. in the horizontal plane of the test device. The designations in the figure have the meanings indicated above. It is apparent from Fig. 4 that the reading zone can be covered from above. In this figure the area where communication between the two zones takes place has been designated E.

The test device is preferably of some inert, material such as plastic or glass and it can be in one piece or made with wells after which possible constricting projections are applied.

In fig. 5 a perspective view of a well in the shape of a key-hole is shown comprising a reagent zone B and a reading zone C. In the reagent zone B a carrier F is applied which is impregnated with the antimicrobial substance, the minimum inhibitory concentration of which is to be determined. A suspension G of microorganisms in a medium is uniformly distributed across the two zones B and C and covering the top of the carrier F. In Fig. 6 the result after incubation is shown, where growth of the microorganism is seen at H in the reading zone whereas there is inhibition of the growth at K. A, B and C are as defined above. The number indiciated in the figure are markings of the carriers. In this case the amount of antibiotic is indicated on the respective carrier. The carriers marked by plus and minus signs are positive and negative controls, respectively.

This invention also relates to a method of characterizing biological materials by means of microbiological, immunological and/or biochemical analyses, according to which the characterization is carried out in two zones communicating with one another, one of which is a reagent zone and the other a reading zone, a reagent on a carrier is applied in the reagent zone and the biologic material to be characterized is distributed across the two zones, after which the whole is mixed, incubated, if required, and a possible reaction is read in the reading zone. The reagent is introduced into the reagent zone on a suitable carrier such as a porous or nonporous disc, a bead coated with the required amount of reagent, or it can be added in the form of a compressed tablet containing the required amount of reagent. The biological material to be characterized is thereafter applied into each well. The biological material is present in or tranferred into liquid form and is preferably applied by means of a pipette, in exact volumes to each well. The test device is thereafter rotated or shaken cautionsly to obtain a uniform mixture of the reagent and biological material in the whole well. De pending on the type of analyses the test device is rotated, shaken, centrifuged and/or incubated for part of or during the whole reaction after which the results in the different wells are read.

The whole procedure can preferably be automated. An apparatus can e.g. dispense the active carriers, and a system can automatically pipette the test sample. The reading can also be conducted using a suitable spectophotometric apparatus illuminating the reading zone.

Thus, test devices according to the present invention can be used when characterizing biological materials such as in microbiological, immunological and biochemical analyses, for example determination of concentrations of substances which are active against microorganisms or other biologic cells. Besides microorganisms biologic materials can for example be other biologic cells, antibodies, antigens, enzymes, blood, serum or other biologic mateial in the form of liquids. The reagent may consist of biologic or chemically active substances such as antimicrobial substances, microbiologically active substances, substances with an inhibiting, lethal, toxic, mutagenic or growth-promoting effect on cells.

Test devices with unlabelled wells can be used for different kinds of reagents and in different concentrations. The latter are applied by the aid of suitable carriers on which the reagent is applied in a desired concentration. Carriers with reagents are packed in separate units and the type of reagent as well as amount is preferably indicated by specific labelling on the carriers itself. Altlernatively, the wells on the test device can be individually labelled for different types of analyses.

The expression "microorganism" includes bacteria such as enterobacteriaceae, staphyloccoci, streptococci, hemophilus, neisseriaceae, bacteriodes and clostridia, mycobacteria, actinomyces, mycoplasma, nocardia, virues and fungi such as mould-fungus, yeast-fungus and Candida.

The expression "biologic cells" comprises cancer cells, normal human cells, animal cells, insect cells and plant cells. Antimicrobial substances are antibiotics, for example aminoglycosides, β-lactam antibiotics, macrolide antibiotics, polymyxins, polypeptides and other chemotherapeut ics such as sulf onamides, antimycot ics, for example 5-fluorocytosine, amphotericin, antiviral agents such as adenine arabinoside (Ara-A), trif luorothymidine, antituberculous agents such as iεoniazide and cycloserine, as well as disinfectants, antiseptics and preservative such as chlorohexidine, ethanol and bensalkonium chloride.

Substances that can have an inhibitory, lethal, toxic or mutagenic effect on cells are anticancer agents, e.g. antracyclines, mitomycins, cancerognic/mutagenic substances such as dimethyl nitrosamine, herbicides such as paraquat and pesticides as well as insecticides such as dicophane (DDT) and chloroquine.

Microbiologically active substances besides the indicated antimicrobial substances are bacteriocin, for example colicin, enzymes, for example peroxidases, various growth factors such as amino acids, vitamins, carbohydrates and other nutrients, various body fluids and components thereof such as serum, blood and specific components in serum, for example β-lysine.

Biologically active substances are interferons such as human interferon, HuIFN-β, IF4N-β, enzymes such as β-lactamases, proteases and ureases, enzyme specific substrates such as nitrates, carbohydrates and proteins, β-lactam antibiotics, aminoglycosides and chloramphenicol, antigens such as immuneglobulins, polysaccharides and toxins from microorganisms. It is for instance possible to determine the minimum inhibitorys concentration of an antimicrobial substance by means of the method of the invention. The determination is carried out in two zones communicating with each other, one being a reagent zone and the other a reading zone. The antimicrobial substance is introduced into the reagent zone on a suitable carrier such as one of those indicated, for example a porous disc. A suspension of the microorganism, the sensitivity of which is to be determined, is thereafter introduced into each well. The suspension is in a suitable culture medium. This is preferably applied by means of a pipette, in exact volumes of microorganism in substrate to each well. The test device is thereafter tilted carefully to achieve a uniform mixture of the substance and the suspension of microorganisms in the whole well. After incubation e.g. at 37°C over night, the results are read, i.e. inhibition of growth of the microorganism in the different wells. As the antimicrobial substance is present in different concentrations on labelled carriers in the different wells it is easy to establish directly the minimum inhibitory concentration of the substance.

One can continue from an MIC-determination as described above to the determination of the minimum bactericidal concentration (MBC). This can be done in different ways. According to one of these, samples are taken from the material existing in the different reading zones after the MIC-determination. These samples are inoculated into fresh culture medium in the wells of another test device or are spread on agar plates. After further incubation the presence of growth is read and the minimum bactericidal concentration (MBC) is determined. Another method of determining the MCB is to remove the carrier with antimicrobial substance from the reagent zone after the MIC determination. A new substanse is then added to the well to inactive the antimicrobial substance used in the MIC-determination, e.g. an enzyme such as a β-lactamase in the cases when a β-lactam antibiotic has been used. The inactivating substance is preferably applied to the reagent zone on a carrier, such as any of those described earlier. After incubation, the presence growth is read and the MCB is determined.

By means of the method of the invention it is also possible to identify microorganisms by determining their biochemical patterns based on fermentation, oxidation or assimilation of different substances. A test substance is introduced into the reagent zone with or without the addition of other reagents for the particular reaction, for example a pH-colour indicator on a suitable carrier, such as a bead coated with the appropriate amount thereof.. A suspension of the microorganism, the biochemical pattern of which is to be determined, is thereafter applied to each well. The supension is made in a suitable culture medium. This is applied for instance with a pipette in exact amounts of microorganisms suspended in culture medium to each well. To detain a uniform mixture of the substances and the suspension of microorganisms in the whole well, the test device is carefully rotated or shaken. After incubation the result is read in different ways depending on the biochemical reaction used. The assimilation pattern is established for instance by detecting growth/no growth, while fermentation or oxidation patterns can be indicated by colour change of a pH-indicator.

Examples of substances used in the determination of assimilation patterns are carbohydrates such as glucose, mannose, lactose, raffinose, melibiose, cellobiose, glycerol, xylitol and gluconate. These substances can be combined with suitable pH-indicators such as phenol red, bromothymol blue, gromocresol purple for fermentation and oxidation studies.

The method of the invention can also be used for characterizing enzymatic patterns of biologic materials (so-called zymograms) by determining enzyme/substrate reactions, in which the determination is carried out in the two zones communicating with one another. A chromogenic substrate, for example a naphthalene derivative on a suitable carrier, for example in the form of a tablet containing the desired amount of the chromogenic substrate, is introduced into the reagent zone. A suspension of the biological material, the zymogram of which is to be determined, is thereafter added to each well. This is preferably applied by means of a pipette, in exact volumes of biological material to each well. The test device is thereafter rotated carefully so that a uniform mixture of chromogenic substrate and the suspension of the biological material is obtained through out the whole well. The test device is then incubated and the result is thereafter read, i.e. colour changes of the enzyme/substrate reactions in the different wells.

Chromogenic substrates that can be used in zymogram studies are for instance chromogenic naphthalene derivatives such as naphthyl phosphate to detect phosphatases, naphthyl butyrate to detect esterases, naphthyl myristate to detect upases, naphthylamides to detect araylamidases and trypsin, naphtyl galactopyranosides to detect galactosidases, naphthyl glucopyranosides to detect glucosidases, nitrocephin to detect β-lactamases etc.

Futhermore, the method of the invention can be used for characterizing and quantifying different biologically active substances based on their immunologic properties. A specific antibody-latex conjugate is applied in the reagent zone and suspension of the biological material, the serologic pattern of which is to be determined, is uniformly distributed across the two zones. The device is thereafter rotated to obtain a uniform mixture of the reagent and the sample. An antigen-antibody reaction resulting in agglutination, i.e. clumping, will take place if the sample contains antigens which are specific to antibody-conjugate present in the reagent. By using a set of different specific antibody-latex conjugates a bacterial species can for example be serotyped and examples of bacterial species that can be classified by means of a serologic pattern are salmonella, shigella and streptococci. Similarly it is possible to determine the amount of antibodies in a biological material by using an antigen-latex conjugate.

Correspondingly one can determine the amount of an active substance that may inhibit an immunologic reaction. Determination of antibiotics in serum can be carried out by this technique. A definite but varying amounts of an antibiotic-antibody conjugate are applied in the reagent zones a suitable carrier. The serum sample or a dilution thereof, the corresponding antibiotic concentration of which is to be determined, is uniformly distributed in the two zones and mixed by means of rotation of the device. A definite amount cf the specific antibiotic-protein conjugate bonded to latex particles (antigen unit) is thereafter applied to the reagent zone and the device is further rotated for the antigen-antibody reaction. The reaction between antigen and antibody units results in agglutination. Addition cf the substances, in this case a specific antibiotic, the concentration of which is to be determined and which have the same or a higher affinity for the antibody unit, prevents or inhibits the reaction between the latex-bonded antigen and the antibody units by bonding to the antibody units resulting in omitted agglutination. Depending on the amount of antibiotic in the sample the agglutination is inhibited in the different wells up to the maximum concentration of antibody units to which the antibiotic sample can be totally bonded. When this concentration of antibody units is exceeded the free antibody units now remaining react with the antigen units and result in agglutination. When reading the sample the result thereof is compared to e.g. a standard curve which has been obtained by determining in advance the minimum amount of antibiotic inhibiting the different concentrations of antibody units by using standard solutions of this antibiotic.

Active substances in biologic liquides that can be quantified by means of an immunologic technique are antimicrobial substances, antibodies, antigens such as toxins and polysaccharides from microorganisms.

Claims

1. A test device for characterization of biological materials by means of microbiological, immunological and/or biochemical analyses, several test wells being included in said test device, each well comprising two adjacent zones, one of which is a reagent zone intended for application of a reagent present on a separate carrier and the other is a reading zone, the two zones communicating with one another so that the biological material to be characterized can be uniformly distributed across the two zones.

2. The test device of claim 1, wherein each test well has a constriction between the reagent zones and reading zones and are alligned in the vertical plane of the test device so that the reagent zone is lying above the reading zone or in the horizontal plane of the test device so that the reagent zone and the reading zone are lying beside one an other.

3. The test device of any of claims 1 and 2, wherein the wells are shaped as key-holes and the circular portion of the well is the reagent zone and the elongated portion thereof is the reading zone or each well is an elongated portion comprising the two zones with constricting projections in between the zones, or each well comprising a square reagent zone and a square reading zone and the two zones are of a different size or that each well has a tapering shape and that the reagent zone is in the wide portion and the reading zone in the narrow portion of the well.

4. The test device of any of claims 1-3 formed in one piece.

5. A method of characterizing a biologic material by means of microbiological, immunological and/or biochemical analyses, wherein the characterization is carried out in two adjacent zones communicating with one another, one being a reagent zone and the other a reading zone, a reagent on a carrier is applied in the reagent zone, the biologic material to be characterized is uniformly distributed across the two zones, all this is mixed and optionally incubated and the reaction is read in the reading zone.

6. The method of claim 5, wherein the minimum inhibitory concentration (MIC) and/or the minimum bactericidal concentration (MBC) is determined, the biologic material comprises microorganisms and the reagent is an antimicrobial substance.

7. The method of claim 5, wherein the biological material comprises microorganisms, their biochemical pattern is determined and the reagent is a substrate.

8. The method of claim 5, wherein the enzymatic pattern of the biological material is determined and the reagent is a chromogenic substrate.

9. The method of claim 5, wherein the immunological pattern of the biological material is determined and the reagent is a specific antibody-latex conjugate or a specific antigen-latex conjugate.

10. The method of claim 5, wherein an active substance in the biologic material is determined by inhibiting a specific antigen-antibody reaction, the reagent partly consists of a specific antibody unit to the substance to be determined and partly of a specific antigen-latex conjugate, the active substance to be determined either being the antibody or the antigen part.

11. The method of any of claims 5-10, wherein the reagent is applied in the reagent zone on a suitable carrier such as a porous or non-porous disc, bead or in a tablet containing the reagent.

12. The method of any of claims 5-11, wherein the determination is carried out by means of a test device according to any one of the claims 1-4.