NL1006680C2 - Method for detecting the presence of an immunologically reactive molecule in a sample. - Google Patents

Description

METHOD FOR DETECTING THE PRESENCE OF AN IMMUNOLOGICALLY REACTIVE MOLECULA IN A SAMPLE

The invention relates to a method for detecting the presence of an immunologically reactive molecule in a sample, comprising contacting the sample with a standard solution of one or more immunologically reactive reference molecules in an incubation mixture to form enable immune complexes between the immunologically reactive reference molecule and the immunologically reactive molecule to be detected, and determining whether immune complexes have been formed by comparing one or more of the physical parameters molecular weight, charge and shape of the components of the incubation mixture with the same physical parameters of the components of the standard solution.

Such a method is important, for example, in determining in a test object, such as a patient or an animal to be examined, the presence of a molecule which can elicit an immunological response. Such molecules are, for example, hapten, macromolecules, pathogens or immune-stimulating parts thereof. Such molecules are in the body when there is an infection. However, it may also be desirable to detect other molecules in other types of samples. Consider, for example, the demonstration of the presence of a certain protein in a plant, or the detection of molecules in soil or water samples.

Infection is usually diagnosed by clinicians or vets based on clinical symptoms and / or anamnesis. In addition, laboratory research into the presence of pathogens or the reaction of the test object to the pathogen is an important part of this. Proven methods for this are, for example, cultivation techniques, serological determinations or DNA testing.

However, a number of diseases have the disadvantage that the pathogens are only present in low concentrations or cannot be isolated from the test object. Herpes viruses, for example, are often not detectable in the bloodstream because they are present in the nervous system and are therefore difficult to access without causing serious damage to the test object. As a result, the detection of such diseases often goes indirectly through the detection of specific antibodies in body fluids. Such antibodies target the causative agent of the disease. The presence of antibodies means that the test object has been in contact with the respective antigen and / or the pathogen.

Such methods to demonstrate the presence of pathogens or antibodies against them in the body of a test object are used in both human health care and veterinary health care. It is crucial that the method is not only fast, but also reliable. It goes without saying that false positive or false negative results can have disastrous consequences in both human health care and veterinary applications.

In particular for veterinary health care, there is further a great and growing need to establish the health status of livestock, in particular cattle, pigs and poultry. This applies not only to classical diagnostics, ie to identify the cause of a prevailing disease or condition, but also to those forms of diagnostics that are more widely applicable for the purpose of monitoring ('monitoring') the course of a (subclinical) infection and prognosis.

Not only in parasitology, but also in virological disorders, such as IBR in cattle and Auzjesky's disease and bladder disease in pigs or Marek's disease in poultry, or in bacteriological disorders, such as Salmonella in poultry, presence of antibodies in serum 1006680 3 of great importance in mitigating or eradicating the associated disease. There is therefore a great need for a diagnostic method for demonstrating seropositivity and then preferably against multiple diseases in one test, whereby the test must be fast, very reliable and inexpensive.

The speed of the method is important, because often a lot of samples have to be tested, sometimes depending on the problem, hundreds or thousands to millions.

The method must be reliable, because missing a percent of these large numbers in the veterinary sector can mean that a large number of sick animals are declared healthy in absolute numbers. 15 These can then again form a source of infection. For human health care, the highest possible reliability speaks for itself.

In the veterinary sector in particular, being cheap is important because margins in that industry are relatively small. Government price control is also important in the human sector.

Currently, for the above-described diagnosis of infections, in particular ELISA techniques and related immunological techniques are used. Although they meet the speed and cost requirements, they have an inherent risk of error due to the methodology used.

In ELISAs, proteins (antibodies or antigens) are adsorbed on the wall of small reaction wells which are then filled with the sample to be tested. Over time, in a subsequent step, the binding of proteins (antigens or antibodies) from the sample to the already bound proteins on the wall of the reaction wells is visualized by adding labeled antibodies specific for the antigen or antibody from the sample.

1006680 4

Crucial to this method is that the reaction wells must be rinsed three times to remove unbound proteins from the sample. The mode and duration of rinsing must be well attuned to the properties of the molecule to be detected from the sample. Insufficient rinsing can lead to false positives, while too much rinsing removes adsorbed proteins and therefore false negatives. Although the risk is minimized by the use of automated and robotized equipment, the inherent risk of error can certainly be a serious methodological disadvantage in large numbers, which may lead to re-infestation, because infestations are not recognized as such.

A completely different method is described in Dutch patent application 93.00965. Herein seropositivity (the presence of an antigen or antibodies against a desired antigen in the body) is determined by means of an electrophoretic method. First, the sample is contacted with a standard solution of one or more immunologically reactive reference molecules in an incubation mixture. This allows the formation of immune complexes between the immunologically reactive reference molecule and the immunologically reactive molecule to be detected in the sample. The reference molecules can be antigens or antibodies.

Determining whether immune complexes have been formed then takes place by comparing the components of the incubation mixture with the components of the standard solution using electrophoretic techniques. The theory behind this is that when a specific antigen is present as a reference molecule in the standard solution, it can react with antibodies present in the sample during the incubation step. The antigen in the standard solution will end up on the electrophoresis gel after electrophoresis. Since binding of the antigen 1006680 5 from the standard solution to one or more antibodies from the sample may occur in the incubation mixture, the physical properties of the antigen will change, as a result of which it will be deposited in a different place on the gel after electrophoresis, than a antigen not part of an immune complex. The reverse situation, where the reference molecule is an antibody and the molecule to be detected is an antigen, works analogously. The physical properties of the antibody will change upon formation of an immune complex and will be observable on the gel.

Electrophoresis is mainly based on the physical parameters of the isoelectric point and molecular weight. Changes in isoelectric point of the reference 15 molecule are determined by isoelectric focusing (IEF), while differences in molecular weight can be determined by native polyacrylamide gel electrophoresis (PAGE). In both methods, the standard solution is included as a reference next to the incubation mixture.

It has now been found that in the detection method described here, IEF electrophoresis has many disadvantages. In the case of IEF, for example, so-called 'flatbed gels' should always be used. These are cast on a horizontal surface without a cover plate, so that it can never be guaranteed that the resulting gel is completely flat. As a result, due to any irregularities in the gel, the bands that arise after staining the separated proteins are not all aligned. The so-called 'melting' of the gel can also result in this.

In principle, the human eye can detect this type of gait deviation. However, if large amounts of samples are to be examined, the electrophoretic separation of the incubation mixtures and reference solutions will preferably be robotized. Readings then take place by means of a computer, which is, however, unable to distinguish between a actually displaced tire and a tire that has moved elsewhere due to an uneven gel or smiling effects. This leads to erroneous readings with all the consequences that entails.

In addition, positioning an application strip on such a flatbed gel is difficult to robotize. Often the sample to be separated will run out under the application strip, which will also give poor results.

In practice, in order to visualize the separated proteins in IEF gels, the reference molecules will often be labeled. Generally this is done on the NH3 * groups by means of a dye. As a result, by shielding the NH3 + groups on the reference molecule, the entire immune complex becomes more acidic and will flow to an acidic pH in the gel upon isoelectric focusing. However, once there, under the influence of the prevailing low pH, the complex between the molecule to be detected and the reference molecule will disintegrate, after which the labeled reference molecule will return to its own isoelectric point. Although an immune complex has been formed in such a situation, it will not be detected. This leads to false negative results.

It has further been found in practice that native electrophoresis, for example due to the aforementioned smiling effects, but also due to the size of immune complexes, does not lead to desired results.

It is therefore the object of the present invention to provide a completely new method for detecting the presence of an immunologically reactive molecule in a sample. This method is based on contacting the sample with a standard solution of one or more immunologically reactive reference molecules in an incubation mixture to allow the formation of immune complexes between the immunologically reactive reference molecule and the immunologically reactive molecule to be detected. after which it is determined whether immune complexes have been formed by comparing one or more of the physical parameters molecular weight, charge and shape of the components of the incubation mixture with the same physical parameters of the components of the standard solution, as in the known detection method. However, it is determined whether immune complexes have been formed by column chromatographic separation of the components of the incubation mixture, column chromatographic separation of the standard solution in the same manner as in the previous separation, and comparison of the elution patterns of both separations, with a change in the elution rate of the immunologically reactive reference molecule in the incubation mixture relative to the elution rate of the same immunologically reactive reference molecule in the standard solution indicates the formation of an immune complex.

The column chromatographic separation preferably takes place by means of gel permeation. Here, only the differences in size between the reference molecule in the standard solution and the reference molecule in the incubation mixture are selected.

In addition, complexation can also change the charge of the reference molecule. In this case, the column chromatography can be based on ion-exchange chromatography.

With electrophoresis techniques it is never clear what causes a deviation. The detection of formed immune complexes according to the invention, on the other hand, is completely independent of the quality of a gel used for the separation or the effect of a dye label on the acidity of the formed immune complex.

The quality of the chromatography column used does not affect the final result of the detection method according to the invention, because each sample and each reference can be tested with the same column.

1006680 8

Any errors in determining a result are extremely minimal, since pollution and other things that negatively affect the functioning of the system do not lead to a wrong diagnosis, but to a recognizable deviating elution pattern, which immediately indicates that the system does not worked properly. If such a deviation is found, the test can be repeated. Instead of false-positive or false-negative results, there are clear disturbances in the elution pattern that can be recognized as deviations in the case of deviations. Both the ELISA and the electrophoresis separation lack such reliability.

The separation of reference molecule and complex can be specifically optimized by choosing different gel permeation materials from the molecule to be detected. A large antigen will require a different column material than a hapten. The expert can choose an appropriate column material based on his knowledge of the molecule to be detected. The flexibility of the system is therefore very great.

The whole column chromatographic separation can be performed quite easily by automated means. Automated chromatographic detection systems with associated recording devices are known per se. All this can therefore be implemented quite easily.

In order to further increase the sensitivity of the method, it is possible to detect formed immune complexes by applying known coloring reactions for proteins. Also, the reference molecule in the standard solution can be labeled. Since only the labeled molecules are detected, irrelevant molecules are excluded. All kinds of labels can be used, such as textile dyes, fluorescent substances or radioactive labels.

The sample to be tested may, for example, consist of serum, blood, plasma or saliva. In addition, 1006 680 1 9, however, materials such as eggs or milk can also be tested.

The samples need not undergo additional treatment.

The immunologically reactive molecule to be detected can be an antigen selected from hapten, macromolecules, pathogens, or immunologically reactive parts thereof, in which situation the immunologically reactive reference molecule is an antibody. The reverse situation can also occur, where the immunologically reactive molecule to be detected is an antibody and the immunologically reactive reference molecule is an antigen selected from haptens, macromolecules, pathogens, or immunologically reactive parts thereof. For example, in the case of an antigen, the formation of an immune complex can be assumed when the molecular weight of the antigen in the incubation mixture has increased by at least 160 kiloDaltons. Namely, IgG antibodies have a molecular weight of 160 kDa. Multiple antibodies can also bind per antigen.

The following definitions are used in the present application.

"Immunologically reactive" means being able to form an "immune complex" with another molecule.

Very generally, this refers to a complex between an anti-25 and an antibody or part thereof. However, reactions between antibodies and anti-idiotype antibodies directed against them are also possible. "Antigen" includes anything that can elicit an immunological response, such as haptens, macromolecules, pathogens or parts thereof. The term "antibody" is used in the usual sense, but can include various types of antibodies, such as IgG, IgM, IgA, IgE etc. or parts thereof.

"Reference molecule" is used to indicate the known molecule that occurs in the standard solution and thus also in the incubation mixture. The reference molecule can be an antigen or an antibody.

1006680 10

The "molecule to be detected" is the molecule to be detected in the sample to be examined. This molecule can also be an antibody or an antigen.

The standard solution 1 is a solution containing at least one or more reference molecules. The 'sample' is the liquid or substance to be tested. When combined, the sample and the standard solution form the 'incubation mixture'. The standard solution as such is also used as a reference.

The present invention will be further illustrated in the examples below, which are given by way of illustration only and not by way of limitation.

15 EXAMPLES

EXAMPLE 1

Detection of antibodies to antigen and Fll of E. coli in a serum sample For the presence of antibodies (seropositivity) against Fll, an antigen of Escherichia coli. in pig serum, 0.5 ml serum sample was mixed with 100 µl of a standard solution of FITC-labeled Fll antigen in PBS. The mixture thus obtained was placed in an autosampler at room temperature after mixing.

10 μΐ of the incubation mixture was then applied to a 1 ml gel permeation column without further treatment. The column was eluted with 30 PBS. The eluate was measured on-line using a fluorescence detector. Both seropositive and seronegative sera (reference) were treated in this way. Figure 1 gives the result.

The first peak in the graph represents seronegative reference serum. The remaining six peaks are from a dilution series of seropositive serum with the same amount of standard solution each time. Comparison of the first peak with the other six shows that in the presence of antibodies in the serum the peak appears next to the peak with antigen, a peak of larger material. This is the antigen-antibody complex. In the presence of more serum, the peak of the complex 5 increases. At one point, only a peak of the complex appears. All the antigen in the standard solution is now bound to antibody in the serum. The shape of the peak is important. In seronegativity, the peak has a Gauss form, in seropositivity, when all the antigen or antibody is bound from the standard solution, the peak has no Gauss form.

EXAMPLE 2 Detection of antibodies to a vaccine

In the same manner as described in Example 1, the presence of antibodies to the vaccine was demonstrated in pig serum from vaccinated pigs. Figure 2 gives the result.

The first peak is the result of the test with serum from an unvaccinated pig. This peak is a clear Gaussian curve. The remaining five peaks indicate a dilution series of the standard with positive serum. The sensitivity of the first seropositive peak corresponds to the sensitivity of the usual ELISA. The four other seropositive peaks with larger dilutions indicate that the sensitivity of the assay of the invention is clearly greater than that of ELISA. The figure also shows that at higher concentrations, the peak falls into two peaks.

J00668 0

Claims (5)

A method for detecting the presence of an immunologically reactive molecule in a sample, comprising contacting the sample with a standard solution of one or more immunologically reactive reference molecules in an incubation mixture in order to form immune complexes between enable the immunologically reactive reference molecule and the immunologically reactive molecule to be detected, and determine whether immune complexes have been formed by comparing one or more of the physical parameters molecular weight, charge and shape of the components of the incubation mixture with the same physical parameters of the components of the standard solution, characterized in that determining whether immune complexes have been formed is accomplished by column chromatographic separation of the components from the incubation mixture, column separation of the component in the same manner as in the previous separation and of the standard solution, and comparing the elution patterns of both separations, where a change in the elution rate of the immunologically reactive reference molecule in the incubation mixture relative to the elution rate of the same immunologically reactive reference molecule in the standard solution indicates the formation of an immune complex. Method according to claim 1, characterized in that the column chromatographic separation is gel permeation. 3. A method according to claim 1, characterized in that the column chromatographic separation is ion exchanger chromatography. 4. A method according to claims 1-3, characterized in that the immunologically reactive molecule to be detected is an antigen selected from hapten, macromolecules, pathogens, or immunologically reactive parts 1006680 thereof, and the immunologically reactive reference molecule is an antibody . 5. A method according to claims 1-3, characterized in that the immunologically reactive molecule to be detected is an antibody and the immunologically reactive reference molecule is an antigen selected from hapten, macromolecules, pathogens, or immunologically reactive parts thereof. 1006660