WO1998039656A1

WO1998039656A1 - Method for detecting the presence of an immunologically reactive molecule in a sample

Info

Publication number: WO1998039656A1
Application number: PCT/NL1998/000125
Authority: WO
Inventors: Alexander Adrianus Moen
Original assignee: Alexander Adrianus Moen
Priority date: 1997-03-04
Filing date: 1998-03-04
Publication date: 1998-09-11
Also published as: NL1006680C2; EP0970373A1; JP2001518183A; AU6638498A

Abstract

The invention relates to a method for detecting the presence of an immunologically reactive molecule in a sample, comprising of placing the sample into contact with a standard solution of one or more immunologically reactive reference molecules in an incubation mixture in order to enable the formation of immune complexes between the immunologically reactive reference molecule and the immunologically reactive molecule for detecting, and determining whether immune complexes have been formed by comparing one or more of the physical parameters of molecular weight, charge and form of the components of the incubation mixture with the same physical parameters of the components of the standard solution, wherein determining whether immune complexes have been formed is brought about by column chromatographic separation of the components of the incubation mixture, column chromatographic separation of the components of the standard solution in the same manner as in the preceding separation and comparison of the elution patterns of both separations, wherein a change in the elution speed of the immunologically reactive reference molecule in the incubation mixture relative to the elution speed of the same immunologically reactive reference molecule in the standard solution indicates the formation of an immune complex.

Description

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

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

Such a method is for instance important in deter- mining the presence of a molecule, which can cause an immunological reaction, in an experimental subject such as a patient or an animal for testing. Such molecules are for instance haptens, macromolecules , pathogens or immune reaction-generating parts hereof. Such molecules are located in the body in the case of an infection. However, it may also be desirable to detect other molecules in other types of samples. It is possible for instance to envisage detection of the presence of a particular protein in a plant, or detection of molecules in soil or water samples. Detection of the presence of determined types of antibodies may also be of importance in determining an allergy or allergic reaction.

An infection is usually determined by physicians or veterinaries on the basis of clinical symptoms and/or anamnesis. In addition, laboratory testing for the presence of pathogens or the reaction of the experimental subject to the pathogen herein forms a significant component. Tried and tested methods herefor are for instance culture techniques, serological determinations or DNA testing. For allergy, further use is also made of skin tests .

A number of diseases have the drawback however that the pathogens (or in the case of allergy, the antibodies) are only present in low concentrations or cannot be isolated from the experimental subject. Herpes viruses are for instance often not detectable in the bloodstream because they are present in the nervous system and therefore not easily accessible without causing extensive damage to the experimental subject. Detection of such diseases consequently often takes place in indirect manner by detecting specific antibodies in bodily fluids. Such antibodies are directed against the originator of the disease. The presence of antibodies means that the experimental subject has been in contact with the relevant antigen and/or the pathogen.

Such methods for detecting the presence of pathogens or antibodies thereto in the body of an experimental subject are used in human medical care as well as in veterinary health care. It is of crucial importance herein that the method is not only quick, but furthermore reliable. It is evident that both in human health care and in veterinary applications false positive or false negative results can have disastrous consequences. Particularly in veterinary health care there is in addition a great and growing need for determining the state of health of livestock, particularly cattle, pigs and poultry. This applies not only to classical diagnostics, i.e. identification of the cause of a prevailing disease or disorder, but also for those forms of diagnostics which can be employed more widely for the purpose of monitoring the progression of a (subclinical) infection and prognosis.

Not only in parasitology, but also in virological disorders, such as IBR in cattle and Auzjesky disease and vesicular disease in pigs or Marek' s disease in poultry, or in bacteriological disorders, such as Salmonella in poultry, determining the presence of antibodies in serum is of great importance in restricting or eliminating the associated disease. There is therefore a great need for a diagnostic method for detecting seropositivity, and then preferably against a plurality of diseases in one test, wherein the test must be quick, very reliable and inexpensive . The speed of the method is important because very many samples must often be tested, sometimes even hundreds or thousands up to millions, depending on the stated problem.

The method must be reliable, because missing a per mil in the case of these large numbers in the veterinary sector can mean that in absolute numbers a large number of diseased animals is declared healthy. These in turn can then form a source of infection. For human medical care the greatest possible reliability is self-evident. Particularly in the veterinary sector a low-cost method is important because the margins in this sector are relatively low. In the human sector government price controls are however also of importance.

At present ELISA techniques and related immunologi- cal techniques are particularly used for the above described diagnoses of infections. While these comply with the speed and cost requirements, they do have an inherent chance of error due to the methodology used.

In ELISAs proteins (antibodies or antigens) are adsorbed on the wall of small reaction wells which are subsequently filled with the sample for testing. After a time the binding of proteins (antigens or antibodies) from the sample to the already bound proteins on the wall of the reaction wells is made visible in a following step by adding labelled antibodies which are specific to the antigen or antibody from the sample.

It is crucial in this method that the reaction wells have to be rinsed up to three times to remove non-bound proteins from the sample. The manner and duration of rinsing must be well adapted to the properties of the molecule for detecting from the sample. Insufficient rinsing could result in false positives, while too much rinsing means the removal of adsorbed proteins and therefore false negative determinations. Although the risk is minimized by the use of automated and robotized equip- ment, the inherent chance of error, certainly in the case of large numbers, can be a serious methodological drawback, whereby infections can reoccur because sources of infection are not recognized as such. An entirely different method is described in the Netherlands patent application 93.00965. Seropositivity (the presence in the body of an antigen or antibodies against a sought antigen) is determined herein by means of an electrophoretic method. The sample is first placed in contact with a standard solution of one or more immunologically reactive reference molecules in an incubation mixture. This enables the formation of immune complexes between the immunologically reactive reference molecule and the immunologically reactive molecule for detecting in the sample. The reference molecules can be antigens or antibodies .

Determining whether immune complexes have been formed subsequently 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 reference molecule in the standard solution, this can react during the incubation step with antibodies present in the sample. After electrophoresis the antigen in the standard solution will come to lie at a determined position on the electrophoresis gel. Because in the incubation mixture binding of the antigen from the standard solution to one or more antibodies from the sample can occur, the physical properties of the antigen will change, whereby after electrophoresis it will come to lie at a different position on the gel than an antigen not forming part of an immune complex. The reverse situation, wherein the reference molecule is an antibody and the molecule for detecting an antigen, functions analogously. The physical properties of the antibody will change after formation of an immune complex and be discernible on the gel.

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

It has now been found that in the detection method described here IEF electrophoresis has a great number of drawbacks. In the case of IEF, so-called "flatbed-gels" must for instance always be used. These are poured onto a horizontal surface without cover plate, so that it can never be guaranteed that the resulting gel is completely flat. The result is then that, due to possible unevenness in the gel, the bands which result after staining of the separated proteins do not all lie in one line. The so- called "smiling" of the gel can also result in this.

The human eye can in principle discern this type of variation in the running pattern. However, when large quantities of samples must be tested, the electrophoretic separation of the incubation mixtures and reference solutions will preferably be robotized. Reading then takes place by means of a computer, which is not however capable of making a distinction between an actually displaced band and a band which has come to lie at a different position under the influence of an unevenness in the gel or smiling effects. This results in incorrect readings with all that this entails.

In addition, positioning of an application strip on such a flatbed gel is difficult to robotize. The sample for separating will often run out from under the applica- tion strip, whereby poor results are likewise obtained. In order to make the separated proteins visible in IEF gels, the reference molecules will often be labelled in practice. This generally takes place on the NH₃ ⁺ groups by means of a dye. The result is that by shielding the NH₃+ groups on the reference molecule the entire immune complex becomes more acidic and during isoelectric focussing will run to an acid pH in the gel. However, having once arrived there, the complex between the molecule for detecting and the reference molecule will separate under the influence of the low pH prevailing there, whereafter the labelled reference molecule will return once again to its own isoelectric point. Although in such a situation an immune complex is indeed formed, it will not be detected. This results in false negative results. It has further been found in practice that native electrophoresis does not lead to the desired results due to said smiling effects, but also due to the size of immune complexes .

It is therefore the object of the present invention to provide a wholly new method for detecting the presence of an immunologically reactive molecule in a sample. This method is based on placing the sample into contact with a standard solution of one or more immunologically reactive reference molecules in an incubation mixture in order to enable the formation of immune complexes between the immunologically reactive reference molecule and the immunologically reactive molecule for detecting, whereafter it is determined whether immune complexes have been formed by comparing one or more of the physical parame- ters of molecular weight, charge and form 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 preceding separation and comparison of the elution patterns of both separations, wherein a change in the elution speed of the immunologically reactive reference molecule in the incubation mixture relative to the elution speed 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. Selection herein takes place exclusively according to differences in size between the reference molecule in the standard solution and the reference molecule in the incubation mixture. In addition, the charge of the reference molecule can also change due to complex formation. In this case the column chromatographic separation can be based on ion exchange chromatography. In the case of electrophoresis techniques it is never clear what has caused a variation. The detection of formed immune complexes according to the invention is on the contrary wholly 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 has no influence on the end result of the detection method according to the invention, because each sample and each reference can be tested with the same column. Possible errors in determining a result are extremely minimal, since contamination and other matters having a negative effect on the functioning of the system do not result in an incorrect diagnosis but to a recognizably different elution pattern which immediately indicates that the system has not functioned as it should. If such a variation is determined, the test can be performed again. Instead of false positive or false negative results, clear disturbances recognizable as variations occur in the elution pattern in the case of variations . Both the ELISA and electrophoresis separation lack such a reliability.

The separation of reference molecule and complex can be optimized specifically by choosing different gel permeation materials, using the molecule for detecting as starting point. A large antigen will require a different column material than a hapten. The skilled person can choose a suitable column material on the basis of his knowledge of the molecule for detecting. The flexibility of the system is thereby very great . The whole column chromatographic separation can be performed very easily in automated manner. Automated chromatographic detection systems with registration equipment coupled thereto are per se known. The various aspects can therefore be implemented in very simple manner. To further increase the sensitivity of the method it is possible to detect formed immune complexes by applying per se known staining reactions for proteins. The reference molecule in the standard solution can also be pro- vided with a label . Because only the molecules with labels are detected, irrelevant molecules are excluded. All types of labels can be used, such as textile dyes, fluorescent substances or radioactive labels. Labelling and staining can take place directly on the molecule for labelling or staining or indirectly with interposing of one or more other molecules .

The sample for testing can for instance consist of serum, semen, blood, plasma or saliva. In addition however, materials such as eggs or milk can also be tested. In addition to the use of samples originating from living animals, it may also be important, for instance in slaughterhouses, to examine the drip liquid of slaughtered livestock. It is further known that fish secrete antibodies. These antibodies can be determined in the water of breeding basins in intensive fish farming. The samples do not have to undergo any additional treatment .

The immunologically reactive molecule for detecting can be an antigen chosen from haptens, macromolecules , pathogens, or immunologically reactive parts thereof, in which situation the immunologically reactive reference molecule is an antibody. The reverse situation can also occur, wherein the immunologically reactive molecule for detecting is an antibody and the immunologically reactive reference molecule is an antigen chosen from haptens, macromolecules, pathogens, or immunologically reactive parts thereof.

In the case of an antigen the formation of an immune complex can for instance be assumed when the molecular weight of the antigen in the incubation mixture has increased by at least 160 kiloDalton, since IgG antibodies have a molecular weight of 160 kDa. A plurality of antibodies can also bind per antigen.

This latter is of importance particularly for small molecules, such as small proteins, peptides and haptens (for instance hormones) . Binding of an antibody to such a 4-1 ι _•_ d W CD rd 1 1 β rH T 1 CQ 1 ^■H tn TJ tn

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thereagainst are also possible. "Antigen" includes everything which can cause an immunological reaction, such as haptens, macromolecules, pathogens or components thereof. The term "antibody" is used in the usual sense, but can comprise different types of antibody, such as IgG, IgM, IgA, IgE etc. or parts thereof.

"Reference molecule" is used to designate the known molecule which occurs in the standard solution and therewith also in the incubation mixture. The reference mole- cule can be an antigen or an antibody.

The "molecule for detecting" is the molecule which must be detected in the sample for testing. This molecule can also be an antibody or an antigen.

The "standard solution" is a solution containing at least one or more reference molecules. The "sample" is the liquid or substance for testing. Added together, the sample and the standard solution form the "incubation mixture" . The standard solution as such is also used as reference . The present invention will be further illustrated in the following examples, which are only given by way of elucidation and not by way of limitation.

EXAMPLES

EXAMPLE 1

Detection of antibodies against antigen Fll of E.coli in a serum sample

In order to detect the presence of antibodies (sero- positivity) against Fll, an antigen of Escherichia coli, in serum of pigs, 0.5 ml serum sample was mixed with 100 μl of a standard solution of FITC-labelled Fll antigen in PBS. The thus obtained mixture was placed after mixing in an autosampler at room temperature . 10 μl of the incubation mixture was then placed without further treatment on a gel permeation column with a content of 1 ml . The column was eluated with PBS . The eluate was measured on-line using a fluorescence detector. Both seropositive and seronegative sera (reference) were treated in this manner. Figure 1 shows the result. Φ ra Φ ,

The concentration of specific IgE is determined after separation on a column by measuring the peak which includes both labels (of allergen and anti-IgE) . Total IgE is measured by adding to the sample only labelled anti- IgE without allergen.

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Claims

1. Method for detecting the presence of an immunologically reactive molecule in a sample, comprising of placing the sample into contact with a standard solution of one or more immunologically reactive reference mole- cules in an incubation mixture in order to enable the formation of immune complexes between the immunologically reactive reference molecule and the immunologically reactive molecule for detecting, and determining whether immune complexes have been formed by comparing one or more of the physical parameters of molecular weight, charge and form 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 brought about by column chromatographic separation of the components of the incubation mixture, column chromatographic separation of the components of the standard solution in the same manner as in the preceding separation and comparison of the elution patterns of both separations, wherein a change in the elution speed of the immunologically reactive reference molecule in the incubation mixture relative to the elution speed of the same immunologically reactive reference molecule in the standard solution indicates the formation of an immune com- plex.

2. Method as claimed in claim 1, characterized in that the column chromatographic separation is gel permeation.

3. Method as claimed in claim 1, characterized in that the column chromatographic separation is ion exchange chromatography.

4. Method as claimed in claims 1-3, characterized in that the immunologically reactive molecule for detecting is an antigen chosen from haptens, macromolecules, patho- gens, or immunologically reactive parts thereof, and the immunologically reactive reference molecule is an antibody.

5. Method as claimed in claims 1-3, characterized in that the immunologically reactive molecule for detecting is an antibody and the immunologically reactive reference molecule is an antigen chosen from haptens, macromolecules, pathogens, or immunologically reactive parts thereof .

6. Method as claimed in claims 1-3, characterized in that the immunologically reactive molecule for detecting and the immunologically reactive reference molecule are both antibodies.