EP0327629A1

EP0327629A1 - Process and device for examining whole blood, other body fluids and other biological fluids

Info

Publication number: EP0327629A1
Application number: EP19880907057
Authority: EP
Inventors: Olaf Thraenhart
Original assignee: Individual
Current assignee: Individual
Priority date: 1987-07-31
Filing date: 1988-07-31
Publication date: 1989-08-16
Also published as: AU2088188A; DE3725504C2; DE3725504A1; WO1989001156A1

Abstract

Un procédé permet de dépister des protéines à propriétés spécifiques de type anticorps ou antigènes dans des fluides corporels ou d'autres matières organiques. On utilise une protéine fixée sur un support qui entre en réaction spécifique avec la protéine que l'on veut dépister dans la solution, rendant cette dernière insoluble, et une deuxième protéine se trouvant dans la solution, pourvue d'un marqueur, qui entre en réaction avec le composant insoluble et devient elle aussi insoluble. On peut ainsi examiner directement du sang, sans centrifugation préalable, afin d'y dépister simultanément une ou plusieurs protéines.A method makes it possible to detect proteins with specific properties of the antibody or antigen type in body fluids or other organic matter. We use a protein fixed on a support which enters into a specific reaction with the protein that we want to detect in the solution, making the latter insoluble, and a second protein found in the solution, provided with a marker, which enters into reaction with the insoluble component and also becomes insoluble. It is thus possible to examine blood directly, without prior centrifugation, in order to simultaneously screen one or more proteins.

Description

Method and device for examining whole blood, other body fluids and other biological fluids

The invention relates to a method for examining whole blood, other body fluids and other biological fluids, and a device for

Detection of antibodies or antigens or in general of substances with antibody-specific or antigen-specific properties.

According to German interpretation 22 06 103

Antibodies and antigens in hormone preparations, in the urine or in sera that have been diluted to reduce the influence of other disturbing factors are examined.

In order to obtain sera, at least 5 ml of blood must first be drawn from the vein using a single syringe. Then the blood has to test for a while hen so that it coagulates, so that the blood cake is deposited below, which is covered by the serum in which blood cells swim. The serum-blood cell mixture is siphoned off with a pipette, pipetted into a centrifuge tube and centrifuged. After centrifugation, the serum is pipetted from the centrifuge tube into another container. This method of obtaining serum is cumbersome, very time-consuming and expensive, and also requires the relatively expensive devices such as centrifuges and pipettes. This method cannot be carried out by the layperson and is also completely unprofitable for a single examination.

Samples from several patients must come together so that a test can be carried out profitably. Carrying out the examination also requires specialist knowledge and the availability of special equipment which, since they are expensive, can only be used expediently in special laboratories. Samples must therefore first be packed and sent to an institute by post, unpacked, registered and handed over to the technical assistants. There, an examination protocol is to be completed from samples from several patients, so that the individual samples can be tested against various antigens or antibodies in several examination approaches. A protocol to test assessment again to create and provide the findings back to the post office ^'then. This results in a period of time from the taking of the sample to the receipt of the finding of at least one, but usually several days. As a result, the finding is only available at a time when the result can often only confirm the therapeutic measures that are immediately necessary when samples are taken in the best case. No result is available in acute emergencies. An example is neurological diseases such as herpes infections, in which the therapeutic use of an anti-herpes agent should be serologically secured. In the case of transplantations as well, values of accident victims who are considered organ donors are available at the earliest hours after the transplantation, although it can be vital for the patient to know whether the accident victim is infected, for example, with the AIDS virus.

The need for the determination of antibodies has increased rapidly in recent years and continues to increase. The desired possibility of mass examinations for HIV virus can serve as an example. This is also the result of the development of microbiological knowledge, the introduction of the enzyme immunoassay by Engvall and Perlman (J. Immunol. 109, 129 (1972)), the offering of ELISA test kits and the progressing automation of antibody and antigen tests, which until now were limited to a few special laboratories due to the high degree of difficulty and the expensive equipment.

In the future, the efforts of

World Health Organization as part of a comprehensive immunization in the Third World ("Expanded Programs on Immunization ") require considerable antibody tests. Simple and, above all, inexpensive test methods are to be developed here (WHO Chronicle, 40, 47 (1986)).

The global spread of HIV infection, like the diagnosis of hepatitis B virus infection, requires mass examinations. Such mass examinations will only be manageable and financially if it is possible to develop methods that can also be carried out by trained laymen outside of special laboratories. The examinations should also be possible in the medical practice or clinic.

With the development of chemotherapy against viral diseases, the virological and immunological diagnostics will be increasingly based on antibody and antigen tests. Short times for the course of the examination are desirable because of therapeutic interventions.

In the context of the examination of high-risk persons, diagnosis of symptoms or serial examinations, the antibody and / or antigen examination for a plurality of pathogens for one person is increasingly being questioned. This is a largely fixed spectrum of the examinations in question. So far, however, the serum of a subject has been examined in different test batches, since the test kits are each made up of 1 antibody or 1 antigen. A serum must therefore have to go through several test approaches in the majority of cases, which leads to delays in the overall assessment.

It is also still necessary to centrifuge blood samples before the antibody test in order to separate the particulate from the liquid components of the blood. This process is time-consuming and requires a centrifuge.

The test kits currently available for the investigation of antibodies and antigens in the ELISA are in principle based on the adsorption of antigens or antibodies on a transparent plastic surface or on corresponding spheres, which are placed in transparent test tubes. This is followed by further binding with an antibody or antigen in the form of an antigen-antibody reaction. Finally, an enzyme-labeled protein is added that reacts specifically with the previous reaction product. The amount of bound antibody or antigen in the unknown sample is visible as a color reaction after the addition of a substrate in the liquid phase and is measured visually or photometrically. _

The determination of antigens on plastic carriers or spheres other than transparent was carried out by Hawkes et al. (Anal. Biochemistry, 119, 142 (1982)) and Paltree and Elliott (J.Immunol. Methods, 52, 395 (1982)). The authors used for the first time

Nitrocellulose paper. Nitrocellulose paper has also been described by Pappas et al (J. Immunol. Methods, 64, 205, 214 (1983)), Walsh et al. (J. Immunol. Methods, 66, 99 (1984)) and Heberling, and Kalter (Develop. Biol. Standard. 64, 199 (1986)) and Schmeer (personal communication, 1986). Herbrink et al. (J. Immunol. Methods, 48, 293 (1982)) compared nitrocellulose and diazobenzyloxymethyl paper for the use of an antibody test. Giallongo et al. (J. Immunol. Methods, 52, 379 (1982)) and Esen et al. (Analytical Biochemistry, 132, 462 (1983)) use chromatography paper as a carrier for protein determinations.

Finally, Lin and Haibert (J. Clin. Microbiology, 24, 7 (1986)) also presented the use of white plastic cards for antibody determinations.

These tests have shown that ELISA tests on non-transparent supports are feasible and give good results. The aforementioned authors have receptor proteins (Palfree et al.), Antibodies against plasma membranes and some virus antigens (Hawkes et al., Heberling and Kalter), murine IgG and IgE (Giallongo et al.), Various proteins (Esen et al., Herbrink et al.) Human allergens (Walsh et al.), Antibodies against leishmaniasis (Pappas et al.), Cytomegalovirus (Lin and Haibert) and Coxiella (Schmeer) were detected on these carriers.

However, none of the authors presents a disease- or problem-related concept of antibody and antigen tests, in which a number of antigens or antibodies are used per patient in accordance with the diagnostic objective. Only Hawkes et al. demonstrate the possibility of multiple virus antigens unite on a carrier, but without discussing a disease-associated antigen selection. Their test approach, as well as that of the other authors, differs in several points in comparison to the new method to be described below, the new method requires, for example, 7 instead of 15 individual steps, the test duration is about 30 minutes instead of 2.5 to 25 hours and the incubation temperature is 37 ° C instead of 20 ° C.

In summary, it should be noted that there is still no cheap, simple and quick method to be carried out, even by trained laymen on the sick bed or in the field, in which a multitude of antibody and antigen determinations are possible at the same time with a drop of blood.

The object of the present invention is to provide a method for the examination of whole blood, other body fluids and other biological fluids, which can be carried out particularly easily, quickly and with minimal expenditure on devices.

This object is achieved in that the liquid is mixed with a surface-active medium and then subjected to test reactions.

The invention first has the advantage that solid components such as cell components of the biological fluid to be examined are lysed or destroyed. This is particularly important in all such examinations in which a reaction on or at one location of a reagent carrier can be used. If solids, for example cell components of the blood, are deposited precisely at this point, they can prevent a reaction of this reagent with the reagent present in the biological body fluid. This risk is eliminated by the present invention.

On the one hand, this method has the great advantage that no devices such as centrifuges are required to prepare the blood. On the other hand, it has the advantage that the biological liquid can be subjected to the test reactions in a very short time after its removal, namely immediately after mixing with a surface or surface-active medium.

The biological fluid can be diluted with a surfactant. In one embodiment of the invention, the medium is a surfactant. It can be a surfactant NP 40 in a concentration of about 0.1%.

The examined biological fluid can expediently be shaken during and or between the incubations. On the one hand, this shaking has the effect of thorough mixing, and on the other hand it prevents solid components of the examined biological fluid from being deposited on the carrier. According to an advantageous development of the invention, the test reaction for determining proteins with antibody or antigen-specific properties comprises the following steps:

Use of one or more proteins fixed to a carrier, which enters into or enters into a specific reaction with the protein to be determined and which is in solution and thereby makes it insoluble, and use of another in

Protein present in solution and provided with an indicator which "reacts" with the insolubilized components and thereby becomes insoluble.

According to another advantageous development of the

The invention comprises the test reaction for the determination of antibodies or antigens the following steps: fixation of one or more first substances with antigen-specific properties on a support, supply of a second substance mixture, the antibody-specific components to be examined with the first substances during a first incubation, can undergo specific test reactions, to the first fabrics; Application of a first indicator which is uniform for the different reactions and which reacts with components of the second substance mixture attached to the first substances during a second incubation period and thus indicates the specific reaction which may have taken place. With these advanced training courses, a surprising number of advantages are achieved:

Since, according to the invention, blood itself is used for the examination, the first advantage is that a small amount of blood is sufficient. This has the further advantage that no disposable syringe is required to draw blood. It also eliminates the difficult process of taking blood from the vein.

Furthermore, the centrifuge that is otherwise required is not required. This means that the method according to the invention can be carried out anywhere, not only in laboratories which are equipped with centrifuges and other devices which are otherwise necessary.

This saves considerable costs in the healthcare sector. Since the determination according to the invention is possible everywhere, the disadvantages of packaging and sending the patient material with the corresponding accompanying documents to an institute and returning the findings from the laboratory to the sender are eliminated. Disadvantages include the delay in the assessment, packaging, postage and unprofitable personnel costs.

There are also no delays in the examination institute due to re-registration etc. before and after the examinations.

Confusion risks, such as those that occur again and again in the above procedure, are excluded in the examination according to the invention, since this begins in the presence of the patient. The time from taking blood to receiving the findings is reduced from days to less than an hour. The finding is still available during the first examination of the patient and can therefore be used therapeutically immediately. The detection method according to the invention thus fulfills all the requirements for immediate emergency diagnostics at the bedside or even in the ambulance. Serological examinations in an emergency are already of importance in brain inflammation caused by herpes virus infections and in organ transplants when organs are taken from accident victims and used in other patients. Serological emergency diagnostics will be required with the expected development of chemotherapeutic agents against viral infections in the near future.

Since the blood to be examined does not have to be processed, because blood itself and not blood serum is being examined, the present method for the determination of antibodies or antigens can also be carried out by the layperson, whereby the costs of the method are considerably reduced.

In addition, the layperson can also use this procedure to investigate whether he is HIV-positive, for example. This advantage is extremely important because the result is not known. This opens up the possibility that many people are now out

Fear of the consequences of getting her HIV status known cannot be examined, the investigation itself can make. It is to be expected that in this way many people who are HIV positive will now also examine this themselves and, if they become aware of their own HIV positive state, will behave differently and responsibly in such a way that they will not be fellow human beings infect. The present invention thus makes a contribution to keeping the further spread of this HIV epidemic and possibly also other epidemics within limits.

Another advantage can be seen in the fact that with the present method, because it is so easy and inexpensive to carry out, serial examinations of entire groups of people are also possible for the first time. Furthermore, the invention allows allergy examinations, which were previously very complex.

Of course, these advantages apply not only to the determination of HIV antibodies, but very generally to the determination of antibodies. It should be pointed out, for example, that around 50,000 people die of rabies in the tropics each year, and the loss of animals annually is in the millions. A correspondingly large number of vaccinations are carried out, but these are not always successful in the tropics, for example because antibodies are not formed due to frequently existing immunodeficiency due to other infections. The present invention enables the serial examination and also the individual examination for antibodies against rabies, so that, if necessary, a re-vaccination can be carried out. In this context, it is pointed out that the series and also individual examinations can only be carried out using the method according to the invention, because the known methods require equipment and specialists who do not exist there.

Another important advantage is that the present method can also be used to test vaccines for their antigen content before they are used. This is particularly important in the tropics, since the cold chain, which is often interrupted there, makes it easy for the vaccines to lose their active ingredient. In addition, tropical countries and vaccines after their manufacture or import u. U. Store for a long time and then do not use later because of the uncertainty of the residual activity. In this way, large resources are wasted. The same applies to immunoglobulin preparations.

According to a development of the invention, capillary blood is used. This further development has the great advantage that the blood does not have to be taken with a syringe, which the layperson is generally unable to do. Rather, it is possible to draw capillary blood with a lancet.

Fingertip or the earlobe. This process is very simple and can be carried out by anyone.

According to another development of the invention

Antibodies and / or antigens in drops of blood, capillary or venous blood are examined directly. Previously, blood had to be centrifuged beforehand so that the corpuscular components interfere with the reaction. The disturbing influence is eliminated by mixing according to the invention with a special buffer.

The disturbing influences in blood drops, capillary or vein blood can be eliminated by mixing with a medium.

The capillary blood can be mixed with the medium in a ratio of at least 1: 2 or 1: 3, preferably 1:50 to 1: 100. The medium can contain a surfactant and a protein. The surfactant can be NP 40. According to one embodiment of the invention, the concentration of NP 40 is about 0.1%.

"The protein can be fetal calf serum, the concentration of which can be, for example, 3%.

According to the present invention, antibodies and / or antigens in body fluids such as urine, cerebrospinal fluid or saliva can be examined directly.

Antibodies or antigens can also be detected in body fluids other than blood. Since HIV also occurs in saliva, for example, tests can also be carried out on these samples. The invention thus has the further considerable advantage that the blood can be dispensed with if the detection sensitivity is sufficient. Antibodies and / or antigens can be examined in prepared body fluids such as blood serum, blood plasma, or others. Antibodies can also be tested in immunoglobulin preparations.

Another important advantage of the present invention is that one or more antigens are tested in vaccines.

Enzyme-linked species-specific anti-immunoglobulins of classes G, M, A, D, E and their subclasses can be used as the first indicator.

Depending on the question and body fluids, antiimmunoglobulins of the different classes or subclasses are then to be used. For the determination of antibodies in saliva e.g. an examination with anti-immunoglobulin-A is carried out.

The first indicator can contain one or more enzyme-coupled antigen-specific antisera. Peroxidase can be used for enzyme coupling.

In another development, the mixture is poured off after the incubation. This shortens the time required to carry out the method, since pouring can be carried out considerably faster than a washing process.

It is expedient to wash after the incubation of the mixture of substances. After the reaction of the second substance mixture with the first first indicator, the carrier is advantageously washed.

According to another embodiment of the invention, the antigens directed against the antibodies are applied crude, partially or highly purified or as recombinant products or as antigen-containing or non-antigen-containing cells or as cell preparations on a non-soluble carrier from which the antigens be absorbed or on which they dry up. The carriers can be materials which, owing to the contrasting color, allow a color reaction to be read directly by the eye. This enables examinations on simple papers (for example typewriter paper). The paper must have contrast (white) and should not be too absorbent in order to optimally design the distribution density of the first material in the carrier / surface unit.

As carriers e.g. Nitrocellulose, Chromatographie¬ papers or filter papers can be used. Transparent materials and plastics can also be used as supports.

According to one embodiment of the present invention, between 1 and 8 al of the first substance can be dropped onto the support. Appropriately, about 1 drop of the first substance is dropped from a cannula onto the carrier. The first substance can be dried briefly at a temperature of more than + 20 ° C. A drying temperature of around 37 ° C is suitable.

Dripping onto a dry support and brief drying at + 37 ° C. has the advantage of the low background after the end of the test. But the remaining steps are also decisive.

The drying time can be 10-15 minutes, depending on the drying temperature.

According to another embodiment of the invention, the supports are coated in the dry state and are used directly after drying or fixing the antigen or antibody.

Such carriers are ready for use very quickly. For mass examination, carriers can be manufactured even more cost-effectively at the examination site. This further advantage of the invention has become possible since a "blocking step", which has hitherto been customary and necessary and requires 30 minutes to 2 hours and which had to be followed by a long drying time, is eliminated.

Phosphate-buffered salt solution with a surfactant and a protein additive, for example NP40 in a concentration of about 0.1%, can be used as the dilution buffer for the second substance mixture and / or the uniform indicator. Fetal calf serum in a concentration of 3% can be used as a protein additive.

The buffer for the washing operations which may be carried out can be a surfactant in phosphate-buffered saline, preferably 0.1% NP40.

The process is advantageously carried out at a temperature between 1 and 100 ° C. The temperature erhöhrung of 20 ° C to 37 ° C can shorten the reaction time to ^'the order of 40-50%, depending on conditions Bedin¬ even stronger.

According to another embodiment of the invention, an antibody or an immune class-specific standard is examined simultaneously for the semi-quantitative determination of the antibody content, and the color reaction of the standard is compared with the result of the patient sample.

This development enables the concentration of the protein to be determined to be estimated even under field conditions.

Simultaneous incubation of sample and 1st indicator further shortens the reaction time. The second incubation period previously required may be omitted. According to another development of the invention, a carrier is used on which several first substances with antigen-specific properties are or are fixed; furthermore, it is not a second substance but one second substance mixture, the antibody-specific constituents to be investigated which can undergo specific test reactions with the first substances during a first incubation, is brought together with the first substances, and a uniform first indicator is applied for the different reactions, which is used during a second incubation period reacts with constituents of the second mixture of substances attached to first substances, and thus indicates the specific reaction that may have taken place.

This development of the invention makes it possible to use a single drop of blood to carry out a large number of antibody and antigen tests in a test batch in a group- or disease-associated manner, by applying a plurality of antigens and / or antibodies to the carrier.

The selection of the antigens and / or antibodies per test batch depends on the requirements that must be met for a screening of specific groups of people, patients and animals. The selection of the antigens or antibodies can also be made according to those for the diagnosis of a clinical picture, a group of symptoms or organ disease.

Examples of group or disease-related examinations are: a) Blood donors at risk

Pregnant women

Tropical returnees Vaccinates

Hospital patients soldiers prostitutes homosexual accident victims prison inmates

b) Group of animals vaccinated

Inventory investigations mass uplift (races, auctions)

c) Disease Associations Virus Infection Screening

Immunity screening Auto immunity screening

Diagnosis of organ diseases, insofar as they can be diagnosed with antigen-antibody tests. STDs Acquired immunodeficiency

According to this further development, antibodies or antigens of one or more pathogens are simultaneously detected which are relevant for the diagnosis of a symptom picture, an organ or systemic disease (disease-associated determination). Furthermore, routine examinations, which must always be carried out in the same way for certain groups of people, and which cover a specific spectrum Antigen-antibody determinations include, can be carried out simplified (target group-associated determination). A major advantage in establishing such a method is the method of testing for a large number of antibodies with a single drop of blood, which is presented here for the first time. Capillary blood can also be used, which is taken in the course of other routine examinations without severe patient nuisance.

This blood collection is also so simple that it can be carried out by trained laypersons. It is also an object of the present invention to provide a device for the detection of antibodies and / or antigens which is simple to produce, economical and easy to use.

According to the invention, this object is achieved by a body with a recess which is adapted in shape to accommodate one or more carriers to which one or more first substances with antigen-specific properties are fixed. The recess can be covered in a water-tight manner and is dimensioned such that its contents can be mixed by shaking the body.

Such a body can be constructed and designed in a very simple manner.

The distance from the surface or surfaces of the carrier can be approximately one and a half times as high or higher than the layer height of the solution to be brought together with the carrier. Further advantages and features of the invention emerge from the subclaims and the drawing.

The invention is described in more detail below with the aid of a few examples in conjunction with the description and the drawing. In detail show:

1 checkerboard titration of sera with and without antibodies against rabies virus,

Fig. 2 antibody detection against rabies virus in whole blood

3 Comparison of different carrier materials for the Blood Drop ELISA

Fig. 4 screening of people

Rabies virus immunity and comparison of the results obtained with this method with those of established methods (neutralization test = RFFIT, and complement formation reaction = KBR)

5 Examination of a whole blood sample (capillary blood) for virus antibodies against viruses of the respiratory tract, childhood diseases and viruses of the herpes virus group (disease-associated examination)

6 Examination of patient blood samples for antibodies against the huma-immune deficiency virus (HIV I), the AIDS pathogen, 7-10 sections through different incubators for the test batch

11 shows a holder with a test strip

Fig. 12 Interpretation aid for the result of the investigation

13 shows a vertical section through an examination device according to the invention,

14 is a plan view of an examination device according to the invention, partially in

Sectional view,

15 shows a section through another embodiment of an examination device according to the invention,

16 is a top view of the device of FIG. 9,

17 is a plan view of an inventive holder with an inserted carrier, and

Fig. 18 is a sectional view of a container with an inserted carrier holder. Example 1: Chessboard titration of three sera for antibodies against rabies virus antigen.

FIG. 1 a shows a graphic representation of the test result with a serum with antibodies against rabies virus. 1 shows 5 strips of nitrocellulose paper, each of which is divided into 6 fields. 2 / al of a concentrated and purified inactivated rabies antigen with 680IU / ml in dilutions of 1:50 to 1: 1200 were dripped into these fields. The 1: 200 dilution of antigen thus contained 0.007 IU / 2 / ul. After drying the strips for 15 minutes at + 37 ° C, 4 ml of the serum dilutions from 1:50 to 1: 600 were each incubated with a strip for 10 minutes at + 37 ° C with gentle shaking. The liquid was then suctioned off and the strips were washed by placing them in about 5 ml of washing buffer for 5 minutes at + 37 ° C. with gentle agitation. This process was repeated 3 times. The last washing buffer is aspirated thoroughly and then 4 ml of an anti-human IgG (conjugate) coupled to peroxidase are incubated in a dilution of 1: 900 (depending on the batch) for 10 minutes at + 37 ° C with gentle shaking. The process is repeated twice with washing buffer and twice with phosphate-buffered saline. During the last • washing process, the substrate (4 chloronaphtol + ^H 2 ° 2) is produced and briefly preheated at + 37 ° C. After the washing process, 3 ml of substrate are added to the strips and 5 minutes with gentle Shake incubated at + 37 ° C. Following the color development, the strips are rinsed under running water and assessed immediately. The result shown here (Fig.la) shows a strong color reaction of the serum dilutions up to 1: 600 when using the

Antigen dilutions 1:50 to 1: 200. The 1:50 serum dilution still has a clear reaction with an antigen dilution of 1: 1200. This is also the case with the serum dilutions 1: 100 and 1: 200. The serum dilution 1: 600 was negative with this antigen dilution, but was still positive with an antigen dilution of 1: 400. The black spots shown only partially broken through in the figures are intended to represent weak color reactions.

Fig. 1b shows the corresponding reaction with a serum without antibodies against rabies virus. The serum dilutions 1:50 to 1: 200 only show a weak non-specific reaction at the antigen dilutions 1:50 and 1: 100. For this reason, a dilution of 1: 200 was used with this antigen in the subsequent studies.

1c shows the result of a rabies immunoglobulin with a known antibody content. A comparison of this strip with the other strips shows an identical color reaction of the serum dilution of the 1: 100 diluted positive serum. This serum therefore has 1/10 of the antibody content of the immunoglobulin preparation. Using the example of the rabies virus, this example shows that there are quantitative dependencies between antigen and antibody with this method and that a semi-quantitative statement about the antibody and antigen content is possible.

Figure 2: Antibody detection in whole blood

FIG. 2 shows the test result that was carried out with the described Blood Drop ELISA. From a clotted, non-centrifuged blood sample, a sample of a test subject, of whom the positive rabies virus antibody status was known, was diluted 1:75 in dilution buffer and then a carrier with rabies virus antigen was immersed in this dilution. After the reaction had continued as shown in Example 1, a positive reaction was detected. Furthermore, a drop of capillary blood of approximately 20 μl from the fingertip of a test subject without rabies virus antibody was dripped directly into a vessel intended for the absorption of the antigen carrier, which contained 1500 / ul dilution buffer. After the course of the reaction described in Example 1, the result was negative.

Example 3: Testing Different Carrier Materials for the Blood Drop ELISA.

FIG. 3 shows the result of 5 rabies virus antibodies positive and 3 negative sera. The antibody status was determined in the established neutralization test. The figure shows that not only nitrocellulose and chromatography paper are suitable as carriers, but also other papers. The pore size should not be too large so that the antigen does not diffuse too strongly and is therefore only present in a relatively low concentration of antigen per unit area, which leads to a weaker coloration.

Example 4: Immunity Screening Against Rabies Virus in Risk Individuals

FIG. 4 shows the results of a screening of 17 risk subjects for rabies immunity using the Blood Drop ELISA. The results were correlated with those of established antibody studies.

Example 5:

In one person, the blood drop ELISA was used to examine in a drop of blood which virus infections had occurred. These results were examined for validity in the conventional complement fixation reaction and in the ELISA of Behring-Werke, Marburg (Enzygnost). With a carrier and the use of a single drop of blood, determinations with 17 antigens were carried out in about 1/2 hour. The result shown in FIG. 5 showed agreement between the results achieved with the conventional methods.

FIG. 6 shows the result of an examination of patient blood samples for antibodies against the human immunodeficiency virus HIV (I), the AIDS pathogen. The HIV antibody status results for these 11 selected individuals are identical in conventional and blood drop ELIZA. The names in the right column mean:

Positive = antibody against HIV present

Negative = no antibodies to HIV present, i.e. not infected

Questionable = probably no antibodies against

HIV, however, additional examinations are recommended.

The annotation digits shown in the figure have the following meaning:

1) The HIV antigen is infected cells (H9 cell strain).

2) the control antigen is uninfected H9 cells.

3) the response is significantly stronger with the infected cells; the reaction with the ^" uninfected cells speaks for additional antibodies against cell components.

4) the reaction with the infected and uninfected cells is equally strong: that

Result is questionable. Further investigations are necessary for clarification. 7 shows a section through an incubator according to the invention for the test batch. The incubator shown essentially consists of a double-walled container 10 which has a recess 12 in which one or more carriers can be arranged. The container 10 is closed by a lid 14, so that the test mixture in which the test strip is located can be shaken. The cover 14 is formed from transparent material, so that a color reaction to the test strip can be recognized through the cover. The lid 14 is also designed so that it can be removed and replaced several times.

The double-walled container 10 is hollow and can be filled with water 18 via a filler neck 16. This embodiment thus allows a water bath at the desired temperature.

8 shows a sectional view through another incubator. The container 20 shown in this Fig. 8 consists of a good heat-conducting material. A test mixture 23, which surrounds a test strip 25, is again contained in the container.

As indicated in this Fig. 8 on the left side, the container has on the outside of its upper region a locking projection 27, under which a locking projection 29, which is formed on the downwardly projecting skirt of the lid 24, projects inwards . In this or another way, the container 20 can be closed by the lid 24 so that the latter snaps onto the container 20.

Such an incubator can easily be shaken by hand without test liquid falling out, and at the same time the lid can be easily removed so that the test mixture can be poured off or the strip can be removed.

This embodiment of the invention has the advantage that it manages without additional heat sources. Such an incubator can first be put in your pocket until it reaches body temperature. The examination can then be carried out. When the carrier is inserted and also when the test mixture is poured in or poured out, the incubator remains at temperature because the test person is holding it in the hand. This incubator also does not cool down during the reaction time: either, the user holds this incubator in hand, e.g. to shake him, or he just puts it back in his pocket. In both cases, the temperature remains essentially the same. If the person; -the the

Carries out the examination, has to go back and forth anyway, this incubator, as long as it is in the trouser pocket, is shaken at the same time, so that the test mixture is thoroughly mixed and the test strip is continuously washed around while the examining person is at the same time doing anything can do another job. FIG. 9 shows another incubator 30, which essentially consists of heat-conducting material 35, in which heating wires 36 are embedded. This embodiment of the invention enables the test to be carried out at constant temperature simply by connecting it to a battery.

FIG. 10 shows a modification of the incubator of FIG. 9. In the exemplary embodiment in FIG. 10, a disposable insert 41 is shown, which lines the recess. This disposable or disposable insert receives the test liquid and the test strip 46. After a test has been carried out, the disposable insert 41 and its contents are removed. The incubator can then be used for the next examination. This incubator 40 also has heating walls or heating wires 47. Furthermore, a thermostat 48 is provided which ensures that the incubator 40 is always kept at a constant temperature. The entire incubator is arranged on a shaker 49.

Fig. 11 shows an illustration for carrying out the AIDS test for lay people. The figure shows a strip or carrier on which 'HIV + cells', marked by 1, and 'HIV cells', marked by 2, are applied. 1 thus denotes virus antigen, and 2 denotes control antigen.

12 shows an interpretation aid for evaluating the result. The result of the investigation is only positive if 1 is colored much more strongly than 2. The Coloring of 1 must be stronger than in the example of Fig. 12 c. The results of the findings are shown in each of the illustrations in FIGS. 12 a, b, c and d, in cases a and b the findings are positive, represented by a "+", in FIG. 12 c the findings are questionable, represented by a " ? ", and in FIG. 12d the result is negative, represented by a" - ". In Fig. 12e the finding is questionable, in 12f it is positive and in 12g and 12h the finding is negative. The identification of a finding by "+" means that antibodies against HIV I are present. The finding "-" means that antibodies against HIV I are not present.

The finding "?" states that the result of the finding is questionable. There are probably no antibodies. In this case, repeating the examination or consulting a doctor is recommended.

FIG. 13 shows another incubator which can also be used by the layperson in a very simple manner. This incubator 50 has a rigid inner container 52 which is surrounded by an outer, flexible plastic bag 54 which is filled with heat-storing material 56. The inner rigid container 52 is thus closed on the left side by a plug-like cover 58.

This embodiment of the invention has the advantage that the incubator can be filled with, for example, granular or pasty material. Such a body will adapt to the hand when it is picked up. If the body is pressed a little, the convex shapes of the fingers will also press into the surface of this incubator, so that a conceivably large contact surface between the outer circumference of the incubator and the hand is achieved. This advantageous result is achieved regardless of whether the person carrying out this examination has a large or a small hand. This ensures optimal heat transfer between the hand and the incubator. In this way, when the material 56 is thermally conductive, the inner container and the test strip arranged in the container and the test mixture are always kept at the same temperature.

In FIG. 13, which shows an 'vertical section, one can also see a holder 58 on which one or more test strips are attached. FIG. 14 shows a top view of the incubator of FIG. 13, partly in section. The same parts again have the same reference numerals. A test strip 60, as shown in FIGS. 11 and 12, is arranged on the holder 58, which is angled and has a handle-like projection 59 at its left end.

The use of the incubator according to FIGS. 13 and 14 and the test strip 60 result from the above descriptions, so that further explanations do not appear necessary for this.

15 shows another embodiment of an incubator which is for sale in an airtight manner final, welded sleeve 79 is packaged. In the upper area of this approximately egg-shaped incubator 70, a recess 72 is again provided, which is closed by a cover 74. As shown at 75, the incubator 70 has, outside the recess 72, recesses 75 which can be designed as a groove. A projection protruding downward from the cover 74 engages in this locking recess 75. In this way, the cover 74 can be snapped onto the incubator 70; the cover can be easily removed from the incubator 70 at any time via its lateral projections 73 which protrude beyond the recesses 75.

Furthermore, it can be seen in the sectional illustration in FIG. 15 that two further, approximately cylindrical recesses 64 are provided in the incubator 70. Containers 86 and 88 are provided in these recesses, which can contain the test mixtures required for the examination. 15 only two such further recesses 82 and 84 are shown. However, as can be seen in FIG. 16, four or more such recesses can be provided.

The liquid containers 86 and 88, which are contained in the recesses 82 or 74, can consist of glass, which has a predetermined breaking point, for example, at its pointed end. In this way, even the layperson can introduce the test solutions or test mixtures into the recess 72 in a very simple manner. According to another development of the invention, for which protection is also sought, these containers 86 and 88 are made of a flexible material that has a predetermined breaking point, for example, at its front tip. This development of the invention has the advantage that the containers 86 and 88 serve multiple uses. First of all, they store the test mixtures. During the examination, they enable the test mixture contained to be brought into the recess 72 with ease and without any specialist knowledge. They also serve the purpose that, after the preliminary reaction, they can aspirate the test mixture out of the recess 12 again, simply by holding it with the open tip in the recess 72, pressed together and then released so that the liquid is released the recess 12 is sucked back into these containers 86 and 88, respectively. The embodiment of FIGS. 15 and 16 thus represents a marketable unit which can be kept indefinitely because it is sealed airtight by the sleeve 79 and contains all the components which are necessary for carrying out an examination.

FIG. 15 also shows a holder 80 which can hold the test strips. This holder is shown in Figures 17 and 18 and is described in more detail with reference to these figures.

17 shows this holder 80 in

Top view. A number of test strips are arranged on the container. On the right edge of holder 80 there is a handle 90 which has a predetermined breaking point at 92. If this holder is inserted into the recess of an incubator, for example in the recess 72, the handle can be easily broken off so that the recess can be closed off by the cover, for example 74. In this way, it is possible in a very simple manner to insert the test strips into the recess.

These test strips are conveniently clamped in the holder 80, e.g. how a slide is clamped in a frame. In this way it is possible to carry out the examinations without touching the nitrocellulose, which essentially consists of the test strip, with the fingers. This is particularly important if the investigations are to be carried out by the layperson.

So far, the test strips or supports made of nitro-cellulose have been offered without such a frame. This is still possible because until now such carrier strips have only been used in laboratories in which the necessary tools such as tweezers and plastic pipettes and the like are available. According to the invention, these tools no longer have to be present, since the holder can be gripped by the handle 90 and the test strips are clamped in the holder. The nitrocellulose does not have to be touched with the fingers to apply the test mixtures either, since, as in the exemplary embodiment in FIG. 15, they can be supplied in plastic containers 86, 86. The holder 80 can be packed in a tub, cf. Fig. 18, which can be inserted into the recess of an incubator. In this way it is ensured that the test strip does not have to be touched with the fingers.

As the average person skilled in the art can see at a glance from FIG. 15, the incubator there can be used as often as desired. The incubator shown there can be used as a finished laboratory device with all

Ingredients and test mixes are sold. Furthermore, it is possible, as additional kits, to have the containers 86 or 88, in the form of vials or small plastic containers, and the tub 94, as shown in FIG. 18, together with the carrier and test strips located thereon, as replacement components to put on the market. The trough 94 in FIG. 18 can of course also be packed in an airtight manner, so that the test strips cannot be influenced subsequently by touching them with fingers or by dirt.

17, an identification aid 96 is also attached to the left edge. ^' This can consist of a printed strip, which is the key to evaluating the test result. Such decryption values are given by way of example by handwriting under the holder 80 in FIG. 17.

Claims

/

Expectations:

5 1. Procedure for testing whole blood, others

Body fluids and other biological liquids, characterized in that the liquid is mixed with a surface-active medium and then subjected to test reactions.

2. The method according to any one of the preceding claims, characterized in that the liquid is diluted with a surfactant. 5

3. The method according to any one of the preceding claims, characterized in that the surface-active medium is a surfactant.

4. The method according to any one of the preceding claims, characterized in that the surface-active medium is a surfactant NP 40 in a concentration of about 0.1%.

5. The method according to any one of the preceding claims, characterized in that the test reaction for the determination of proteins with antibody- or antigen-specific properties comprises the following steps: Use of one or more on a carrier

30 fixed proteins, which enter into or enter into a specific reaction with the protein to be determined and which is in solution, making it insoluble makes or make, and use of another protein in solution and provided with an indicator that reacts with the insolubilized components and thereby becomes insoluble.

6. The method according to any one of claims 1 to 4, characterized in that the test reaction for the determination of antibodies or antigens comprises the following steps: fixing one or more first substances with antigen-specific properties on a support,

Feeding a second mixture of substances, the 'antibody-specific constituents to be investigated of which may undergo specific test reactions with the first substances during a first incubation, to the first substances;

Application of a first indicator which is uniform for the different reactions and which reacts with components of the second substance mixture attached to the first substances during a second incubation period and thus indicates the specific reaction which may have taken place.

7. The method according to any one of the preceding claims, characterized in that capillary or venous blood is used.

8. The method according to any one of the preceding claims, characterized in that is shaken during and / or between the incubations.

9. The method according to any one of the preceding claims, characterized in that antigens or antibodies are examined in a drop of blood.

10. The method according to any one of the preceding claims, characterized in that capillary blood is mixed with the medium in a ratio of at least 1: 2 or 1: 3, preferably 1:50 to 1: 100.

11. The method according to any one of the preceding claims, characterized in that the protein is fetal calf serum.

12. The method according to any one of the preceding claims, characterized in that the concentration of the fetal calf serum is about 3%.

13. The method according to any one of the preceding claims, characterized in that antibodies and / or Antigen¬ ne in body fluids such as urine, cerebrospinal fluid or saliva are examined directly.

14. The method according to any one of the preceding claims, characterized in that antibodies and / or Antigen¬ ne in processed body fluids such as blood serum, blood plasma, or others are examined.

15. The method according to any one of the preceding claims, characterized in that antibodies are examined in immunoglobulin preparations.

16. The method according to any one of the preceding claims, characterized in that one or more antigens are examined in vaccines.

17. The method according to any one of the preceding claims, characterized in that enzyme-coupled species-specific anti-immunoglobulins of classes G, M, A, D, E and their subclasses are used as the first indicator.

18. The method according to any one of the preceding claims, characterized in that the first indicator contains one or more enzyme-linked antigen-specific antisera.

19. The method according to any one of the preceding claims, characterized in that peroxidase is used for the enzyme coupling.

20. The method according to any one of the preceding claims, characterized in that the further indicator is 4-chloronaphtol plus H2O2.

21. The method according to claim 1 or 2, characterized gekenn¬ characterized in that is poured after the incubation of the mixture of substances.

22. The method according to any one of the preceding claims, characterized in that washing is carried out after the incubation of the mixture of substances.

23. The method according to any one of the preceding claims, characterized in that after the reaction of the second substance mixture with the first first indicator, the carrier is washed.

24. The method according to any one of the preceding claims, characterized in that the antigens directed against the antibodies crude, partially or highly purified or as recombinant products or as antigen-containing or non-antigen-containing cells or as Zellaufar¬ processing are applied to a non-soluble carrier, by where the antigens are absorbed or on which they dry up.

25. The method according to any one of the preceding claims, characterized in that the carriers are materials which, because of the contrasting color, allow a direct reading of a color reaction with the eye.

26. The method according to any one of the preceding claims, characterized in that papers are used as a carrier.

27. The method according to any one of the preceding claims, characterized in that e.g. Nitro cellulose, chromatography papers or filter papers can be used.

28. The method according to any one of the preceding claims, characterized in that the carrier is transparent

Materials are used.

29. The method according to any one of the preceding claims, characterized in that plastics are used as a carrier.

30. The method according to any one of the preceding claims, characterized in that optical aids are used to compare the sample with the standard.

31. The method according to any one of the preceding claims, characterized in that the auxiliary paper with

Imprint of the color reaction and the interpretation are.

32. The method according to any one of the preceding claims, characterized in that the carrier is provided with an amount of about or at least 0.05 ul of the first substance.

33. The method according to any one of the preceding claims, characterized in that about 1-8 ul of the first

Drip substance onto the carrier.

34. The method according to any one of the preceding claims, characterized in that about 2-4 / ul of the first substance are dripped onto the carrier. - •

35. The method according to any one of the preceding claims, characterized in that about 1 drop of the first substance is dropped from a cannula onto the carrier.

36. The method according to any one of the preceding claims, characterized in that the first substance for a short time is dried at a temperature of more than + 20 ° C.

37. The method according to any one of the preceding claims, characterized in that the temperature for drying is 20-80 ° C.

38. The method according to any one of the preceding claims, characterized in that the temperature for drying is about 37 ° C.

39. The method according to any one of the preceding claims, characterized in that the drying time is 10-15 minutes.

40. The method according to any one of the preceding claims, characterized in that the carrier is coated in the dry state and can be used directly after drying or fixing the antigen or antibody.

41. Method according to one of the preceding claims, characterized in that phosphate-buffered saline solution with a surfactant and a protein additive is used as the dilution buffer for the second substance mixture and / or the uniform indicator.

42. The method according to any one of the preceding claims, characterized in that NP40 is used as the surfactant in a concentration of about 0.1%.

43. The method according to any one of the preceding claims, characterized in that fetal as a protein additive Calf serum is used in a concentration of 3%.

44. The method according to any one of the preceding claims, characterized in that the buffer for the washing operations which may be carried out is a surfactant in phosphate buffered saline, preferably 0.1% NP40.

45. The method according to any one of the preceding claims, characterized in that the method at a

Temperature between 1 and 100 ° C is carried out.

46. The method according to any one of the preceding claims, characterized in that the method is carried out at a temperature between 20 ° and 60 ° C.

47. The method according to any one of the preceding claims, characterized in that the method is carried out at a temperature of about 37 ° C.

48. The method according to any one of the preceding claims, characterized in that the sample to be examined is incubated simultaneously with the first indicator according to claim 9.

49. The method according to any one of the preceding claims, characterized in that for the semi-quantitative determination of the antibody content, an antibody or an immune class-specific standard is examined at the same time, and the color reaction of the standard is compared with the result of the patient sample.

50. Device for the detection of antibodies or antigens, according to one of the preceding claims, characterized by a body with a, in the shape, adapted recess for receiving one or more carriers, to which one or more first substances with antigen-specific properties are fixed, thereby characterized in that the recess is waterproof and dimensioned so that its contents can be mixed by shaking the body.

51. Apparatus according to claim 50, characterized gekennzeich¬ net that the distance from the or the surfaces of the carrier is about one and a half times as high or higher than the layer height of the solution to be brought together with the carrier.

52. Device according to one of claims 50 to 51, characterized in that the recess is flat on its base.

53. Device according to one of claims 55 to 52, characterized in that the bottom of the recess has elevations or depressions which allow washing of the inserted carrier.

54. Device according to one of claims 50 to 53, characterized in that the recess contains a holder for the carrier such that it can be washed around from all sides.

55. Device according to one of claims 50 to 54, characterized by an insert into the recess bar and removable holder for the carrier.

56. Device according to one of claims 50 to 55, characterized in that the holder holds the carrier at edges and thus allows wetting with liquids on both sides of the carrier.

57. Device according to one of claims 50 to 56, characterized in that the holder is made of plastic.

58. Device according to one of claims 50 to 57, characterized in that the carrier is clamped between two parts of the holder.

59. Device according to one of claims 50 to 58, characterized in that the holder has a handle so that the carrier can be removed from reaction vessels.

60. Device according to one of claims 50 to 59, characterized in that the handle is bent so that the handle protrudes from the reaction vessel. _,

61. Device according to one of claims 50 to 60, characterized in that there is a predetermined breaking point between the holder and the frame of the carrier in order to break off the handle in order to archive the holder with the carrier for documentation purposes.

62. Device according to one of claims 50 to 61, characterized in that the holder with the carrier in the reaction vessel or the recess or a compartment vacuum-packed i.

63. Device according to one of claims 50 to 62, characterized in that the frame of the holder has an identification field.

64. Device according to one of claims 50 to 63, characterized in that the frame of the holder has elevations on the underside, which allow washing of the inserted carrier.

65. Device according to one of claims 50 to 64, characterized in that the device is flat.

66. Device according to one of claims 50 to 65, characterized in that the device is adapted to its outer shape of the human hand.

67. Device according to one of claims 50 to 66, characterized in that it consists of a good heat-conducting material.

68. Device according to one of claims 50 to 67, characterized in that it rests on the palm and on the fingers, with correspondingly shaped depressions.

69. Device according to one of claims 50 to 68, characterized in that device according to one of the device is designed as a body which has one or more further recesses.

70. Device according to one of claims 50 to 69, characterized in that the or further Ausnehmun¬ gene are designed to accommodate substances that can be used for the desired detection.

71. Device according to one of claims 50 to 70, characterized in that the further recesses are designed to receive removable containers which contain the usable substances.

72. Device according to one of claims 50 to 71, characterized in that the containers are small vials or tubes.

73. Device according to one of claims 50 to 72, characterized in that the containers consist of a deformable material.

74. Device according to one of claims 50 to 73, characterized in that the container consists of an elastic or another material which resumes its original shape.

75. Device according to one of claims 50 to 74, characterized in that the containers are designed to be torn open by hand by means of predetermined breaking points or peelable or rotatable ends.

76. Device according to one of claims 50 to 75, characterized in that the containers have a pierceable membrane.

77. Device according to one of claims 50 to 76, characterized in that the body consists of a good heat-conducting material.

78. Device according to one of claims 50 to 77, characterized in that the body consists of heat-storing material.

79. Device according to one of claims 50 to 78, characterized in that the body is a hollow

Contains volume into which material with a high heat capacity brought to a predetermined temperature can be introduced or is contained.

80. Device according to one of claims 50 to 79, characterized in that the body consists of a heat-in, but isolating against heat leakage material or materials.

81. Device according to one of claims -50 to 80, characterized in that the cover is prepared in such a way that it can be repeatedly taken up and down, e.g. B. by gluing.

82. Device according to one of claims 50 to 81, characterized in that the cover is partially complementary to the outer shape of the recess and is thereby coverable and removable.

83. Device according to one of claims 50 to 82, characterized in that the body is designed so that it can be heated in the pocket.

84. Device according to one of claims 50 to 83, characterized in that the substances are largely protected against inactivation.