DE3725504C2 - - Google Patents

Description

The invention relates to a method for determining Proteins with antibody or antigen specific Properties in body fluids.

According to German interpretation 22 06 103 Antibodies and antigens in hormone preparations, in the urine or in sera that have been diluted to influence from reducing otherwise disruptive factors will.

To get sera you must first use a single-use syringe at least 5 ml of blood drawn from the vein will. Then the blood has to go for a while protrude so that it coagulates so that the bottom of the  Separates blood cake from the serum in which Blood cells swim, is covered. The Serum-blood cell mixture is with a pipette siphoned off, pipetted into a centrifuge tube and centrifuged. After centrifugation, the serum from the centrifuge bowl into another container pipetted. This method of serum extraction is cumbersome, very time-consuming and expensive, and also requires the relatively expensive devices like Centrifuge and pipettes. This procedure is by the layperson not feasible and for a one-time examination also completely unprofitable.

Samples from several patients must come together so that a test can be carried out profitably can. Conducting the investigation also requires expertise and the availability of special equipment, which, since they are expensive, only in special laboratories can be used sensibly. Need rehearsals therefore first packed and mailed to an institute sent there, unpacked, registered and be handed over to the technical assistants. There is an examination protocol from samples from several patients finish so the individual Samples in several test approaches against different Antigens or antibodies can be tested can. Then again after test assessment to draw up a protocol and the findings back To give mail. This results in a period of Sampling until receipt of the finding of at least one, but usually several days.  

This means that the finding is only available at a point in time to which the result is often only the Sampling immediately necessary therapeutic Measures at best can confirm. In the acute No result is available in an emergency. As an an example are neurological diseases like herpes infections to name, in which the therapeutic application of a Anti herpes remedies should be serologically secured. Values of accident victims also exist in transplants, who can be considered an organ donor, at the earliest Available hours after the transplant, although it may be vital for the patient can know whether the accident victim z. B. with the AIDS virus is infected.

The need for the determination of antibodies has in has increased rapidly in recent years and is also increasing continue to grow. As an example, the desired possibility of mass examinations Serve HIV virus. This is also the result of the development microbiological knowledge, introduction of the enzyme immunoassay by Engvall and Perlman (J. Immunol. 109, 129 (1972)), the offering of ELISA test kits and advancing automation of antibody and antigen studies up to because of the high level of difficulty and the expensive devices limited to a few special laboratories were.

In the future, the efforts of the World health organization as part of a comprehensive Immunization in the Third World ("Expanded  Program on Immunization ") extensive antibody studies make necessary. Here are simple and above all to develop cheap test methods (WHO Chronicle, 40, 47 (1986)).

The global spread of HIV infection requires just like the diagnosis of the hepatitis B virus Injection mass examinations. Personnel and financially such mass examinations are only too to be able to cope if it is possible to develop methods those outside of special laboratories also from trained laypersons can be carried out. The Examinations should also be in medical practice or clinic.

With the development of chemotherapy for viral diseases is the virological and immunological diagnostics increasingly on antibody and antigen tests be supported. Short times for the Examination process are due to therapeutic interventions to strive for.

As part of the investigation of risk groups, diagnosis of symptom pictures or screening examinations increasingly the antibody and / or antigen test for a plurality of pathogens for one person questioned. This is largely a fixed spectrum of the possible Investigations. So far, however, the serum has become one Subjects examined in different test approaches, because the test kits are assembled to 1 Antibody or 1 antigen based. A serum must  therefore, in the vast majority of cases, several Go through test approaches, causing delays in the Overall assessment leads.

Also, it is still necessary to have blood samples taken before Centrifuge antibody to determine the particulate from the liquid components of the blood to separate. This process is time consuming and requires a centrifuge.

The z. Test kits currently offered for the investigation of Antibodies and antigens in the ELISA are based in principle on the adsorption of antigens or antibodies a transparent plastic surface or an equivalent Spheres placed in transparent test tubes will. Then there is another Binding with an antibody or antigen in the form of a Antigen-antibody reaction. Eventually a added enzyme-labeled protein that specifically with the previous reaction product reacts. The amount of bound antibody or antigen in the unknown Sample is used as a color reaction after adding a substrate visible and visual in the liquid phase or measured photometrically.

Determination of antigens other than transparent Plastic carriers or balls have been developed by Hawkes et al. (Anal. Biochemistry, 119, 142 (1982)) and Paltree and Elliott (J. Immunol. Methods, 52, 395 (1982)) published The authors are using for the first time Nitrocellulose paper. Nitrocellulose paper was also made 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. Default. 64, 199 (1986)) and Schmeer (personal Communication, 1986). Herbrink et al. (J. Immunol. Methods, 48, 293 (1982)) compared nitrocellulose and diazobenzyloxymethyl paper for use an antibody test. Giallongo et al. (J. Immunol. Methods, 52, 379 (1982)) and Esen et al. (Analytical Biochemistry, 132, 462 (1982)) set chromatography paper as a carrier for protein determinations. Finally, Lin and Halbert (J. Clin. Microbiology, 24, 7 (1986)) also the use of white plastic cards presented for antibody determinations.

These studies have shown that ELISA tests cannot be carried out on transparent supports are and deliver 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 Halbert) and Coxiella (Schmeer) captured these carriers.

However, none of the authors presents a disease or problem-related concept of antibody and antigen tests where a number of antigens or Antibodies according to the diagnostic objective is used per patient. Only Hawkes et al. demonstrate the possibility of multiple virus antigens  to unite on one carrier but without one to discuss disease-associated antigen selection. Their test approach as well as that of the other authors differs from that to be described below new method in several points, so needed the new method z. B. 7 instead of 15 individual steps The test takes about 30 minutes instead of 2.5 to 25 hours and the incubation temperature is 37 ° C 20 ° C.

In summary, it should be noted that there is still none cheap, easy and fast, even by semi-skilled Lay people to be performed on the sick bed or in the field Method there, with a drop of blood Numerous antibody and antigen determinations are possible at the same time.

The object of the present invention is a method for the examination of whole blood to create that particularly easily, quickly and with minimal effort on devices is feasible.

This object is achieved in that a Whole blood sample with a surfactant is mixed and then the antibody or is subjected to antigen-specific test reaction.

The invention first has the advantage that fixed Components such as cell components of the test item biological fluid are lysed or destroyed. This is particularly important in all such investigations, where there is a reaction to or at a  location of a reagent supporting carrier can be used. If solids, e.g. B. Cell components of the blood are exactly on this Deposit, they can have a reaction to this Reagent with that in the biological body fluid prevent existing reagent. This danger will eliminated by the present invention.

On the one hand, this method has the great advantage on that no devices such as centrifuges for reprocessing of the blood are required. Second, it points the advantage that the biological fluid in conceivably shortly after their removal, namely immediately after mixing with an interface or surface-active medium, the test reactions can be subjected.

The biological fluid can with a surface-active medium can be diluted. After a Embodiment of the invention is the medium Surfactant. There can be one surfactant NP 40 in one Concentration of about 0.1%.

The examined biological can expediently Liquid during and or between the Incubations are shaken. This shake has on the one hand the effect of thorough mixing, and on the other hand, it prevents deposits from becoming more solid Components of the examined biological fluid on the carrier.  

According to an advantageous development of the invention be one or more spaced apart proteins fixed to a carrier.

According to another advantageous development of the invention, the test reaction comprises the following steps:
Fixing one or more first substances with antigen-specific properties on a carrier,
Supplying the sample mixed with the surfactant to a first incubation;
Application of an indicator which reacts with components of the sample attached to the first substances during a second incubation period and thus indicates the specific reactions which may have taken place.

With these advanced trainings will be an amazing one A variety of advantages achieved.

Because according to the invention blood itself for examination the advantage to be mentioned first is that a small amount of blood is enough. That brings the other Advantage with it that no disposable syringe is necessary is to take blood. Furthermore, the difficult blood collection from the vein.

Furthermore, the centrifuge that is otherwise required is not required. The means that the inventive method everywhere can be done, not just in laboratories using Centrifuges and other devices necessary are equipped.

This can result in significant healthcare costs save on. Since the determination according to the invention is possible everywhere, the disadvantages of Packaging and dispatch of patient material with the corresponding accompanying documents to an institute as well as the return of the findings from the laboratory to Sender. The disadvantages are the time delay of findings, packaging and postage as well to mention unprofitable personnel costs. There are also no time delays in the Investigation institute through re-registration etc. before and after the examinations.

There are also risks of confusion, such as those mentioned above. Procedure occur again and again with the invention Investigation excluded, since this in The patient's presence begins.  

The time from taking blood to receiving the Findings are shortened from days to less than one Hour. The finding is still in the period of the first Examination of the patient available and therefore immediately therapeutically usable. The detection method according to the invention thus fulfills all requirements for immediate emergency diagnostics at the bedside or even in the ambulance. Serological examinations in an emergency they already have their meaning for brain inflammation caused by herpes virus infections and in organ transplants when organs from Accident victims are taken from other patients should be used. Serological emergency diagnostics will be expected in the near future Development of chemotherapy drugs against viral infections be required.

Because the blood to be examined cannot be processed must, because blood itself is examined and not blood serum the present method can be used to determine of antibodies or antigens, even from the layperson be carried out, reducing the cost of the procedure be significantly reduced.

Add to that the layperson with this procedure too can examine whether he is HIV-positive, for example is. This advantage is very important, because the result is not known. This opens the possibility that many people are now out Afraid of the consequences of their HIV status becoming known not to be examined, the investigation itself  can make. It is expected that on this So many people who are HIV positive do that now also examine yourself and yourself if you like them own HIV positive state is known accordingly different, behave responsibly, that they don't infect other people. The The present invention thus contributes to the further spread of this HIV epidemic and possibly also to keep other epidemics within limits.

Another advantage is the fact that with the present procedure because it is so simple and cheap to be carried out for the first time, also serial examinations entire ethnic groups become possible. Furthermore, the Invention allergy tests, so far very were expensive.

Of course, these advantages do not only apply to the determination of HIV antibodies, but quite general for the determination of antibodies. So that's on it to point out that, for example, in the tropics 50,000 people die each year from rabies that Animal losses per year are in the millions. Although there are a corresponding number of vaccinations, that are not always successful in the tropics, for example because of frequent immune deficiency no antibodies formed due to other infections will. The present invention enables Screening and also the individual examination Antibodies against rabies, so that a possibly Vaccination can be done.  

In this context it is pointed out that the Series and individual examinations only with the methods according to the invention can be carried out, because the well-known process equipment and specialists require that does not exist there.

Another important advantage is that with the present Procedures also include vaccines before they are used their antigen content can be examined. This is of importance especially in the tropics because of the there often broken cold chain a loss of active ingredient vaccines are easily given. Come in addition, that tropical countries have vaccines after their manufacture or import u. U. store for a long time and because of Then uncertainty of the residual activity will not later use. This way, great resources wasted. The same applies Immunoglobulin preparations.

After a further binding of the invention, capillary blood used. This training brings the big one Advantage with it that the blood collection is not with a Syringe must be performed, for which the layperson in is generally unable. It is much more possible to draw capillary blood with a lancet from the Fingertip or the earlobe. This The process is very simple and can be done by everyone be performed.

According to another development of the invention Antibodies and / or antigens in blood drops, capillary or venous blood examined directly.  

Previously, blood had to be centrifuged beforehand because the corpuscular components interfere with the reaction. By mixture according to the invention with special buffer the disruptive influence is eliminated. Conveniently, can be shaken between the individual incubations.

The disturbing influences in the drop of blood, capillary or Venous blood can be mixed with a medium be eliminated.

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

The protein can be fetal calf serum Concentration z. B. can be 3%.  

Antibodies and / or antigens can be prepared in Body fluids such as blood serum, blood plasma, or others are examined. Antibodies can also be found in Immunoglobulin preparations are examined.

The first indicator can be enzyme-linked species-specific class G anti-immunoglobulins, M, A, D, E and their subclasses can be used.

Depending on the question and body fluids are then Antiimmunoglobulins of different classes or Use subclasses.

The first indicator can be one or more enzyme-linked contain antigen-specific antisera. For the Enzyme coupling, peroxidase can be used.  

According to another embodiment of the invention are the antigens directed against the antibodies crude, partially or highly purified or as recombinant Products or as antigenic or non-antigenic Cells or as cell refurbishments on one Insoluble carrier applied from which the antigens be absorbed or on which they dry up. The carrier can be materials that are due to the Contrast staining a direct reading of a color reaction allow with the eye. This makes examinations on plain papers (e.g. typewriter paper) possible. The paper must be in contact (white) and should not be too absorbent to the Distribution density of the first substance in the carrier / unit area optimally designed.

As a carrier z. B. nitrocellulose, chromatography papers or filter papers are used. Also Transparent materials and also plastics can can be used as a carrier.

According to an embodiment of the present invention can contain between 1 and 8 µl of the first substance dropping the carrier. Expediently is about 1 drop of the first substance from a cannula onto the Carrier dripped.  

The first substance can briefly at a temperature of more than + 20 ° C. One is suitable Drying temperature of around 37 ° C.

Drip onto dry medium and briefly dry at + 37 ° C has the advantage of the low background after the end of the test. But the rest of the steps are as well a decisive factor in this.

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

According to another embodiment of the invention the carrier coated in the dry state and after Drying or fixation of the antigen or antibody used directly.

Such carriers are ready for use very quickly. For Mass exams can make carriers even more cost effective be made at the site of the examination. This further advantage of the invention has become possible because a previously customary and necessary 30 minutes to 2 hours requiring "blocking step", the one long drying time had to follow.  

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

The process is advantageously carried out at a temperature performed between 1 and 100 ° C. The temperature increase The reaction time can change from 20 ° C to 37 ° C Shorten by an order of 40-50%, depending on the conditions even stronger.

According to another embodiment of the invention for the semi-quantitative determination of the antibody content at the same time be antibody or more immune-specific Standard examined, and the color reaction of the Standards will result in the patient test compared.

This training enables you even under field conditions an estimate of the concentration of the determining protein.

Simultaneous incubation of sample and 1st indicator shortens the response time further. The so far The 2nd incubation period may be necessary omitted.

According to another development of the invention, a Carrier used on which several first fabrics with antigen-specific properties can be fixed or are; furthermore, not a second substance, but one  second mixture of substances, the antibody-specific ones to be examined Ingredients with the first substances specific test reactions during a first incubation can come in, brought together with the first fabrics, and for the different reactions a uniform first indicator applied, the during a second incubation period with the first Constituents of the second mixture of substances responds, and so, if any, has taken place indicates specific reaction.

This development of the invention makes it possible to a single drop of blood in one at a time Test approach a variety of antibody and antigen testing Perform group or disease-associated, by placing several antigens on each of the carriers and / or antibodies are applied.

The selection of antigens and / or antibodies per Test approach depends on the needs of a screening of specific people, patients and Animal groups are to be provided. The selection of the antigens or antibody can also be used for diagnosis a clinical picture, a group of symptoms or Organ disease occur.

Examples of group or disease associated Investigations are:

• a) Risk person
  Blood donor
  Pregnant women
  Tropical returnees
  Vaccinates
  Hospital patients
  soldiers
  prostitute
  Homosexual
  Accident victim
  Prison inmates
• b) group of animals
  Vaccinates
  Inventory surveys
  Mass buoyancy (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 immune deficiency

After this advanced training, antibodies become simultaneous or antigens of one or more pathogens, which for the diagnosis of a symptom picture, an organ or systemic disease are relevant (disease-related Determination). Routine exams, as they are in for certain groups of people must always be carried out in the same way, and which a certain spectrum  Antigen antibody determinations include, simplified be carried out (target group-associated Determination). A major advantage for establishment Such a process is the first time here presented method of examining a variety of antibodies with a single drop of blood. To Application can also come from capillary blood that is in the frame from other routine examinations anyway without strong ones Harassment of the patient is removed.

This blood draw is also so simple that it is from trained laypersons can be carried out.

The object of the present invention is also a Device for performing the above method create.

This object is achieved according to the invention by a body with a recess adapted in shape to accommodate one or more carriers to which one or more first substances with antigen or antibody-specific properties are fixed, wherein the bracket has a recess for the carrier contains such that it is washed from all sides can be.

Such a body can be constructed in a very simple and simple way be trained.

The distance from the surface (s) of the support can be about one and a half times as high or higher than that Layer height of those to be brought together with the carrier Solution.  

Developments of the invention are based the subclaims.

The invention is based on some embodiments in connection with the description and the drawing described in more detail. Show in detail

Fig. 1 as a comparative example, a checkerboard of sera with and without antibodies against the rabies virus,

Fig. 2 antibody detection against rabies virus in whole blood,

Fig. 3 Investigation of patients blood samples for antibodies against the human immune deficiency virus (HIV I), the AIDS virus,

Fig. 4-7 sections through various incubators for the assay,

Fig. 8 shows a holder with a test strip,

Fig. 9 aid in interpreting the result of the examination,

Fig. 10 is a vertical section through an inventive analysis device,

Fig. 11 is a plan view of an inventive assay device, partly in section,

Fig. 12 is a section through another embodiment of an assay device according to the invention,

Fig. 13 is a plan view of the apparatus of FIG. 9,

Fig. 14 is a plan view of a holder according to the invention with an inserted carrier, and

Fig. 15 is a sectional view of a container with an inserted carrier holder.

Example 1: Checkerboard titration of three sera Antibodies against rabies virus antigen

FIG. 1a is a diagrammatic representation of the test result is shown with a serum containing antibodies to rabies virus. Fig. 1 shows 5 nitrocellulose strips of paper that are each divided into 6 fields. 2 μl of a concentrated and purified inactivated rabies antigen at 680 IU / ml in dilutions of 1:50 to 1: 1200 were dripped into these fields. The amount of antigen at a dilution of 1: 200 thus contained 0.007 IU / 2 µl. 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 suctioned off 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 wash buffer and twice with phosphate buffered saline. During the last washing process, the substrate (4-chloronaphtol + H₂O₂) is produced and preheated briefly at + 37 ° C. After the washing process is complete, 3 ml of substrate are added to the strips and incubated for 5 minutes with gentle shaking at + 37 ° C. After the color development, the strips are rinsed under running water and assessed immediately. The result shown here ( FIG. 1a) shows a strong color reaction of the serum dilutions up to 1: 600 when using the antigen dilutions 1:50 to 1: 200. The serum dilution 1:50 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 in the figures, which are only partially broken, represent weak color reactions.

FIG. 1b shows the corresponding reaction with a serum without antibodies to rabies virus. The serum dilutions 1:50 to 1:200 only show a weak non-specific reaction with 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.

Fig. 1c shows the result of rabies immunoglobulin with 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.

The example of the rabies virus shows that using this method, quantitative dependencies between Antigen and antibody exist and that a semi-quantitative Statement on the antibody and antigen content is possible.

Fig. 2: Detection of antibodies in whole blood

Fig. 2 shows the test result was performed by the method described Blood drop ELISA. From a clotted, non-centrifuged blood sample, a sample of a subject known to be positive for rabies virus antibody status 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 approx. 20 μl from the fingertip of a test subject without rabies virus antibody was dripped directly into a vessel intended for receiving the antigen carrier, which contained 1500 μl 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

Table 1 (see p. 38) shows the result of 5 Rabies virus antibodies positive and 3 negative Sera. The antibody status was established in the Neutralization test raised. The table shows that not just nitrocellulose and chromatography paper as Carriers are suitable, but also other papers. The Pore size should not be too large for the antigen not diffused too much and therefore per unit area only in a relatively low antigen concentration is present, which leads to a weaker coloring.

Example 4: Immunity Screening for Rabies Virus in Risk takers

In Table 2 (see p. 39) the results are one Screening of 17 risk groups Rabies immunity was examined with the Blood Drop ELISA. The results were more established with those Antibody tests correlated.  

Example 5

One person should use the Blood Drop ELISA in a drop of blood to examine what viral infections had expired. These results were published in the conventional complement fixation reaction and in ELISA of the Behring-Werke, Marburg (Enzygnost) for validity examined. With a carrier and using a only drops of blood were determined in about 1/2 hour performed with 17 antigens. The table 3 (see p. 40) The result shown showed agreement of Results achieved with traditional methods.

FIG. 3 shows the result of an examination of patient blood samples for antibodies against the human immune deficiency virus HIV (I), the AIDS pathogen.

The results on HIV antibody status are with these 11 selected people in conventional and Blood drop ELIZA identical. The designations in the right column mean:

Positive = antibody against HIV present
Negative = no antibodies to HIV present, ie not infected
Questionable = probably no antibodies against HIV, however additional examinations are recommended.

Table 1

Comparison of different carrier materials for the Blood Drop ELISA

Table 2

Screening of individuals for rabies virus immunity (immunity screening) with the Blood Drop ELISA and comparison of the results obtained with this method with those of established methods (neutralization test = RFFIT, and complement fixation reaction = KBR)

Table 3

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

The annotation numbers shown in FIG. 3 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: the result is questionable. Further investigations are required for clarification.

FIG. 4 shows a section through an inventive incubator for the assay. 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.

FIG. 5 shows a sectional view through another incubator. The container 20 shown in this Fig. 5 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. 5 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 is poured off or the strip can be removed.

This embodiment of the invention has the advantage that it does not need any additional heat sources. A Such an incubator can be in your pocket be plugged in until it reaches body temperature  assumes. After that, the exam can be done will. When inserting the carrier and also when inserting or pouring the test mixture, the incubator remains open Temperature, because the test person holds it in his hand holds. This incubator cools during the reaction time also not from: either, the user keeps this incubator in hand, e.g. B. to shake him or else, he just puts it back in Pocket. In both cases, the temperature remains in the get essential. If the person who Makes an examination, has to go back and forth anyway, will this incubator as long as it is in your pocket is shaken at the same time, so that a good mixing of the test mixture and a good one there is constant washing around the test strip, while the examiner is any one can do other work.

FIG. 6 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 a constant temperature simply by connection to a battery.

FIG. 7 shows a modification of the incubator of FIG. 6. In the exemplary embodiment of FIG. 7, 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. 8 shows an illustration for carrying out the AIDS test for lay people. The figure shows a strip or carrier on which "HIV + cells", denoted by 1 , and "HIV cells", denoted by 2 , are applied. 1 denotes virus antigen and 2 denotes control antigen.

The Fig. 9 shows an interpretation aid for the evaluation of the result. The result of the investigation is only positive if 1 is markedly more strongly colored than 2 . The coloration of 1 must be stronger than in the example in FIG. 9c. , B among the representations of FIGS. 9a, c the finding result and d are each shown in the cases A and B the result is positive, represented by a "+" in Fig. The finding 9c is questionable, represented by a " ? ", and in FIG. 9d the result is negative, represented by a" - ". In Fig. 9e the finding is questionable, in 9f it is positive and in 9g and 9h 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 none Antibodies are present. A repetition of the Examination or consultation with a doctor are recommended in this case.

Fig. 10 shows another incubator, which can also be used by the layman and 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 with z. B. granular or pasty material can be filled. 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 beneficial result is achieved regardless of whether the person performing the 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.

FIG. 10, which shows a vertical section, also shows a holder 58 on which one or more test strips are attached. FIG. 11 shows a top view of the incubator of FIG. 10, partly in section. The same parts again have the same reference numerals. A test strip 60 , as shown in FIGS. 8 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. 10 and 14 and the test strip 60 result from the above descriptions, so that further explanations do not appear to be necessary.

FIG. 12 shows a further embodiment of an incubator which is packaged for sale in an airtight, sealed sleeve 79 . 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 recesses 75 outside the recess 72 , which can be designed as a groove. A projection projecting 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. 12 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. In the sectional view of FIG. 12, only two such further recesses 82 and 84 are shown. However, as can be seen in FIG. 13, four or more such recesses can be provided.

The liquid container 86 or 88 , which are contained in the recesses 82 or 74 , may consist of glass, which, for. B. has a predetermined breaking point at its tip 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 which, for. B. has a predetermined breaking point 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 suck the test mixture out of the recess 12 again, simply by holding it with the open tip in the recess 72 , squeezing it and then releasing it so that the liquid from the Recess 12 is sucked back into these containers 86 and 88 , respectively. The embodiment of FIGS. 12 and 13 thus represents a marketable unit which can be kept indefinitely because it is hermetically sealed by the casing 79 and contains all the components which are necessary for carrying out an examination.

FIG. 12 also shows a holder 80 which can hold the test strips. This holder is shown in FIGS. 14 and 15 and is described in more detail with reference to these figures.

In FIG. 14 one sees this holder 80 in a plan view. A number of test strips are arranged on the container. On the right edge of the 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, e.g. B. in the recess 72 , the handle can be easily broken off, so that the recess through the lid, for. B. 74 , can be completed. In this way, it is possible in a very simple manner to introduce the test strips into the recess.

These test strips are expediently clamped in the holder 80 , e.g. B. 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 nitrocellulose 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. Also, to apply the test mixtures, the nitrocellulose does not have to be touched with the fingers, since, as in the exemplary embodiment in FIG. 12, they can be supplied in plastic containers 86 , 88 .

The holder 80 can be packed in a tub, cf. Which can be inserted into the recess of an incubator Fig. 15. This ensures 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. 12, the incubator there can be used as often as desired. The incubator shown there can be sold as a finished laboratory device with all ingredients and test mixtures. Furthermore, it is possible, as additional kits, to use the containers 86 or 88 , in the form of vials or small plastic containers, and the tub 94 , as shown in FIG. 15, together with the carrier and test strips thereon, as replacement components in the To bring trade. The trough 94 in FIG. 15 can of course also be packaged in an airtight manner, so that the test strips cannot be influenced subsequently by touching them with fingers or by dirt.

In Fig. 14 an identification aid 96 is also mounted even on the left margin. 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. 14.

Claims (55)

1. A method for the determination of proteins with antibody- or antigen-specific properties in body fluids, characterized in that a whole blood sample is mixed with a surface-active medium and then subjected to the antibody- or antigen-specific test reaction. 2. The method according to claim 1, characterized in that that the surfactant is a surfactant. 3. The method according to any one of the preceding claims, characterized in that the surfactant  Medium a surfactant NP 40 in a concentration of about Is 0.1%. 4. The method according to any one of the preceding claims, characterized by the use of an or several, spaced from each other on one carrier fixed fabrics with antibody or antigen-specific properties associated with that determining protein a specific reaction come in. 5. The method according to claim 4, characterized by the following steps:
Fixing one or more first substances with antigen or antibody-specific properties on a support,
Supplying the sample mixed with the surfactant to a first incubation;
Application of an indicator which reacts with components of the sample attached to the first substances during a second incubation period and thus indicates the specific reactions which may have taken place. 6. The method according to any one of the preceding claims, characterized in that capillary blood or venous blood is used. 7. The method according to any one of claims 5 and 6, characterized characterized in that during and / or between the Incubations are shaken.   8. The method according to any one of the preceding claims, characterized in that a drop of blood as a sample is used. 9. The method according to any one of the preceding claims, characterized in that whole blood with the surfactant medium in the ratio of at least 1: 2 or 1: 3, preferably 1:50 to 1: 100 is mixed. 10. The method according to any one of the preceding claims, characterized in that the surfactant Medium contains a surfactant and a protein. 11. The method according to any one of the preceding claims, characterized in that the protein fetal calf serum is. 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 in immunoglobulins containing samples are examined. 14. The method according to any one of the preceding claims, characterized in that one or more antigens be examined in vaccines. 15. The method according to any one of the preceding claims, characterized in that the indicator is a or  several enzyme-linked antigen-specific antisera contains. 16. The method according to any one of the preceding claims, characterized in that as an indicator enzyme-linked species-specific anti-immunoglobulins of classes G, M, A, D, E and their subclasses be used. 17. The method according to any one of claims 15 and 16, characterized in that as the enzyme peroxidase is used. 18. The method according to any one of the preceding claims, characterized in that against the antibodies targeted antibodies crude, partially or highly purified or as recombinant products or as antigenic or cells not containing antigens or as cell preparations applied to a non-soluble support from which the antigens are absorbed or on which they dry up. 19. The method according to any one of claims 4 to 18, characterized in that the carrier materials are a direct one due to a contrast coloring Allow a color reaction to be read by eye. 20. The method according to any one of claims 4 to 19, characterized in that as a carrier papers be used.   21. The method according to any one of claims 4 to 20, characterized in that the carrier is nitrocellulose, Chromatography papers or filter papers are used will. 22. The method according to any one of claims 4 to 21, characterized in that as a carrier transparent Materials are used. 23. The method according to any one of claims 4 to 22, characterized in that plastics as a carrier be used. 24. The method according to any one of claims 4 to 23, characterized in that as a dilution buffer for the indicator phosphate buffered saline with a Surfactant and a protein additive is used. 25. The method according to any one of the preceding claims, characterized in that for those possibly carried out Washes a surfactant in phosphate buffered Saline solution, preferably 0.1% NP40, is used. 26. The method according to any one of the preceding claims, characterized in that the method at a Temperature of about 37 ° C is carried out. 27. The method according to any one of the preceding claims, characterized in that the sample to be examined in a first reaction step with a first Indicator is incubated.   28. The method according to any one of the preceding claims, characterized in that for semi-quantitative Determination of the antibody content of the sample at the same time an immune class-specific antibody standard is examined, and the color reaction of the standard with the result of the patient sample is compared. 29. Device for carrying out the method according to one of the preceding claims, characterized by a body ( 10 , 20 ) with a, in the form, adapted recess ( 12 ) for receiving one or more carriers to which one or more first substances with antigen - Or antibody-specific properties are fixed, the recess ( 12 ) containing a holder for the carrier such that it can be washed around from all sides, and is tightly closed. 30. The device according to claim 29, characterized in that the distance between the surface or surfaces of the carrier and the inner surface of the body ( 10 , 20 ) is at least one and a half times as high as or higher than the layer height of the solution which washes around the carrier . 31. The device according to one of claims 29 and 30, characterized in that the bottom of the recess ( 12 ) has elevations or depressions which allow washing around the inserted carrier. 32. Device according to one of claims 29 to 31, characterized by a in the recess ( 12 ) insertable and removable holder ( 58 ) for the carrier. 33. Apparatus according to claim 32, characterized in that the holder ( 58 ) holds the carrier at edges and thus allows wetting with liquids on both sides of the carrier. 34. Device according to one of claims 32 and 33, characterized in that the holder ( 58 ) is made of plastic. 35. Device according to one of claims 32 to 34, characterized in that the holder ( 58 ) has a handle ( 59 ) so that the carrier can be removed from reaction vessels. 36. Apparatus according to claim 35, characterized in that the handle ( 59 ) is bent so that the handle protrudes from the reaction vessel. 37. Device according to one of claims 35 and 36, characterized in that the handle ( 59 ) can be broken off by a predetermined breaking point in order to archive the holder ( 58 ) with the carrier for documentation purposes. 38. Device according to one of claims 32 to 37, characterized in that the holder ( 58 ) is vacuum-packed with the carrier. 39. Device according to one of claims 32 to 38, characterized in that the frame of the holder ( 58 ) has an identification field. 40. Device according to one of claims 32 to 39, characterized in that the frame of the holder ( 38 ) has elevations on the underside, which allow washing around the inserted carrier. 41. Device according to one of claims 29 to 40, characterized in that the device of their outer shape of the human hand is adapted. 42. Device according to one of claims 29 to 41, characterized in that they are made of good heat-conducting Material. 43. Device according to one of claims 41 and 42, characterized in that it is on the palm of the hand and on the fingers with appropriately shaped hollows issue. 44. Device according to one of claims 29 to 43, characterized in that the device has a further or more recesses ( 82 , 84 ). 45. Apparatus according to claim 44, characterized in that the further recesses ( 82 , 84 ) are designed to receive substances which can be used for the desired detection. 46. Device according to one of claims 44 and 45, characterized in that the further recesses ( 82 , 84 ) are designed to receive removable containers ( 86 , 88 ) containing the usable substances. 47. Apparatus according to claim 46, characterized in that the containers ( 86 , 88 ) consist of a deformable material. 48. Device according to one of claims 46 and 47, characterized in that the containers ( 86 , 88 ) consist of an elastic or another material which re-assumes its original shape. 49. Device according to one of claims 46 to 48, characterized in that the containers ( 86 , 88 ) are designed to be torn open by hand by predetermined breaking points or ends that can be pulled off or turned off. 50. Device according to one of claims 46 to 49, characterized in that the containers ( 86 , 88 ) have a pierceable membrane. 51. Device according to one of claims 29 to 50, characterized in that the body ( 10 , 20 , 30 ) consists of heat-storing material. 52. Device according to one of claims 29 to 51, characterized in that the body ( 10 , 20 , 30 ) is hollow and in it a material brought to a predetermined temperature with high thermal capacity can be penetrated or is contained therein. 53. Device according to one of claims 29 to 52, characterized in that the body ( 10 , 20 , 30 ) consists of a material which allows heat to enter, but which is insulating against the escape of heat. 54. Device according to one of claims 29 to 53, characterized in that the closable cover ( 14 , 56 , 74 ) of the recess is prepared in such a way that it can be repeatedly removed and removed. 55. Device according to one of claims 29 to 54, characterized in that the body ( 54 , 70 ) is designed in the form so that it can be inserted into a pocket, where it can be warmed up and shaken by going back and forth.