DE2424465C3 - Method for the simultaneous detection of antigens and their antibodies in the solid-state radioimmuno test - Google Patents

Description

45

The invention relates to bring antigens and their antibodies by a solid-state radioimmunoassay, in which a sample of an unknown serum with the walls of a container in contact to a method of ^ ₀ detection, have subsequently incubated for a coating with a known antigen content, Sucks off and washes, then adds a radioactively labeled, highly pure antigen, incubates again, sucks off and washes and then measures the radioactivity of the coated parts in a γ-radiation meter. _to

It is known that the presence of antigens or their antibodies in an unknown sample can be detected by direct or indirect radioimmune testing (cf. e.g. C a 11, K. and T regear, GW, Science 158 (1967) , Pages 1570-1572, "Solid-Phase _{(1 <j} Radioimmunoassay in Antibody-Coated Tubes"). A test device for direct radioimmuno testing for antigens or their antibodies is also known from DT-OS 22 62 479. However, it has been known to date Antigens or their antibodies can be detected radioimmunologically in a single test method at the same time. The task was therefore to detect antigens and their antibodies simultaneously in a relatively short-term method using a combined radioimmuno test.

Based on the known method of the type mentioned at the outset, this object is achieved by that one for the simultaneous detection of antigens with at least one binding site or of their Antibodies that have at least two binding sites in the molecule, the sample of the unknown serum first incubated with a known amount of the antibody and only then with the mixture obtained Brings walls of the container into contact, which have a coating with known antigen content.

The so-called "unknown" serum to be examined according to the invention can be a non-coagulable one Whole blood, plasma, serum or other body fluid, e.g. B. urine or saliva.

For the first incubation stage of the process, 10 to 15% of the maximum amount of the antibody that can be fixed by the coating of the reaction container used is preferably used. For example, microtiter plates, test tubes or other suitable devices can be used as reaction containers. One incubates e.g. B. 1 to 16 hours within a temperature range of 20 to 50 ⁰ C, preferably 1 hour was 37 ° C or 12 hours at 20 ⁰ C. Longer incubation times did not interfere with the process.

Antigen and antibodies which can be detected by the method according to the invention, are z. B. Viruses and virus sub-units such as hepatitis B antigens or antibodies and the Coxsackieviruses A. and B, bacteria, membranes, cell walls, various Hormones, e.g. B. insulin, drugs such as antibiotics, gamma globulins, etc.

The liquids used for washing or rinsing should preferably have a neutral pH exhibit.

Since a close relationship between the HB antigen and hepatitis B can be considered proven and HB antigen positive As is well known, blood can transmit the pathogen that causes hepatitis B in the recipient if it does not is immune, leads to a clinically detectable or also to a clinically undetectable infection, it is It is of great importance to determine whether HB antigen is present in the blood of donors. The HB antibody cr is of clinical importance because it occurs in a high percentage in the convalescence phase of a hepatitis B infection can be proven. It is also not yet known whether blood products whose serum 11B antibodies also contain the pathogen causing hepatitis B can transfer. The detection of the IIB antibody as sensitive as possible is therefore also one important concern in the clinic. The method according to the invention is for example based on a Methods for the detection of HB antigen and antibody illustrated.

example

A. Direct radioimmunoassay for HB antibodies, whose basic procedural features are known

Cutable microtiter plates. for example from Cooke, are adjusted with a mixture

HB antigens of both subtypes D and Y (S ch ο be r. A., Thomssen, R. and U. K ab ο th, Deutsche Medizinische Wochenschrift 97 (1972), pp. 1579-1583, »Serological subtypes des Hepaiitis- B antigen [ALStralia antigen] «) coated. The mixture is; alkaline (0.01 M-Tns buffer, pH 9.5) and contains about 300 ng HB antigen protein per ml.

The HB antigen was purified from full SeruTi using the so-called R ₀ -S method (R _u = density, S = sedimentation), ie a combined density equilibrium and sedimentation gradient centrifugation carried out in cesium chloride. The criteria for the purity of the preparations are their lack of reaction with anti-human serum in the immunodiffusion test and the non-reaction of antibodies generated in the test animal with these preparations with human serum proteins.

The HB antigen used for the coating is left in the wells of the microtiter plates for at least 6 hours at 20 ° C., then suctioned off. After rinsing five times with neutral buffer (0.01 M-Tris buffer, pH 7.5), samples of a dilution series of an EicS-HB antiserum, which if possible only contains the specific subtype endeterminant Anti-a, are pipetted in. After a ! cubation of z. B. 1 hour at 37 ° C or 12 hours at 20 "C, the contents are again suctioned off and the plate is rinsed. Then labeled HB antigen (prepared according to WM Hunter and I \ C. Greenwood. Nature 194 [ 1962], p 495). also a mixture of both subtypes, which provides 20,000 to 40,000 counts per minute (LPM) / 0, l ml was added and 1 hour at 37 ⁿ C or 12 hours at 20 ¹ C incubated After renewed suction

Table 1

and rinsing, the microliler plates are cut and the radioactivity in a radiation meter measured. With low serum dilutions of the cloth! I get a high impulses. which decreases in the white diluted samples and finally the pulse values of the simultaneously carried negative controls achieved (Fig. 1). The specificity this direct HB antibody eletesis hay itself secure on the one hand by inhibition attempts. This will be the case

ίο material to be examined for H B antibodies is mixed with a certain amount of HB antigen and incubated, e.g. B. 1 hour at 37 ° C. using tubes or microtiter plates! that are not coated with IIB alloys. After this incubation, the mixture is incubated in HB antigen-coated plates, then suctioned off and washed. Then ^{125 I} -labeled HB-Aniigen is added and incubated again. After suction and washing, the radioactivity is measured. If HB antibodies are present in the material to be examined, then these antibodies are bound by the added antigen and can therefore no longer be bound to the antigen present on the solid surface in the second incubation. (Hence the name »inhibition test«)

It was also possible to show that antiglobulins or anti-lipoproteins possibly present in the test serum do not lead to false positive results. Research has shown that all received Pulse values are to be regarded as specific for HB antibodies which are reproducible above 2.0 times the Mean values of the negative control are as shown in Table 1 below:

Relationship between specificity and mean X of the negative controls

factor

Number of sera Specific

<x 183 0 0 X- 1.5 X 169 0 ö \ J5X-2X 17th 6th 33 1X-15X 5 5 100 > 2.5 X 48 48 100

422

B. The test described under A is suitable for combined HB-antigen and antibody detection, when performing a pre-incubation of the serum to be examined with a predetermined, relatively low HB antibody level. If the test serum contains HB antigen, then in the solid-state radioimmuno test carried out according to A, a reduction in impulses compared to the negative controls is observed, while if sufficient quantities of HB antibodies are present, an increase in impulses can be determined because the impulse count is added to the given antibody (cf. Fig. 3).

Zui ¹ pre-incubation is preferably used an amount of antibody which provides 10-15% of the maximum fixable radioactivity that is present at low serum dilution in the saturation region of the respective calibration antibody. About 3500 batches with negative controls have shown that the standard deviation of 1 sigma is approximately 10% if the 10 to 15% antibody binding mentioned above produces pulses between 500 and 1500 per minute. A slightly higher standard deviation of the negative X controls (> 10%) results with pulse numbers below 500 per minute. 3 sigma were chosen as the safety threshold, ie HB antigen is present when the pulse reduction is reproducibly more than 30% of the negative controls, HB antibodies when a pulse increase of more than 30% is recorded (FIG. 2). Here, too, an incubation period of 1 hour at M "C has given excellent results in the various incubation steps.

ho As already stated above, the described test clearly specific results. The difference / between the combined test and that direct antibody radioimmuno test is that the antibody present in the test serum is

(15 added / added to the given antibody. Da HB antigen detection is carried out in the combined test by reducing the specified amount of antibody, is the already described specificity of the HB antibodies

perbestimniung at the same time the basis of the specific HIJ antigen detection. [a unspecific anliqueur reduction by other antibodies can be excluded, the exceptions are those in the sample material possibly the presence of the target body directed against the HB target body itself. The residence tour is shown in the following table 2, lis should also be noted that for a reliable mean value formation for the negative cone rolls at least H sera should be carried out should.

/ was it in the practical application de combined tests are particularly advantageous because! dii reaction (without measurement) completed in gill J hours can be done, but if sufficient / urgent Sees available, it is advantageous to the Inkiibalionsdauei after adding the labeled antigen extend to 90 to I2 (minutes / u.

For this purpose 3 sheets of drawings

Claims (5)

Patent claims: 1. Method for the detection of antigens and their antibodies by means of a solid-state radioimmune test. In which a sample of an unknown serum is brought into contact with the walls of a container that has a coating with a known antigen content, then incubated, suctioned off and washed, then a radioactively labeled, highly pure antigen is added, incubated again, suctioned off and washed and then d ^; e measuring the radioactivity of the coated parts in a j'-radiation measuring device, characterized in that for the simultaneous detection of antigens with at least one binding site or their antibodies which have at least two binding sites in the molecule, the sample of the unknown serum is first carried out with a known amount of the antibody is incubated and the mixture obtained is only then brought into contact with the walls of the container, which have a coating with a known antigen content. 2. The method according to claim I, characterized in that for the first incubation 10 to 15% of the maximum from the coating of the reaction vessel fixable amount of the antibody is used. 3. The method of claim I or 2, characterized in that I to 16 hours at Temperaluren of 20 to 50 "C, preferably 1 ^ o hour at 37 ° C or 12 hours at 20 ⁰ C. 4. The method according to any one of claims I to 3, characterized in that when using from microtiter plates these cut up at the end of the process and the radioactivity of the individual parts measures. 5. The method according to any one of claims 1 to 4, characterized in that one is to be examined Material used that does not coagulate whole blood, plasma, serum, or other body fluids.