DE2424465A1 - PROCEDURE FOR THE SIMULTANEOUS DETECTION OF ANTIGENS AND THEIR ANTI-BODIES IN THE SOLIDS RADIO-IMMUNOTEST - Google Patents

Description

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

The invention relates to a method for simultaneous detection of antigens and their antibodies by solid-state radioimmunoassay.

It is known that the presence of antigens or of their Antibodies in an unknown sample by direct or indirect radioimmune testing (see e.g. Catt, K. and Treqr, G.W., Science 158 (1967), pages 1570-1572 "Solid-Phase Radioimmunoassay in Antibody-Coated Tubes".) However, it still is failed to find antigens or their antibodies radioimmunologically in a single To demonstrate test procedures. Surprisingly, it is possible according to the invention in a relatively short-term procedure using a combined radioimmuno test Detect antigens and their antibodies.

The invention relates to a method for simultaneous detection of antigens and their antibodies through a solid-state radioimmunoassay, which thereby is characterized in that one for the detection of antigens with at least one binding site or from their An antibodies which have at least two binding sites in the molecule need to take a sample of an unknown serum with a known amount of an antibody incubated and the resulting mixture then with the walls of a container in Brings contact, which have a coating with known antigen content, then incubated again, then aspirated and washed, a radioactively labeled> high purity Adds antigen, incubates again, sucks off and washes again and finally the Radioactivity of the coated parts in a y-radiation eater of known type measures.

The so-called unknown serum to be examined according to the invention a non-coagulable whole blood, plasma, serum or other body fluid, i.B. Urine or saliva.

It is preferred to use for the first incubation stage of the process 10 to 15% of the maximum that can be fixed by the coating of the reaction container used Amount of antibody. For example, microtiter plates, Test tube or other suitable devices are used.

Incubate for 1 to 16 hours within a temperature range from 20 to 50 ° C, preferably 1 hour at 37 ° C or 12 hours at 2D0C. Longer Incubation times do not interfere with the process.

Antigen and antibodies produced by the method according to the invention can be detected are e.g.

Viruses and virus subunits such as hepatitis B antigens or antibodies and the Coxsackieviruses A and B, bacteria, membranes, cell walls, various hormones, e.g. insulin, drugs such as antibiotics, gamma globulins, etc.

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

Since a close relationship of the HB antigen to hepatitis B has been proven Can apply and HB-antigen-positive blood is known to be the causative agent of hepatitis B that can be transferred to a clinically in the recipient, if he is not immune detectable or clinically undetectable infection, It is of great importance to determine whether there is HB antigen in the blood of donors is available. The HB antibody is of clinical importance because it is in a high Percentage detected in the recovery phase of a hepatitis B infection 0 It is also not yet known whether blood products whose serum HB antibodies contains, can also transmit the pathogen that causes hepatitis B. Also that if possible sensitive detection of the HB antibody is therefore a major concern in the Clinic. The method according to the invention is for example based on a Method for the detection of HB antigen and antibody explained.

Example A. Direct Radioimmunoassay for HB Antibodies: Cuttables Microtiter plates, for example from Cooke, are cleaned with a mixture XB antigens of both subtypes D and Y (Schober, A., Thomssen, R. And U. Kaboth, German Medical Wochenschrift 97 (1972), pp.1579-1583 "Serological subtypes of the hepatitis B antigen (Australia antigen) ".). The mixture is alkaline (0.01 M-Tris buffer, pH 9.5) and contains about 30-0 ng HB antigen protein per ml.

The HB antigen was purified from full serum using the so-called Ro-S method (Ro = density, S = sedimentation), i.e. its combined density equilibrium - and sedimentation gradient centrifugation performed in cesium chloride. criteria The purity of the preparations is their lack of reaction with anti-human serum in the immunodiffusion test as well as the non-reaction of the antibodies generated in the test animal with these preparations with human serum proteins.

The HB antigen used for coating is used for at least 6 hours left at 20 ° C. in the wells of the microtiter plates, then suctioned off. To Samples are rinsed five times with neutral buffer (0.01 M Tris buffer, pH 7.5) a dilution series of a calibration HB antiserum, if possible only the specific Contains subtype determinant anti-a, pipetted in.

After an incubation of e.g. 1 hour at 37 ° C or 12 hours at 20 ° C. the contents are again suctioned off and the plate is rinsed. It will then 125J-labeled HB antigen (manufactured by W.M. Hunter and F.C. Greenwood, Naturo 194 (1962) 5.495), also a mixture of both subtypes, that between 20,000 and 40,000 pulses per minute (IpMt / 0.1 ml supplies, added and 1 hour at 37 0C or incubated for 12 hours at 20 ° C.

After suctioning off and rinsing again, the microtiter plates are cut up and the radioactivity measured in a y-ray MeE device. At low serum dilutions of the calibration HB antibody, a high number of pulses is obtained, which is further diluted in the rehearse decreases and finally the impulse values of the simultaneously entrained negative Controls achieved. (Illustration 1). The specificity of this direct HB antibody test could be secured on the one hand by inhibition attempts. This is based on HB antibodies material to be examined mixed with a certain amount of HB antigen and incubated, e.g. 1 hour at 37 0C, using tubes or microtiter plates that are not coated with HB antigen. After this incubation, the The mixture is incubated in HB antigen-coated plates, then suctioned off and washed. Then 125 labeled HB antigen is added and incubated again.

After suctioning off 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 be found in the second incubation no longer adhere to the antigen present on the solid surface (hence the name inhibition test).

It was also possible to show that possibly present in the test serum Antiglobulins or anti-ß-tipoproteins do not lead to false positive results.

Investigations have shown that all pulse values obtained as specific for HB antibodies that are reproducible over 2.0 fold of the mean of the negative control are like the following Table 1 shows: Table 1 -relation between specificity and mean value X of the negative Controls.

Factor number of sera specific ru 183 0 0 X - 1.5 X 169 0 0 1.5 X - 2X 17 6 33 2 # - 2.52 5 5 100) 2.5 2 48 - 48 100 422 B. The one described under A. Test is suitable for combined HB antigen and antibody detection if you have a Pre-incubation of the serum to be examined with a predetermined, relatively low HB antibody amount carries out. If the test serum contains HB antigen, then the A solid-state radioimmune test carried out according to A shows a pulse reduction compared to the negative controls are observed while in the presence of sufficient amounts of HB antibodies a pulse increase can be determined, since the pulse number is the the given antibodies are added (see Figure 3).

An amount of antibody is preferably used for preincubation which supplies between 10 and 15% of the maximum fixable radioactivity, ie. in the saturation range of relevant calibration antibody is present at low serum dilution. Around 3500 Approaches with negative controls have shown that the standard deviation is 1 sigma approximately 10% if the mentioned 10 to 15 antibody binding pulses between Deliver 500 and 1500 per minute. A slightly higher standard deviation of the negative Controls () 10%) results from pulse numbers below 500 per minute. As a security threshold 3 sigma were chosen, i.e. HB antigen is present if the impulse reduction is reproducible Above 30 of the negative controls, HB antibody if a pulse increase is observed of over 30 is recorded (Figure 2). Here, too, has an incubation period of 1 hour at 37 ° C. in the various incubation steps Results delivered.

As already stated above, the test described delivers unambiguously specific results. The difference between the combined test and the direct one Antibody radioimmuno test is based on the fact that the antibody present in the test serum added to the given antibody. Because the HB antigen detection is carried out in the combined test by reducing the specified amount of antibody the already described specificity of the HB-antibody determination at the same time basis the specific HB antigen detection. A non-specific antibody reduction by other antibodies can be excluded. Exceptions are those in the sample material possibly present antibodies that are directed against the HB antibody itself are. The performance of the test is graphically recorded in Table 2 below. It should also be noted that for a reliable averaging at At least 8 sera should be included in the negative controls.

It is special when it comes to the practical application of the combined test advantageous that the reaction (without measurement) can be completed in a good 3 hours however, if there is sufficient time, it is advantageous to use the Extend the incubation period to 90 to 120 minutes after adding the labeled antigen.

TABLE 2 Test execution negative control: Ag-positive serum Ak-positive serum k and Ag: X Ak: 1 + Calibration ab serum; xxxx xxAxx xxxx xx d: I of the 1st Incu- 7 in -I vl -s. , Incubation; I e washing; 30 Addition of 125J-mar- - ~~~~ I Ct Ag: Ä Incubation; 0: ' C 5 ; ra I: pt - X - 'I>H> ecc X <O counting ~ ~. ~ a A ss s: « QI ax d QI < <ng S o « H. ~ av Q u I ~ Q «N to vaa YXX s: on Hz wv de «<o «So U @«> N ~> = Q : s XQX, qw HH ~ X : J 4 a Q h Q nauan Q oX v nX o £ t <too ¢ o rX « C) Sz> QE çr X w o Q lg: CJ Q «> C) Q t O @ OR n 2 v R nn>'« n n. ,, .D Sz = «« hnd X «~ Q l «YO # IJ Vl QQC Vl X h 4 - @ gh ct bl: n 3 «« n OXQ d . H AsQ ¢ SD. z Ag = antigen, Ak = antibody

Claims (5)

Claims: 1.) Method for the simultaneous detection of antigens and their antibodies by a solid-state radioimmunoassay, characterized in that that one for the detection of antigens with at least one binding site or of their antibodies, which have at least two binding sites in the molecule, one Sample of an unknown serum incubated with a known amount of an antibody and then the mixture obtained in contact with the walls of a container brings that have a coating with known antigen content, then again incubated, then aspirated and washed, a radioactively labeled, highly pure antigen admits, incubated again, again aspirated and washed and finally the radioactivity of the coated parts in a y-radiation measuring device of known type. 2.) The method according to claim 1, characterized in that for the first incubation 10 to 15% of the maximum that can be fixed by the coating of the reaction container Amount of antibody used. 3.) Process according to Claims 1 and 2, characterized in that 1 to 16 hours at Temperaturon from 20 to 50 C, preferably 1 hour at 37 ° C or 12 hours at 20 ° C incubated. 4.) Process according to Claims 1 to 3, characterized in that when using microtiter plates, these are cut up at the end of the process and measure the radioactivity of the individual parts. 5.) Process according to Claims 1 to 4, characterized in that the material to be examined is non-coagulable whole blood, plasma, serum or other body fluids used.