DE4228395A1 - Immuno-absorbent for allo-antibody determn. - comprising magnetic carrier particles coated with monoclonal antibody bonding with target antigen - Google Patents

Abstract

Immunoabsorbents (I) consist of magnetic carrier particles surface-coated with antibodies which are themselves treated with monoclonal antibodies (MAB). On incubating with whole blood or with pre-purified cell suspension MAB bond with animal or human cell surface antigens (target antigens) (CSA). CSA are pref. lymphocyte, thrombocyte, monocyte or granulocyte antigens. USE/ADVANTAGE - The use of (I) is claimed for detection and quantitative determn. of allo-antibodies (AAB), esp. in serum. Specifically (I), after bonding with CSA, is used in an immunoassay as solid phase to which AAB binds, the bonded AAB subsequently being determined using labelled antibodies. AAB are e.g. blood group antibodies (formed e.g. after transfusion and leading to incompatibility or even haemolysis) or antibodies against thrombocyte surface glycoproteins (formed e.g. after transfusion or during pregnancy, and leading to severe diseases such as thrombopenia). (I) can be used for early detection of these dangerous antibodies, by selective screening. (I) can be reacted directly with whole blood, without previous isolation of CSA, and provide sensitive, rapid and selective assays.

Description

The invention relates to immunoadsorbents for antibody detection tion. Immunoadsorbents are antibodies fixed to carriers, which can be used to complex antigens.

Antigen-antibody reactions also play a role in medicine important role. For example, the surfaces of the Ery throcytes are known to contain antigenic substances determine a person's blood type. Against this blood group Penantigens are targeted to blood group antibodies, so-called Alloantibodies z. B. are formed after transfusions and can lead to intolerance or even hemolysis.

Alloantibodies can also be used against surface glycoproteins of platelets (HPGP), so that after a blood test transfusion or serious illnesses during pregnancy can occur (thrombopenia). In such cases it happens to recognize as early as possible whether dangerous antibodies are present or their formation is expected got to.

Various tests for the detection of thrombocyte-specific antibodies have also been developed (Blumberg, N., Masel, D. Mayer, T., Horan P. and Heal, J. (1984) Removal of HLA-A, B antigens from platelets. Blood 63, 448; Dunstan, RA and Rosse, WF (1985) Posttransfusion purpura. Report of a case with anti PL ^A1 masked by HLA antibodies. Transfusion 25, 219; Ishida, F., Saji, H., Maruya , E. and Furihata, K. (1991) Human platelet-specific antigen, Siba, is associated with the molecular weight polymorphism of glycoprotein Ibα. Blood 78, 1722; Kiefel, v., Santoso, S., Weisheit, M. and Mueller-Eck hardt, C. (1987) Monoclonal antobody-specific immobilization of platelet antigens (MAIPA): A new tool for the identification of platelet-reactive antibodies. Blood 70, 1722.), which are, however, quite complex to use.

From DE-PS 30 14 036 are magnetic carriers for the solid phase known immunological analysis with which a quantification  which is possible from antigens or from antibodies. For a Screening of cell surface-specific antibodies is required however, the carriers are loaded with a target antigen, the Isolation if possible with the carrier itself.

The object of the present invention is therefore Immunad to provide sorbents with which such an antibody Test can be done easily.

According to the invention, the task is solved by Immunad sorbents with monoclonal antibodies after incubation with whole blood, serum, plasma or pre-cleaned cell suspensions sions on animal or human cell surface antigens (Target antigens) are bound.

The particular advantage of these adsorbents is that targeting by binding monoclonal antibodies (MoAb) antigens for the selective screening of surface-specific Antibodies first isolated and then for quantification tion can be used.

In the following, the production is based on selected examples of the immunoadsorbents according to the invention.

Manufacture of immunoadsorbents Antisera and monoclonal antibodies

Monoclonal antibodies against
1. HLA (human leukocyte antigen) class I-Beta II microglobelin
2. CD41a (HPGP IIb / IIIa)
3. CD42b (HPGP Ib) and
4. CD49b (HPGP IIa)
were developed by Immunotech Corp. (Marseille, France). They were dissolved in sterile water with 0.1% NaN ₃ until a MoAb concentration of 0.2 mg / ml was established.

Human polyvalent antisera against HLA class I and HPA5 -b- were obtained from Transfu's sera by conventional methods sion recipients. An HPA1 -a antiserum came from the Institute for Clinical Immunology and Transfusion Medicine the University of Gießen, FRG.

Immobilization of monoclonal antibodies on immunomagneti cal carrier

Immunomagnetic carriers (Dynabeds M-280, Dynal Corp., Oslo, Norway), which were already coated with covalently bound sheep antibodies against mouse / rat immunoglobulins, were suspended in saline and counteracted at 20 ° C with monoclonal antibodies HPGP incubated for one hour (100 µl carrier material, 10 µl MoAb, 50 µl H ₂ O) and then kept at 4 ° C.

Isolation of platelet antisera (target antigens)

After the platelet antigens had been determined beforehand, donor blood was fractionally centrifuged after EDTA treatment. The platelets obtained in this way or via a cell separator (Spectra, Cobe; AS104, Fresenius GmbH, FRG) were then washed three times, resuspended to 2.5 × 10 ⁹ platelets / ml and at 4 ° C. in saline with 0.1 % (w / v) NaN ₃ stored.

5-10 µl of the carrier loaded with MoAb were treated with 100 µl EDTA treated blood or platelet concentrate for 8-10 min incubated with a shaker and then twice with cook saline solution and concentrated in the magnetic field (MPC-96, Dynal Corp., Oslo, Norway). The platelets isolated in this way were then resuspended in 100 µl of 0.01 M Tris in 0.15 M NaCl (TBS), pH 7.2, 0.5% Tween 20, 0.5% Triton X-100 and 0.1 mM Proteinase blocker (Pefabloc SC, Boeringer-Mannheim GmbH, FRG) and shaken for 20-30 min. The monoclonal antibodies be loaded carriers attached themselves to the target antigens sought and could be isolated and cleaned.  

Incubation at 4 ° C overnight had no effect on the Antigenicity of platelet glycoproteins.

Use of immunoadsorbents

The immunoadsorbents according to the invention can basically be use for all immunological reactions. Compared to conventional solid phase immunoassays, however, allow using monoclonal antibodies loaded carrier that the to be bound Target antigen directly from whole blood without prior centrifuge gation can be won. By binding the target antigen will also increase sensitivity when using large blood quantities and a high selectivity with regard to the Antibody reached. This makes the immunoadsorbents in particular suitable for quick and easy antibody detection.

Detection of antibodies to HLA and HPA (human platelet antigen) glycoproteins

The immunoadsorbents according to the invention were first loaded with HLA or HPA glycoproteins (target antigens) and then incubated in prediluted serum (1: 6 TBS) for 15-20 min at room temperature in microtiter plates on a shaker. The immunomagnetic carriers thus obtained were then washed twice with TBS and concentrated in the magnetic field. Then 100 μl of an enzyme-labeled antibody (for example F _ab fragments of a goat antibody labeled with alkaline phosphatase against the Ig class to be examined) were added to each well of the microtiter plate. After an incubation period of 20 min at room temperature, the mixture was washed twice with TBS. The activity of the enzyme-labeled antibodies, ie the bound phosphate, was then determined under standard conditions after adding the substrate (100 μl p-nitrophenyl phosphate in glycine buffer, pH 9.5). Under the action of phosphatase p-nitrophenyl phosphate was converted to yellow-colored p-nitrophenol. The enzyme reaction was stopped after 30 min by adding 3 N NaOH. 100 µl of the supernatant were removed and measured in the photometer at 405 nm.

Table 1

Absorbance values after enzyme immunoassay

Human antisera against HLA class I or HPA1-a antigens were incubated with HPGP antigens bound to immunoadsorbent. The table shows specific differences for each Target antigens.

Table 2

Results from enzyme immunoassays performed

Antisera against various HPA antigens were identified with Immunad sorbent-bound HPGP antigens from different subjects A-D incubated.

Fig. 1 detection sensitivity of the invention Immunoadsorbents.

5 μl of the immunoadsorbents directed against HPA (CD41a) were incubated in individual dilution stages with blood or purified thrombocytes. It can be seen that 2.5 × 10 ⁵ thrombocytes are sufficient to give a significant detection reaction.

Claims (4)

1. Immunoadsorbents consisting of particulate, magneti , carriers coated with antibodies, thereby ge indicates that the antibodies are monoclonal Antibody acts after incubation with whole blood or pre-cleaned cell suspensions on animal or human cell surface antigens (target antigens) are bound. 2. Immunoadsorbents according to claim 1, characterized in that that the cell surface antigens are thrombo-, Lympho-, mono- or granulocyte antigens. 3. Use of the immunoadsorbents according to claim 1 or 2 for Detection and quantification of alloantibodies in particular others in sera. 4. Use of the immunoadsorbents according to claim 3, characterized ge indicates that the immunoadsorbents after binding of the Target antigens used as a solid phase in an immunoassay to which the alloantibodies bind, the bound then alloantibodies with labeled antibodies be detected.