2.1. Absorption of Interfering Antiiso/Allotypic Human Anti-Mouse Antibodies
1. Mouse IgG-agarose suspension: polyclonal mouse IgG coupled to cyanogen bromide-activated agarose (Sigma-Aldrich, Deisenhofen, Germany). Resuspend settled gel by repeated shaking immediately before filling the pipet (see Note 1).
2. Phosphate-buffered saline with Tween (PBS-T): prepare 10X stock with 0.027 M KCl, 1.37 MNaCl, 0.1 M Na2HPO4, 0.018 M KH2PO4, and 1% Tween-20. Store at 4°C. Prepare working solution by dilution of one part with nine parts water.
3. Conical 4.5-mL polystyrene test tubes (Sarstett, Nümbrecht, Germany) (see Note 2).
2.2. Determination of Human Antiidiotypic Anti-OC125 Antibodies
1. Anti-human IgG-agarose suspension: Fc-specific anti-human IgG goat antibodies coupled to cyanogen bromide-activated agarose (Sigma-Aldrich). Resuspend settled gel by repeated shaking immediately before filling the pipet (see Note 3).
2. Conical 4.5-mL polystyrene test tubes (Sarstett) (see Note 2).
3. PBS-T: prepare 10X stock with 0.027 M KCl, 1.37 M NaCl, 0.1 M Na2HPO4, 0.018 M KH2PO4, and 1% Tween-20. Store at 4°C. Prepare working solution by dilution of one part with nine parts water.
4. Mouse IgG solution: polyclonal mouse IgG (reagent grade; Sigma-Aldrich) is dissolved at 12 mg/mL in PBS-T and stored in single use (0.4 mL) aliquots in 10 mL tubes at -20°C.
5. Detector antibody solution: the therapeutic antibody is used as detector antibody (see Note 4). These instructions assume the use of 125I-labeled OC125 detector antibodies provided as tracer of the CA-125-II 125I IRMA, DiaSorin, Stillwater, MN (see Note 5). The original tracer solution is diluted 10-fold with PBS-T and supplemented with 0.8 mg/mL polyclonal mouse IgG by addition 0.6 mL of the tracer solution to a 0.4-mL aliquot of mouse IgG solution together with 5 mL PBS-T (see Note 6).
6. Standard solutions: a serum pool with elevated levels of antiidiotypic antibodies can serve as arbitrary standard material. In the present protocol we use a serum pool made of samples drawn from two ovarian cancer patients treated repeatedly with OC125 antibodies. After absorption of antiiso/allotypic antibodies (see Note 7) the serum supernatant is stored as master standard stock solution in single use (60 pL) aliquots at -80°C. Working standard solutions are prepared by 1:2, 1:4, 1:8,1:16, and 1:32 dilution of master standard with PBS-T. The concentration of the antiidiotypic antibodies is expressed as arbitrary units per milliliter (arb.units/mL). A nominal value of 1 X 103 arb.units/mL of antiidiotypic anti-OC125 antibodies is assigned to the master standard stock solution.
7. Diluent: a serum pool free of antiiso/allotypic human anti-mouse antibodies (HAMA) and antiidiotypic antibodies diluted 1:5 with PBS-T serves as diluent. The diluent is stored in single use (500 pL) aliquots at -80°C.
1. Antigen solution: A high amount of the therapeutic antibody is directed against the antigen and is used to inhibit binding of the detector antibodies to "internal image" antibodies. Thus, these instructions assume the use of the cancer antigen 125 (CA-125) which is the corresponding antigen to the OC125 antibody (see Note 8). Two-hundred fifty microliters of CA-125 stock solution (1 X 106 arb.units/mL, Fitzgerald Industries International, Concord, MA) is diluted 10-fold with 2.25 mL PBS-T and stored in single use (0.4 mL) aliquots at -20°C.
2. Detector solution A (without antigen): the detector solution A corresponds to the detector antibody solution described in Subheading 2.2. The original OC125 tracer provided with the CA-125-II 125I IRMA, DiaSorin is diluted 10-fold with PBS-T and supplemented with 0.8 mg/mL polyclonal mouse IgG by addition 0.6 mL of the tracer solution to a 0.4-mL aliquot of mouse IgG solution together with 5 mL PBS-T (see Note 6).
3. Detector solution B (with 2000 arb.units/mL CA-125): 0.3 mL of the original OC125 tracer is mixed with 0.2 mL of mouse IgG solution, 60 pL antigen solution, and 2.44 mL PBS-T (see Note 6).
4. Detector solution C (with 10,000 arb.units/mL CA-125): 0.3 mL of the original OC125 tracer is mixed with 0.2 mL of mouse IgG solution, 300 pL antigen solution, and 2.2 mL PBS-T (see Note 6).
The serum concentration of human antibodies newly formed in response to the application of a therapeutic antibody can be measured in serum samples prepared in the common way from blood collected by venipuncture into plain tubes. A serum volume of 1 mL is adequate for analysis. Serum samples can be stored at -20°C for several months without any effect on antibody concentration.
Antiiso/allotypic anti-murine antibodies (HAMAs) are found in a high percentage of patients treated with murine antibodies. For direct determination of the concentration of such HAMAs, specific assays are commercially available (see Note 9). HAMAs generally can falsify the results of immunometric assays that involve murine test antibodies by cross-linking capture and detector antibodies (19,20). To avoid those interferences, HAMAs can be removed from serum samples by absorption onto mouse-IgG-agarose gel before measurement of antiidiotypic antibodies. Furthermore, HAMA activity can be blocked by addition of nonspecific murine antibodies to the assay buffer (21).
Human antiidiotypic antibodies formed by patients after antibody therapy are measured by an immunometric assay procedure involving the applied therapeutic antibody as detector antibody. These instructions describe the determination of human antibodies developed after application of the anti-CA-125 antibody OC125. They can easily be adapted to other therapeutic antibodies if they are available in labeled form (see Note 5). Fc-specific anti-human antibodies immobilized on agarose gel are used as capture antibodies (see Note 10). To exclude interferences by serum components such as the original antigen that may inhibit binding of the detector antibody, a two-step assay design was chosen with a separate detection step after removal of unbound serum components by washing (see Note 11).
Internal image antibodies can be quantified indirectly as a subgroup of anti-idiotypic antibodies. To this end, binding of detector antibodies is measured in the presence of a high concentration of the original antigen, which inhibits binding of the detector antibodies to "internal image" antibodies (see Note 12).
3.1. Absorption of Interfering Antiiso/Allotypic Human Anti-Mouse Antibodies
1. For absorption of HAMA from serum samples, 200 pL of mouse IgG-agarose suspension (equivalent to 100 pL gel) is pipetted into a conical 4.5-mL polystyrene test tube (see Note 13).
2. Add 2 mL of PBS-T, mix each tube and centrifuge at 3500g for 15 min (see Note 14).
3. Carefully aspirate the buffer supernatant. Pay attention that the sedimented pellet is not destroyed (see Note 15).
4. Add 400 pL PBS-T to the washed agarose pellet followed by 100 pL of the respective serum sample.
5. Mix each tube and incubate for 2 h at room temperature on an orbital shaker with intensive agitation (see Note 16).
6. Stop incubation by centrifugation at 3500g for 15 min. The serum supernatant can be stored at 4°C until further processed for measurement of specific antiidiotypic antibodies (see Note 17).
3.2. Determination of Human Antiidiotypic Anti-OC125 Antibodies
1. For binding of human serum IgG, 100 pL of anti-human IgG-agarose suspension (equivalent to 50 pL gel; see Note 18) is pipetted into a conical 4.5-mL polystyrene test tube (see Note 13).
2. Add 2 mL of PBS-T, mix each tube and centrifuge at 3500g for 15 min (see Note 14).
3. Carefully aspirate the buffer supernatant. Pay attention that the sedimented pellet is not destroyed (see Note 15).
4. Add 200 pL of PBS-T to the washed agarose pellet followed by 10 pL of the serum supernatant obtained after absorption of HAMA (as described in Subheading 3.1.).
5. Mix each tube and incubate for 1 h at room temperature on an orbital shaker with intensive agitation (see Note 16).
6. Stop incubation by addition of 1 mL PBS-T followed by centrifugation at 3500g for 15 min.
7. Carefully aspirate the buffer supernatant and wash the agarose pellet by resuspension in 2 mL PBS-T. After centrifugation at 3500g for 15 min aspirate the buffer supernatant.
8. Add 300 pL of detector antibody solution, mix each tube and incubate over night at room temperature on an orbital shaker with intensive agitation (see Note 16).
9. Stop incubation by addition of 1 mL PBS-T followed by centrifugation at 3500g for 15 min.
10. Carefully aspirate the buffer supernatant and wash the agarose pellet two times by resuspension with 2 mL PBS-T, centrifugation at 3500g for 15 min and aspiration of the buffer supernatant (see Note 19).
11. The radioactivity bound to the washed pellet is measured in a y-counter (see Note 20).
12. The respective antiidiotypic antibody concentration of a sample is calculated by interpolation from a standard curve measured in the same assay as the sample. The standard curve is generated processing the working standard solutions such as the samples. The radioactive counts measured for each working standard solution is plotted against the standard concentration (see Note 21). If samples have concentrations greater than the highest standard, they must be reanalysed after adequate dilution with the diluent.
1. For indirect determination of internal image antibodies, the human IgG of the serum supernatant obtained after absorption of HAMA (as described in Subheading 3.1.) is bound to anti-human IgG agarose according to the procedure described in Subheading 3.2., steps 1-7. Three replicates are performed for each serum sample.
2. Add 300 pL of one of the detector solutions A, B, and C to one of the three replicates, mix all tubes and incubate over night at room temperature on an orbital shaker with intensive agitation (see Note 16).
3. Stop incubation by addition of 1 mL PBS-T followed by centrifugation at 3500g for 15 min (see Note 14).
4. Furthermore the buffer supernatant is carefully aspirated, the agarose pellet is washed and the bound radioactivity is measured as described in Subheading 3.2., steps 10-11.
5. The apparent antiidiotypic antibody concentrations of the three replicates are calculated by interpolation from a standard curve generated using the detector solution A (without antigen). The concentration of "internal image" antibodies is calculated as the difference between the apparent concentrations measured with (detector solution C) and without (detector solution A) addition of antigen (see Note 22).
1. For complete binding of HAMAs directed against the different iso/allotypic epitopes a polyclonal mouse IgG preparation is optimal (21). We found this agarose gel suitable for HAMA absorption but numerous competitive reagents are available from other commercial sources.
2. Clear conical test tubes are used to have better control when the buffer supernatant is aspirated from sedimented agarose pellet.
3. We found this agarose gel suitable for binding human serum IgG but numerous competitive reagents are available from other commercial sources.
4. Humanized antibodies cannot be used as detector antibodies because they directly bind to the anti-human IgG antibodies employed as capture antibodies. To determine antiidiotypic antibodies developed in response to therapy with a humanized antibody, the original murine MAb, the humanized MAb is derived from, should be used as detector antibody.
5. This protocol can easily be adapted for other therapeutic antibodies. Instead of radio-labeling antibodies also can be conjugated to biotin using commercial available biotinylation kits (e.g., Biotin Tag™, Micro Biotinylation Kit, Sigma-Aldrich).
6. HAMAs may interfere with the determination of specific antiidiotypic antibodies by cross-linking capture and detector antibodies. Thus, a high amount of polyclonal mouse IgG is added to the detector antibody solution as a target for HAMA binding to prevent binding to the test antibodies. A polyclonal mouse IgG preparation is the best agent for blocking HAMA interferences because all allotypic epitopes are presented (21). The supplementation with mouse IgG can be omitted when the antiiso/allotypic antibodies are completely removed by previous absorption with mouse IgG-agarose (see Subheading 3.1.).
7. Absorption of antiiso/allotypic antibodies can be performed as described (see Subheading 3.1.). To get a sufficient volume of master standard, a greater serum volume can be absorbed with an accordant amount of gel (e.g., 1 mL serum diluted with 4 mL PBS-T is absorbed with 1 mL gel).
8. For inhibition of the binding of detector antibodies to the "internal image" antibodies the relevant antigen, the therapeutic antibody is developed against, should be used. The antigen concentration of the detector solutions B and C should be high enough to achieve a complete inhibition of "internal image" antibody binding.
9. For determination of human antibodies developed against murine iso/allotypic epitopes (HAMA) various test kits are commercially available (e.g., ImmuSTRIP HAMA Test Kit, Immunomedics, Morris Plains, NJ or HAMA-ELISA medac, MEDAC, Wedel, Germany). In our experience the HAMA-ELISA medac, which use polyclonal mouse IgG as both capture and detector antibodies is a reliable test for simple quantification of those HAMAs.
10. We use capture antibodies immobilized on agarose gel instead by coating on 96-well plates to get a high binding capacity for human serum IgG, which is a limitation of assay sensitivity. Insufficient binding capacity of the solid phase can be compensated in part by increasing the amount of gel used in the first incubation step.
11. There are alternative assay formats, in which the antiidiotypic antibodies are captured by the immobilized therapeutic antibody. Subsequently the bound antiidio-typic antibodies are detected by incubation with labeled anti-human immuno-globulin G antibodies (17,22). In a homologous "sandwich"-assay format the therapeutic antibody can be used as both capture and detector antibodies (23). However, because in these assay formats the immobilized therapeutic antibodies are incubated with whole serum, possible interferences are to be expected for serum samples containing the original antigen. In contrast, the assay procedure described here allows the correct quantification of antiidiotypic antibodies in the presence of the original antigen because in a first step the human serum IgG is selectively extracted and interfering serum components including circulating antigen, which may react with the detector antibodies are removed before specific detection of the anti-idiotypic antibodies by labeled therapeutic antibodies.
12. Itisawell-accepted principle that "internal image" antibodies are identified by inhibition of their binding to the original antibody by the corresponding antigen. However, also binding of antiidiotypic antibodies recognizing paratope associated idiotopes, which do not present an internal image of the antigen can be inhibited by the antigen, probably owing to alteration of the three-dimensional structure of the paratope (24).
13. Distribution of agarose gel in test tubes may be done with a multidispensing device (e.g., Eppendorf Multipipette), but pipetting time should not exceed 10-15 s to avoid sizable sedimentation of gel.
14. Conditions of centrifugation can be modified to assure the formation of a pellet compact enough for removal of buffer supernatant.
15. Alternatively, tubes can be decanted to remove the buffer supernatant, but in our hand, supernatant can be aspirated more exhaustively.
16. To prevent sedimentation of agarose gel, the incubation is performed with agitation at 1600 rounds per min. We use the orbital shaker IKA Vibrax VXR with the suitable attachment VX2 for 16-mm test tubes.
17. The absorbed supernatants may be stored at 4°C for 2 d. For prolonged storage (up to 1 wk) they should be kept at -20°C.
18. To ensure complete binding of human IgG from 2 pL serum we use 50 pL antihuman IgG agarose gel with a binding capacity of 3 mg human IgG per mL. We found this agarose gel suitable for IgG binding but numerous competitive reagents are available from other commercial sources.
19. This washing step is very important. Insufficient removal of the detector solution grossly affects accuracy and precision of test results.
20. If a biotin labeled detector antibody is used, determination of the bound biotin can be done using commercial available detection kits (e.g., Avidin-Peroxidase and SigmaFast™OPD, Sigma-Aldrich).
21. For data reduction we use the computer assisted data reduction program of the y-counter LB 2111 (Berthold, Bad Wildbad, Germany), but other programs appropriate to reduction of immunoassay data also can be used.
22. The replicate with the detector solution B is not used for calculation. It only serves as control to indicate how far the antigen concentration of the detector solution C is sufficient to inhibit the binding of "internal image" antibodies completely.
The author would like to thank Dietlind Ackermann for excellent technical assistance.
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