Haptens, hapten conjugates, compositions thereof and method for their preparation and use

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Recently, Citrina and Venus have been developed, two bright versions of a yellow light GFP mutant (YFP) that mature effectively.

Two recently developed varieties of DsRed, known as T1 and E57, display ripening

enhanced, which makes them preferable for use in two-color experiments.

5 The fluorescence of some GFP variants can be "photoactivated" by specific illumination, which offers the advantage that the fluorescence can be activated at a selected time point. Three fluorescent proteins have been developed that undergo photochemical modification on or near the chromophore, PA-GFP, Kaede and KFP1, which allow selective activation of fluorescence signals after specific illumination and which can be used to fluorescently mark individual cells, organelles or proteins.

10 Table 1 provides other examples of signal generating and conjugate fractions comprising these fractions.

Table 1

Examples of antibody detectable label conjugates

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<dl><dt>Antibody conjugate label </dt><dd>Recommended for Emitted color Tag excitation Label Issuance </dd></dl>

<dl><dt>Lake Placid Blue (EviTag ™ Quantum Dot) </dt><dd>Flow cytometry, immunoblots and fluorescence microscopy &lt;450 490 </dd></dl>

<dl><dt>Fluorescein (i.e., FITC) </dt><dd>Flow cytometry, incl. BD FAC and Guava System systems, and fluorescence microscopy 494 518 </dd></dl>

<dl><dt>Adirondack Green (EviTag ™ Quantum Dot) </dt><dd>Flow cytometry, immunoblots and fluorescence microscopy &lt;450 520 </dd></dl>

<dl><dt>Rhodamine Green </dt><dd>Fluorescence microscopy 502 527 </dd></dl>

<dl><dt>Catskill Green (EviTag ™ Quantum Dot) </dt><dd>Fluorescence microscopy &lt;450 540 </dd></dl>

<dl><dt>Rhodamine 6G </dt><dd>Flow cytometry, immunoblots and fluorescence microscopy 525 555 </dd></dl>

<dl><dt>Hops Yellow (EviTag ™ Quantum Dot) </dt><dd>Flow cytometry, immunoblots and fluorescence microscopy &lt;450 560 </dd></dl>

<dl><dt>Amersham Cy3 </dt><dd>Fluorescence microscopy 550 565 </dd></dl>

<dl><dt>R-Phycoerythrin (PE) </dt><dd>Flow cytometry, Luminex® and Guava systems, FRET assays, capillary electrophoresis and use (495) 565 575 </dd></dl>

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<dl><dt>with FITC for double labeling </dt><dd /></dl>

<dl><dt>Rhodamine Red </dt><dd>Flow cytometry, fluorescence microscopy 560 580 </dd></dl>

<dl><dt>Birch Yellow (EviTag ™ Quantum Dot) </dt><dd>Fluorescence microscopy &lt;450 580 </dd></dl>

<dl><dt>Amersham Cy3.5 </dt><dd>Fluorescence microscopy 581 596 </dd></dl>

<dl><dt>Fort Orange (EviTag ™ Quantum Dot)</dt><dd>Flow cytometry, immunoblots and fluorescence microscopy &lt;450 600 </dd></dl>

<dl><dt>SulfoRhodamine (Alias ​​Texas Red®) </dt><dd>Flow cytometry and fluorescence microscopy 596 615 </dd></dl>

<dl><dt>Amersham Cy5 </dt><dd>Immunoblot, incl. Amersham Typhoon System, and immunofluorescent applications 650 670 </dd></dl>

<dl><dt>Allophycocyanin (APC) </dt><dd>FRET tests and HTRF tests 652 670 </dd></dl>

<dl><dt>Amershani Cy5.5 </dt><dd>Immunoblot, especially LI-COR Odyssey systems 675 694 </dd></dl>

<dl><dt>Biotin </dt><dd>Flow cytometry and other fluorescent applications -</dd></dl>

Many of these tags can be used with multiple antibodies that do not interreact to create personalized multiplexed assays.

VII. Processes for the formation of hapten conjugates - Reaction schemes

5 The following schemes provide examples of embodiments of a method useful for producing conjugates of the present embodiment. Other synthetic methodologies are also useful for producing these conjugates and it should not be construed that the following schemes limit the method to the specific synthetic methodologies illustrated.

1. Nitropyrazole Conjugates

Scheme 1 illustrates a suitable method for coupling examples of nitropyrazol haptens to an alkylene oxide linker 10 and subsequently to a protein vehicle.

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phosphate buffer (PBS) 0.15 molar. The concentration of BSA-dPEG8-benzofurazano was 2 µg / ml, and the concentration per well of 50 µL. These samples were incubated at 4 ° C until the next day. 1% skimmed milk powder (NFDM) (10 mg / mL, 300 µL / per well) was used as a blocking reagent, followed by incubation at 37 ° C for 120 minutes. The plates were washed, when deemed necessary, using 0.15 M PBS which

5 It contained 0.05% Tween 20. Next, each hapten tested was used to produce mouse antiserum. A total concentration of 80 [micro] l per well of mouse antiserum diluted with 1% NFDM in 0.15 M PBAS was used, using the dilution and plate design protocol indicated below in Table 6 The plates were then incubated at 37 ° C for 150 minutes. An anti-mouse goat radish peroxidase conjugate (Gt-α-Mu-HRP, Pierce) was used as a secondary antibody at a concentration of 1: 10,000

10 in 0.15 M PBS with 0.05% Tween 20 to obtain a total volume of 50 µL / per well. Next, the plates were incubated for 60 minutes at 37 ° C. The ELISA configuration and results are summarized in Table 2 below.

Table 2

ELISA test results

<dl><dt>Accession: </dt><dd>Client Sample tested Anti-mouse serum </dd></dl>

<dl><dt>500421 </dt><dd>Window </dd></dl>

<dl><dt>Test parameters </dt><dd /></dl>

<dl><dt>He passed </dt><dd>Reagent Serial dilution Dilution buffer solution Concentration Volume / well Incubation </dd></dl>

<dl><dt>Ag coating. </dt><dd>VMSI1357-98 0.15M PBS 2µg / ml 50µl ON @ 4C </dd></dl>

<dl><dt>Blocking </dt><dd>1% NFDM 0.15M PBS 10µg / ml 300 µl 2hr @ 37C </dd></dl>

<dl><dt>Sample dilution </dt><dd>See below 5 x 1% NFDM at 0.15m PBS Game @ 1.50 80 µl 2,5hr. @ 37C</dd></dl>

<dl><dt>Secondary ac </dt><dd>Gt-α-Mu-HRP , 15M PBS w / 0.05% Tween 20 1 (10,000) 50µl 1hr @ 37c </dd></dl>

<dl><dt>Plate design </dt><dd /></dl>

<dl><dt>Sample dilution </dt><dd> 1:50 1:250 1:1250 1:6250 1:31250 1:156250 </dd></dl>

<dl><dt>1 </dt><dd> 2 3 4 5 6 7 8 9 10 11 12 </dd></dl>

<dl><dt>Mu # 1 </dt><dd>TO 1.8 9 0.0 1 1.8 1 0.0 0 1.8 2 0.0 0 1.60 0.00 1.06 0.0 0 0.5 6 0.0 0 </dd></dl>

<dl><dt>Mu # 2 </dt><dd>B 1.7 6 0.0 1 1.8 7 0.0 0 1.7 3 0.0 0 1.25 0.00 0.60 0.0 0 0.2 5 0.0 0 </dd></dl>

<dl><dt>Mu # 3 </dt><dd>C 1.7 0 0.0 1 1.8 3 0.0 0 1.8 7 0.0 0 1.50 0.00 0.95 0.0 1 0.3 7 0.0 0 </dd></dl>

<dl><dt>Mu # 4 </dt><dd>D 1.7 5 0.0 1 1.7 3 0.0 0 1.8 3 0.0 0 1.49 0.01 0.98 0.0 0 0.4 2 0.0 0 </dd></dl>

<dl><dt>Mu # 5 </dt><dd>AND 1.6 2 0.0 1 1.5 8 0.0 0 1.7 2 0.0 0 1.35 0.00 , 074 0.0 0 0.3 3 0.0 0 </dd></dl>

<dl><dt>Concentrate or premix (PbP) </dt><dd>F 0, 06 0.0 1 0.0 1 0.0 0 0.0 0 0.0 1 0.00 0.00 0.00 0.0 0 0.0 0 0.0 0 </dd></dl>

<dl><dt>x </dt><dd /><dt>x </dt><dd /><dt>x </dt><dd /><dt>x </dt><dd /><dt>x </dt><dd /><dt>x </dt><dd /></dl>

<dl><dt>Antigen </dt><dd>VMSI-1357-98 Lot # C05081610 x = blank (no antigen) </dd></dl>

<dl><dt>Buffer solution </dt><dd>0.15M PBS with 0.05% Tween 20 NFDM = skimmed milk powder </dd></dl>

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1992 ; Pluckthun, Biotechnology 9: 545, 1991; Huse et al., Science 246: 1275, 1989 and Ward et al., Nature 341: 544, 1989, all incorporated herein by reference.

Often, the functional heterologous proteins of E. coli or other bacteria are isolated from the inclusion bodies and require solubilization using potent denaturants and subsequent withdrawal. During the solubilization step, as is known in the art, a reducing agent must be present to separate the disulfide bonds. An example of buffer solution with a reducing agent is the following: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). The reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in a reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, incorporated herein by reference, and especially as described in Buchner et al., supra.

Renaturation is typically performed by dilution (for example, 100 times) of the denatured and reduced protein in refolding buffer solution. An example of a buffer solution is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

As a modification of the double chain antibody purification protocol, the heavy and light chain regions are solubilized and reduced separately and then combined in the refolding solution. An example of yield is obtained when these two proteins are mixed in a molar ratio so that a 5-fold molar excess of one protein is not exceeded with respect to the other. It is recommended to add excess oxidized glutathione or other low molecular weight oxidizing compounds to the refolding solution once the exchange-redox has been completed.

In addition to the recombinant methods, the antibodies disclosed herein can also be produced, in whole or in part, using standard peptide synthesis. Solid phase synthesis of polypeptides less than about 50 amino acids in length can be performed by joining the C-terminal amino acid of the sequence with an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. The techniques for solid phase synthesis are described in Barany &amp; Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol.

2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Longer proteins can be synthesized by condensation of the amino and carboxyl terms of the shorter fragments. Methods for forming peptide bonds by activation of a carboxy-terminal end (such as by using the coupling reagent N, N'-dicyclohexylcarbodiimide) are known in the art.

XII. Antigens

Some examples of antigens of interest include those listed below:

Table 1

Examples of antigens of interest (target antigens)

<dl><dt>Viral target antigens </dt><dd>Examples of target antigen sequences of the target antigens SEC. ID. :</dd></dl>

<dl><dt>Bk </dt><dd>TLYKKMEQDVKVAHQ GNLPLMRKAYLRKCK TFSRMKYNICMGKCI 1 22 23 </dd></dl>

<dl><dt>JC </dt><dd>SITEVECFL two </dd></dl>

<dl><dt>Epstein-Barr (EBV) </dt><dd>QPRAPIRPI 3 </dd></dl>

<dl><dt>cytomegalovirus (CMV) </dt><dd>NLVPMVATV 4 </dd></dl>

<dl><dt>HPV </dt><dd>YMLDLQPET (T) 5 </dd></dl>

<dl><dt>Influenza A </dt><dd>GILGFVFTL 6 </dd></dl>

<dl><dt>Target tumor antigens and their derived peptides </dt><dd /></dl>

<dl><dt>PRAME </dt><dd>LYVDSLFFL 7 </dd></dl>

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<dl><dt>WT1 </dt><dd>RMFPNAPYL 8 </dd></dl>

<dl><dt>Survivin </dt><dd>ELTLGEFLKL 9 </dd></dl>

<dl><dt>AFP </dt><dd>GVALQTMKQ 10 </dd></dl>

<dl><dt>ELF2M </dt><dd>ETVSEQSNV eleven </dd></dl>

<dl><dt>Proteinase 3 and its PR1 peptide </dt><dd>VLQELNVTV 12 </dd></dl>

<dl><dt>neutrophil elastase </dt><dd>VLQELNVTV 13 </dd></dl>

<dl><dt>MAGE </dt><dd>EADPTGHSY 14 </dd></dl>

<dl><dt>MART </dt><dd>AAGIGILTV fifteen </dd></dl>

<dl><dt>tyrosinase </dt><dd>RHRPLQEVYPEANAPIGHNRE 16 </dd></dl>

<dl><dt>GP100 </dt><dd>WNRQLYPEWTEAQRLD 17 </dd></dl>

<dl><dt>NY-That-1 </dt><dd>VLLKEFTVSG 18 </dd></dl>

<dl><dt>Herceptin </dt><dd>KIFGSLAFL 19 </dd></dl>

<dl><dt>carcinoembryonic antigen (CEA) </dt><dd>HLFGYSWYK twenty </dd></dl>

<dl><dt>PSA </dt><dd>FLTPKKLQCV twenty-one </dd></dl>

<dl><dt>Fungal target antigen </dt><dd /></dl>

<dl><dt>Blastomyces dermatitidis </dt><dd>CELDNSHEDYNWNLWFKWCSGHGR TGHGKHFYDCDWDPSHGDYSWYLW DPSHGDYSWYLWDYLCGNGHHPYD DYLCGNGHHPYDCELDNSHEDYSW DPYNCDWDPYHEKEKYDWDLWNKWCY KY 24 25 26 27 28 29 </dd></dl>

Table 2 Examples of tumors and their tumor antigens

<dl><dt>Tumor </dt><dd>Target antigens associated with the tumor </dd></dl>

<dl><dt>Acute myelogenous leukemia </dt><dd>Wilms 1 tumor (WT1), preferably expressed melanoma antigen (PRAME), PR1, proteinase 3, elastase cathepsin G </dd></dl>

<dl><dt>Chronic Myelogenous Leukemia </dt><dd>WT1, PRAME, PR1, proteinase 3, elastase, cathepsin G </dd></dl>

<dl><dt>Myelodysplastic Syndrome </dt><dd>WT1, PRAME, PR1, proteinase 3, elastase, cathepsin G </dd></dl>

<dl><dt>Acute lymphoblastic leukemia </dt><dd>PRAME </dd></dl>

<dl><dt>Chronic lymphocytic leukemia </dt><dd>Survivor </dd></dl>

<dl><dt>Non-Hodgkin lymphoma </dt><dd>Survivor </dd></dl>

<dl><dt>Multiple myeloma </dt><dd>Esophageal New York 1 (NY-That-1) </dd></dl>

<dl><dt>Malignant melanoma </dt><dd>MAGE, MART, Tyrosinase, PRAME GP100 </dd></dl>

<dl><dt>Breast cancer </dt><dd>WT1, Herceptin </dd></dl>

<dl><dt>Lung cancer </dt><dd>WT1 </dd></dl>

<dl><dt>Prostate cancer </dt><dd>Prostate specific antigen (PSA) </dd></dl>

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