A method for quantitatively detecting the content and activity of RIPK1 based on single molecule immunology technology

Abstract

The present application relates to a kind of for detecting the standard sample of RIPK1 content and activity, the standard sample can specifically recognize RIPK1 and phosphorylated RIPK1.The standard sample of the present application has excellent stability in the application of various antibodies, different blockers and various dyes, and also shows excellent stability and sensitivity in actual sample (serum blood sample), can meet the detection needs of low concentration sample, and can respond to a variety of detection scenarios.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry and molecular biology, specifically to a method for quantitatively detecting the content and activity of RIPK1 based on single-molecule immunoassay. Background Technology

[0002] Receptor-interacting serine / threonine-protein kinase 1 (RIPK1) is a key regulatory protein that regulates physiological processes such as apoptosis, necrosis, and inflammation. RIPK1 has a molecular weight of approximately 76 kDa and contains an N-terminal kinase domain (KD), a C-terminal death domain (DD), and an intermediate domain.

[0003] The physiological and pathological functions of RIPK1 have been extensively studied in the tumor necrosis factor α (TNFα) signaling pathway. After TNFα binds to its receptor TNFR1, it recruits downstream signaling molecules such as TRADD and RIPK1, thereby activating a series of complex cascade reactions. Ubiquitinated RIPK1 acts as a scaffold to recruit downstream IKKα / β-NEMO and TAK1-TAB2 / 3 complexes, activating downstream mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling, promoting inflammation. Furthermore, activated RIPK1 can form apoptosis or necrosis body complexes with FADD or RIPK3, thereby inducing apoptosis or necrosis. Phosphorylation at RIPK1S166 (pRIPK1) is a marker of RIPK1 kinase activation.

[0004] Numerous studies have demonstrated that RIPK1 levels and activity play a crucial role in the pathological processes of various diseases, including autoimmune diseases and neurodegenerative diseases. In animal models, extensive research has also shown that inhibiting RIPK1 kinase activity can alleviate the pathological progression of related diseases. Currently, multiple clinical trials are underway for RIPK1 kinase inhibitors to explore their broad potential applications in treating a range of human diseases.

[0005] Currently, there is a lack of a rapid and accurate method for quantifying RIPK1 content and kinase activity. Summary of the Invention

[0006] The purpose of this invention is to provide a standard for the quantitative detection of RIPK1 content and activity.

[0007] Another object of the present invention is to provide a method for quantitatively detecting the content and activity of RIPK1.

[0008] In a first aspect, the present invention provides a standard, the standard comprising a first standard comprising a polypeptide as shown in formula (I):

[0009] Z1-L1-Z2-Z3 (I)

[0010] In the formula,

[0011] Z1 is the amino acid sequence that specifically recognizes the pRIPK1 antibody, containing FKMW-{pS}-KLNN;

[0012] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0013] Z2 is an unlabeled or untagged sequence;

[0014] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8.

[0015] In another preferred embodiment, the standard further includes a second standard comprising a polypeptide as shown in formula (II):

[0016] Z4-L1-Z2-Z3 (II)

[0017] In the formula,

[0018] Z4 has the amino acid sequence shown in SEQ ID NO:1;

[0019] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0020] Z2 is an unlabeled or untagged sequence;

[0021] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8.

[0022] In another preferred embodiment, Z1 has an amino acid sequence as shown in SEQ ID NO: 2.

[0023] In another preferred embodiment, the tag sequence is selected from the group consisting of His6 tag, GST tag, MBP tag, FLAG tag, HA tag, and Myc tag.

[0024] In another preferred embodiment, the first standard comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO:6.

[0025] In another preferred embodiment, the second standard comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO:5.

[0026] In another preferred embodiment, the standard is used to prepare RIPK1 detection reagents, detection plates, or kits.

[0027] In another preferred embodiment, the standard further includes a solid support on which the polypeptide is modified.

[0028] In another preferred embodiment, the solid support is selected from the group consisting of magnetic beads, membrane supports, resins, gels, and fluorescent microspheres.

[0029] In a second aspect, the present invention provides a reagent kit comprising standards as described in the first aspect of the present invention.

[0030] In another preferred embodiment, the kit includes a first standard and a second standard, the first and second standards being defined as described in the first aspect of the invention.

[0031] In another preferred embodiment, the first standard and the second standard are located in the same or different containers.

[0032] In a third aspect, the invention provides the use of the standard as described in the first aspect or the kit as described in the second aspect for detecting RIPK1 or Pripk1.

[0033] In a fourth aspect, the present invention provides a polypeptide conjugate comprising:

[0034] (a) The polypeptide portion, said polypeptide being as shown in formula (I) or formula (II):

[0035] Z1-L1-Z2-Z3(I)

[0036] In the formula,

[0037] Z1 is the amino acid sequence that specifically recognizes the pRIPK1 antibody, and its sequence contains FKMW-{pS}-KLNN;

[0038] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0039] Z2 is an unlabeled or untagged sequence;

[0040] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8;

[0041] Z4-L1-Z2-Z3(II)

[0042] In the formula,

[0043] Z4 has the amino acid sequence shown in SEQ ID NO:1;

[0044] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0045] Z2 is an unlabeled or untagged sequence;

[0046] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8;

[0047] and

[0048] (b) A conjugate selected from the group consisting of: detectable markers, therapeutic agents, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0049] In another preferred embodiment, the (a) portion is coupled to the coupling portion by a chemical bond or a connector.

[0050] In another preferred embodiment, the radionuclide includes a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof.

[0051] In another preferred embodiment, the coupling portion is a detectable marker.

[0052] In another preferred embodiment, the detectable marker is a chemical marker, a biological marker, or a combination thereof.

[0053] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0054] In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.

[0055] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles.

[0056] In another preferred embodiment, the polypeptide conjugate is modified on a solid support.

[0057] In another preferred embodiment, the solid support is selected from the group consisting of magnetic beads, membrane supports, resins, gels, and fluorescent microspheres.

[0058] In a fifth aspect, the present invention provides a method for detecting p-RIPK1, comprising the steps of:

[0059] The sample to be tested is contacted with a peptide in the first standard of the kit described in the first aspect of the present invention or the peptide conjugate described in the kit described in the second aspect of the present invention, or the peptide conjugate described in the fourth aspect of the present invention, and the formation of a peptide p-RIPK1 complex is detected, wherein the formation of a complex indicates the presence of p-RIPK1 in the sample to be tested.

[0060] In another preferred embodiment, the method further includes the step of:

[0061] The sample to be tested is contacted with a peptide in the standard as described in the first aspect of the present invention or the second standard in the kit as described in the second aspect of the present invention, or the peptide conjugate as described in the fourth aspect of the present invention, and the formation of a peptide RIPK1 complex is detected, wherein the formation of a complex indicates the presence of RIPK1 in the sample to be tested.

[0062] In another preferred embodiment, the polypeptide or polypeptide conjugate further includes a detectable marker.

[0063] In another preferred embodiment, the detectable marker includes fluorescein.

[0064] In another preferred embodiment, the method includes the steps of:

[0065] (s1) Contact the sample to be tested with a standard as described in the first aspect of the present invention or a first standard in the kit described in the second aspect of the present invention;

[0066] (s2) Contact the sample to be tested with a standard as described in the first aspect of the present invention or a second standard in the kit described in the second aspect of the present invention;

[0067] Steps (s1) and (s2) can be performed in any order.

[0068] In another preferred embodiment, the method further includes the step of detecting the formation of the standard with RIPK1 or p-RIPK1 by means of a fluorescence signal.

[0069] In another preferred embodiment, the detected fluorescence signal is converted into the content of RIPK1 or p-RIPK1 according to a standard curve.

[0070] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0071] In another preferred embodiment, the sample to be tested includes serum, plasma, or a combination thereof, preferably a human serum or plasma sample.

[0072] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0073] Figure 1 A linear graph comparing different magnetic beads for Human Total RIPK1 is displayed.

[0074] Figure 2 A linear graph comparing different magnetic beads of Human p-RIPK1 is shown.

[0075] Figure 3 A linear graph comparing different blockers of Human total RIPK1 is shown.

[0076] Figure 4 Linear graphs comparing the effects of Human p-RIPK1 antibody on different blocking agents are shown.

[0077] Figure 5 A linear graph comparing the Human total-RIPK1 antibody against different dyes is shown.

[0078] Figure 6 A linear graph comparing different antigen dilutions for Human total-RIPK1 is shown.

[0079] Figure 7 A linear graph comparing different antigen dilutions of Human p-RIPK is shown. Detailed Implementation

[0080] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a standard for the quantitative detection of RIPK1 content and activity. This standard specifically recognizes RIPK1 and phosphorylated RIPK1. Experiments show that the standard of this invention exhibits excellent stability in applications with various antibodies, different blocking agents, and various dyes, and also demonstrates excellent stability and sensitivity in actual samples (serum blood samples). The detection limit for p-RIPK1 using the standard of this invention is as low as 1 pg / ml, and the cross-reactivity with other related proteins is less than 1%. Based on these findings, this invention was completed.

[0081] the term

[0082] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0083] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0084] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0085] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0086] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0087] SortA protein

[0088] Sortase A (SrtA) is a transpeptidase derived from Gram-positive bacteria. The SrtA transpeptidase from Staphylococcus aureus is the most widely used. This enzyme recognizes the LPXTGG sequence of a protein (where X is any amino acid residue), cleaves the peptide bond between threonine and glycine residues to form an enzyme-substrate intermediate, and then the oligoglycine peptide bridge located in peptidoglycan attacks the intermediate, forming a new peptide bond with the C-terminus of the target protein, thereby anchoring the target protein to the cell wall surface. Its mechanism of action involves recognizing the LPXTG motif at the C-terminus of the donor protein and cleaving it between threonine (T) and glycine (G), subsequently linking the C-terminus of the donor protein to the oligoglycine sequence (e.g., GGG) at the N-terminus of the recipient protein.

[0089] RIPK1 protein

[0090] RIPK1 (Receptor-Interacting Protein Kinase 1) is a serine / threonine kinase whose amino acid sequence contains multiple functional domains, such as the N-terminal kinase domain, the intermediate domain, and the C-terminal death domain (DD). The kinase domain is responsible for catalytic activity; the intermediate domain participates in protein-protein interactions; and the death domain (DD) mediates interactions with other proteins that have death domains.

[0091] RIPK1 plays a key role in cell survival, apoptosis, and necroptosis: Cell survival: It promotes cell survival by activating the NF-κB pathway; Cell apoptosis: Under certain conditions, it participates in caspase-8-mediated apoptosis; Necropsy: It forms a complex with RIPK3 and MLKL, triggering necroptosis.

[0092] Phosphorylation regulates the functions of RIPK1: 1. Kinase activity: Phosphorylation can activate or inhibit its kinase activity. 2. Protein-protein interactions: Phosphorylation affects interactions with other proteins. 3. Cell fate determination: The phosphorylation state determines whether a cell survives, undergoes apoptosis, or undergoes necroptosis.

[0093] The standard of the present invention

[0094] The standard of the present invention includes a first standard, the first standard comprising a polypeptide as shown in formula (I):

[0095] Z1-L1-Z2-Z3(I)

[0096] In the formula,

[0097] Z1 is the amino acid sequence that specifically recognizes the pRIPK1 antibody, containing FKMW-{pS}-KLNN;

[0098] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0099] Z2 is an unlabeled or untagged sequence;

[0100] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8.

[0101] In a preferred embodiment, the standard further includes a second standard comprising a polypeptide as shown in formula (II):

[0102] Z4-L1-Z2-Z3(II)

[0103] In the formula,

[0104] Z4 has the amino acid sequence shown in SEQ ID NO:1;

[0105] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0106] Z2 is an unlabeled or untagged sequence;

[0107] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8.

[0108] In a preferred embodiment, Z1 has the amino acid sequence shown in SEQ ID NO: 2. In a preferred embodiment, the tag sequence is selected from the group consisting of His6 tag, GST tag, MBP tag, FLAG tag, HA tag, and Myc tag.

[0109] In a preferred embodiment, the first standard comprises a polypeptide with the amino acid sequence shown in SEQ ID NO:6. In a preferred embodiment, the second standard comprises a polypeptide with the amino acid sequence shown in SEQ ID NO:5.

[0110] The reagent kit of the present invention

[0111] The kit of the present invention includes the standards of the present invention. In a preferred embodiment, the kit includes a first standard and a second standard, the first and second standards being defined as described in the first aspect of the present invention.

[0112] In a preferred embodiment, the first standard and the second standard are located in the same or different containers.

[0113] The polypeptide conjugate of the present invention

[0114] The polypeptide conjugate of the present invention contains:

[0115] (a) The polypeptide portion, said polypeptide being as shown in formula (I) or formula (II):

[0116] Z1-L1-Z2-Z3(I)

[0117] In the formula,

[0118] Z1 is the amino acid sequence that specifically recognizes the pRIPK1 antibody, and its sequence contains FKMW-{pS}-KLNN;

[0119] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0120] Z2 is an unlabeled or untagged sequence;

[0121] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8;

[0122] Z4-L1-Z2-Z3(II)

[0123] In the formula,

[0124] Z4 has the amino acid sequence shown in SEQ ID NO:1;

[0125] L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue);

[0126] Z2 is an unlabeled or untagged sequence;

[0127] Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8;

[0128] and

[0129] (b) A conjugate selected from the group consisting of: detectable markers, therapeutic agents, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0130] In a preferred embodiment, the (a) portion is coupled to the coupling portion via chemical bonds or a connector.

[0131] In a preferred embodiment, the coupling portion is a detectable marker.

[0132] The main advantages of this invention include:

[0133] 1. The standards of the present invention have good specificity and stability, and the cross-reactivity rate with other related proteins is less than 1%, which can cope with a variety of detection scenarios.

[0134] 2. The method of the present invention has high sensitivity and can detect p-RIPK1 as low as 1 pg / ml, which can meet the detection requirements of low concentration samples.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0136] method

[0137] 1. Synthetic RIPK1 and pRIPK1 detection standards

[0138] Because the full-length RIPK1 protein contains an intermediate domain, it is prone to aggregation and precipitation during protein purification, making it impossible to obtain the complete full-length RIPK1 protein. Furthermore, the fully phosphorylated RIPK1 protein at the s166 site is also difficult to prepare. To achieve absolute quantification of RIPK1 and its phosphorylated form at the s166 site (pRIPK1), we designed and synthesized corresponding detection standards based on the specific recognition sites of RIPK1 and pRIPK1 antibodies at the KD domain. Since the detection method is based on a double-layer sandwich assay, we also utilized an antibody that recognizes the RIPK1 DD domain to establish the method. The wild-type DD domain is prone to aggregation; therefore, we expressed a mutant DD protein that can be recognized by the DD antibody but does not aggregate. We used Sortase A transpeptidase to couple the DD protein with the synthesized KD domain peptide to obtain the detection standards.

[0139] a. Human total RIPK1 peptide:

[0140] AIFANKEPYENAICEQQLIMCIKSGNRPDVDDITEYCPREIISLMKLCWELPE TGG(SEQ ID NO:11)

[0141] b. Human pRIPK1 peptide:

[0142] KPENILVDNDFHIKIADLLGLASFKMW-{pS}-KLNNEEHNELREVDGTALPET GG(SEQ ID NO:12)

[0143] cEcoli expresses the RIPK1-DD domain

[0144] RIPK1 DD protein sequence:

[0145] GGSTNTNFKEEPAAKYQAIFDNTTSLTDKHLDPIRENLGKHWKNCARKLG FTQSQIDEIDHDYERDGLKEKVYQMLQKWVMREGIKGATVGKLAQALHQCSRI DLLSSLIYVS(SEQ ID NO:13)

[0146] The RIPK1 DD sequence was constructed in a PET vector, expressed and purified in Ecoli, and the expressed bacterial culture was transferred from a 16°C shaker to a centrifuge bottle. The centrifuge was balanced and centrifuged at 4000 rpm for 30 min at 4°C. 20 ml of PBS was prepared, and PMSF / protease inhibitor was added at a 1:100 ratio. The mixture was then cooled on ice. After centrifugation of the bacterial culture, remove the supernatant and resuspend the precipitate in pre-cooled PBS containing inhibitors in a 50ml centrifuge tube. Place the tube on ice and sonicate for 5-10 minutes, then centrifuge at 12000 rpm and 4 degrees Celsius for 30 minutes. After centrifugation, pour the supernatant into a nickel column (His column) and react for 30 minutes to 1 hour, stirring constantly to ensure the protein in the supernatant binds fully to the His beads. After binding, filter out the supernatant and wash the beads with binding buffer for 2-3 column volumes to remove impurities. Then soak the beads in 5ml of Elute buffer for 5-10 minutes to wash the protein off the beads. Collect the Elute buffer and concentrate it to 1ml using a 10KD concentrator. Filter the solution through a 0.22um filter membrane and purify the protein using a molecular sieve column. Aliquot the protein, name it pet-RIPK1 DD, and flash freeze it in liquid nitrogen at -80 degrees Celsius.

[0147] dEcoli expresses Sortase A (SrtA) protein

[0148] Sortase A (SrtA) is a transpeptidase derived from Gram-positive bacteria. The SrtA transpeptidase from Staphylococcus aureus is the most widely used. This enzyme recognizes proteins... LPXTGG The sequence (X is any amino acid residue) is used to cleave the peptide bond between threonine and glycine residues to form an enzyme-substrate intermediate. Subsequently, the oligoglycine peptide bridge located in peptidoglycan attacks the intermediate and forms a new peptide bond with the C-terminus of the target protein, thereby anchoring the target protein on the cell wall surface.

[0149] Sortase A protein sequence:

[0150] QASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK(SEQ ID NO:4)

[0151] To link the synthesized peptide with RIPK1 DD, we constructed the Sortase A protein sequence into the pET28a vector and expressed and purified it using Ecoli.

[0152] The final Human Total RIPK1 standard sequence obtained:

[0153] AIFANKEPYENAICEQQLIMCIKSGNRPDVDDITEYCPREIISLMKLCWELPE T-GGGKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLA EITPDKAFQDKLYPFTWDAVRYNGKLIAYP-TNTNFKEEPAAKYQAIFDNTTSLT DKHLDPIRENLGKHWKNCAEKLGFTQSQIDEIDHDYERDGLKEKVYQMLQKWVMREGIKGATVGKLAQALHQCSRIDLLSSLIYVSQN(SEQ ID NO:5)

[0154] Human pRIPK1 standard sequence:

[0155] KPENILVDNDFHIKIADLGLASFKMW-{pS}-KLNNEEHNELREVDGTALPET-GGGKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAE ITPDKAFQDKLYPFTWDAVRYNGKLIAYP-TNTNFKEEPAAKYQAIFDNTTSLTD KHLDPIRENLGKHWKNCAEKLGFTQSQIDEIDHDYERDGLKEKVYQMLQKWV MREGIKGATVGKLAQALHQCSRIDLLSSLIYVSQN(SEQ ID NO:6)

[0156] After peptide synthesis, the peptides were dissolved in 1×TBS solution. One tube of pet-RIPK1 DD protein was taken from -80°C, with 20 μL used as a control for subsequent experiments. The remaining peptides and Sortase A (SrtA) ligase were added at a 1:10 ratio, followed by 10 mM CaCl2, and incubated at room temperature for one day for ligation. After ligation, Coomassie brilliant blue staining was used to distinguish the control and experimental groups. A shift in the molecular weight of the ligated peptide indicated successful ligation. The protein was then purified using a molecular sieve to remove unligated enzymes and other proteins. The purified protein was collected as a protein standard and flash-frozen in liquid nitrogen at -80°C.

[0157] 2. Capture antibody biotin labeling

[0158] We are preparing to develop our own RIPK1 DD capture antibody using SiteClick. TM The Biotin Antibody Labeling Kit (Invitrogen, S20033) was used to biotinylate RIPK1 DD capture antibody, which was then added to glycerol and stored at -20°C. The purified antibody concentration was 0.5 mg / ml.

[0159] 3. ELISA method to validate detection of antibody pairs

[0160] Prepare 96-well plates. Dilute streptavidin to 1 μg / ml with coating buffer, then coat each well with 100 μL / well. Incubate overnight at 37°C and 450 rpm. Wash wells 2-3 times with 5‰ Tween 20 solution. Add 200 μL / well of 2% BSA prepared in PBS, incubate at 37°C and 450 rpm for 1 hour, and wash wells 2-3 times with 5‰ Tween 20 solution. Dilute biotin-labeled DD antibody 1:1000 with BSA prepared in 1% PBS and add 100 μL / well to each well. Incubate at 37°C and 450 rpm for 1 hour, and wash wells 2-3 times with 5‰ Tween 20 solution. Dilute protein samples with PBS to 0.2 mg / ml, 0.1 mg / ml, 0.01 mg / ml, 0.001 mg / ml, and 0.0001 mg / ml, respectively. Add 100 μL / well to each well and incubate at 37°C and 450 rpm. After 1 hour, wash the wells 2-3 times with 5‰ Tween 20 solution; dilute the paired antibody with BSA solution prepared in 1% PBS at a ratio of 1:300, add 100 μL / well to the plate, incubate at 37°C and 450 rpm for 1 hour, and wash the wells 2-3 times with 5‰ Tween 20 solution; dilute the enzyme-labeled secondary antibody with BSA solution prepared in 1% PBS at a ratio of 1:10000, add 100 μL / well to the plate, incubate at 37°C and 450 rpm for 30 minutes, and wash the wells 2-3 times with 5‰ Tween 20 solution; add the chromogenic substrate at 100 μL / well to the plate, incubate at 37°C and 450 rpm for 15 minutes, and add the stop solution at 50 μL / well. Read the values ​​immediately using an ELISA450 microplate reader.

[0161] 4. Single-molecule immunofluorescence

[0162] A kit for detecting RIPK1 protein includes magnetic beads coated with capture antibodies and detection antibodies labeled with fluorescent dyes, wherein the capture antibodies and detection antibodies can bind to different sites of the RIPK1 protein, forming a sandwich structure.

[0163] The magnetic beads have a diameter of 0.3–5 μm, and their working concentration is 0.01 mg / mL or higher and less than 0.5 mg / mL. Preferably, the diameter of the magnetic beads is 1–2.8 μm. The working concentration of the magnetic beads is preferably 0.1–0.2 mg / mL. The surface of the magnetic beads is modified with one or more of carboxyl, amino, or toluenesulfonyl groups, or the surface of the magnetic beads is modified with streptavidin protein, preferably toluenesulfonyl groups and surface modification with streptavidin protein.

[0164] The magnetic beads were coated with a coating solution at a concentration of 20 μg antibody / mg magnetic beads, resulting in a stock solution concentration of 10 mg / ml. The solution was stored at 4°C. During the experiment, the magnetic beads were diluted 100-fold to 0.1 mg / ml using magnetic bead dilution buffer. The coated RIPK1 DD capture antibody was diluted in 30 mM PBS. The dye-labeled detection antibody was diluted in 30 mM PBS containing 0.65% NaCl, 5% trehalose, 4% BSA, 0.1% Tween 20, and 0.05% PC300. The antigen was diluted to different concentrations using antigen dilution buffer. 25 μL of magnetic bead coating solution + 25 μL of diluted antigen were incubated at 37°C for 6 min. Then, 10 μL of detection antibody was added, and the mixture was incubated at 37°C for 4 min before washing. The results were then read using a single-molecule immunofluorescence instrument.

[0165] result

[0166] 1. ELISA method to validate detection of antibody pairs

[0167] Our first step was to use commercially available antibodies to validate the reliability of the detection standards. The commercially available antibodies were RIPK1 antibody (CST, 3493s) and pRIPK1 antibody (CST, 44590S, S166 site). We performed a double-antibody sandwich ELISA experiment using a self-developed antibody that recognizes the RIPK1 DD domain. 293T cells were overexpressed with the full-length Human RIPK1 plasmid. Lysate was used as a positive control, and PBS as a negative control. The RIPK1 DD capture antibody concentration was 0.5 mg / ml, and it was used at a 1:1000 dilution. The results are as follows:

[0168] Table 1a Pairing of RIPK1 DD capture antibodies with commercially available total RIPK1 detection antibodies

[0169] Positive control negative control 200ug / ml 100ug / ml 10ug / ml 1ug / ml 0.1ug / ml RIPK1 1.19 0.112 3.171 3.126 3.091 3.085 2.645

[0170] Table 1b Pairing of RIPK1 DD capture antibody with commercial p-RIPK1 detection antibody

[0171] Positive control negative control 200ug / ml 100ug / ml 10ug / ml 1ug / ml 0.1ug / ml p-RIPK1 1.089 0.142 3.165 3.151 3.143 2.859 1.642

[0172] The results in the table show that all commercially available antibodies can pair with DD antibodies and can be measured using ELISA. According to the experimental results, the lowest protein concentration in this experiment was 0.1 μg / ml, with all values ​​being high. These values ​​represent the lowest protein concentration detectable by the total RIPK1 and p-RIPK1 microplate readers; therefore, the protein concentration was further diluted from 0.1 μg / ml for measurement. High-concentration standards were used as positive controls in this experiment, and PBS was used as a negative control.

[0173] Table 1 shows the minimum concentration of total RIPK1 protein standard detected by the microplate reader.

[0174] Positive control negative control 100ng / ml 50ng / ml 25ng / ml 1ng / ml 0.1 ng / ml RIPK1 3.154 0.698 2.911 1..608 0.696 0.6 0.533

[0175] Table 1 shows the minimum concentration of p-RIPK1 protein standard detected by the ELISA reader.

[0176] Positive control negative control 100ng / ml 50ng / ml 25ng / ml 1ng / ml 0.1 ng / ml p-RIPK1 3.160 0.433 1.165 0.606 0.489 0.496 0.416

[0177] The two ELISA tests showed that, using commercially available antibodies paired with DD, different concentrations of the total RIPK1 protein standard could be detected, with the minimum detectable concentration range between 50 ng / ml and 25 ng / ml. Similarly, the minimum detectable concentration range for the p-RIPK protein standard was between 100 ng / ml and 50 ng / ml. This experiment demonstrates that the synthesized protein standards can be normally recognized by DD antibodies and either total RIPK1 or p-RIPK1 antibodies, further validating the feasibility of the protein standards.

[0178] 2 Comparison of different detection antibodies

[0179] To further investigate the reliability of our self-developed RIPK1 antibody and pRIPK1(s166) antibody in detecting RIPK1 content and activity, we tested two human RIPK1 antibodies (5-8 and 5-10) and five human pRIPK1(s166) antibodies (YJY-10-1, YJY-10-2, YJY-10-3, YJY-10-4, and YJY-10-5) developed in our laboratory. All capture antibodies were RIPK1 DD antibodies.

[0180] The ELISA results are as follows:

[0181] Table 2a Comparison of two different total RIPK1 detection antibodies

[0182] Positive control negative control 100ng / ml 50ng / ml 25ng / ml 1ng / ml 0.1 ng / ml 5-8 3.054 0.753 1.358 1.588 0.654 0.708 0.623 5-10 0.868 0.526 0.989 0.754 0.656 0.653 0.545

[0183] The results in the table show that the 5-8 human total RIPK1 antibody developed by the laboratory can successfully pair with RIPK1 DD and successfully detect protein standards of different concentrations. Therefore, 5-8 was chosen to be used for total RIPK1 detection in later experiments.

[0184] Table 2b Comparison of 5 different p-RIPK1 detection antibodies

[0185] Positive control negative control 100ng / ml 50ng / ml 25ng / ml 1ng / ml 0.1 ng / ml YJY-10-1 3.151 0.433 0.765 0.566 0.689 0.696 0.616 YJY-10-2 2.956 0.463 0.886 0.643 0.568 0.659 0.542 YJY-10-3 3.287 0.495 0.856 0.687 0.456 0.589 0.654 YJY-10-4 3.061 0.433 1.154 0.855 0.432 0.459 0.489 YJY-10-5 2.985 0.488 0.986 0.768 0.463 0.432 0.455

[0186] The results in the table show that the YJY-10-4 human p-RIPK1 antibody developed in the laboratory can successfully pair with RIPK1DD and successfully detect protein standards at different concentrations. Moreover, the effect is significantly better than the other four antibody pairs. Therefore, YJY-10-4 was chosen for p-RIPK1 detection in later experiments.

[0187] 3. Comparison of antibodies captured by different magnetic beads

[0188] To achieve better and more stable experimental results, we need to conduct experiments and optimize the process. Based on the double-antibody sandwich principle of ELISA, we first tested different magnetic bead coating processes for the capture antibody. Initially, we coated the capture antibody at a concentration of 20 μg antibody / mg magnetic beads, resulting in a stock solution concentration of 10 mg / mL. When using the magnetic beads, we used a 1:100 ratio, meaning the working solution concentration was 0.1 mg / mL.

[0189] For dye-labeled detection antibodies, use 200 μg antibody / ml dye, and use the dye at a ratio of 1:10, i.e., 20 μg antibody / ml dye working solution.

[0190] Table 3a Comparison of different magnetic beads for Human total RIPK1

[0191]

[0192]

[0193] Table 3b Linear formula for comparing different magnetic beads in Human total RIPK1

[0194] Linear formula R-squared value <![CDATA[y1=26.037x+1044.2]]> <![CDATA[R1 2 =0.9991]]> <![CDATA[y2=71.985x+581.54]]> <![CDATA[R2 2 =0.9998]]> <![CDATA[y3=9.1212x+2597.4]]> <![CDATA[R3 2 =0.9928]]> <![CDATA[y4=68.54x+987.75]]> <![CDATA[R4 2 =1]]> <![CDATA[y5=75.487x+2851.6]]> <![CDATA[R5 2 =0.9989]]>

[0195] according to Figure 1 Tables 3a and 3b show the area results and linear formulas. Magnetic bead 4 has the best linearity. Therefore, in subsequent experiments, magnetic beads 4 were selected to be used as the coating of the human total RIPK1 antibody with the captured antibody.

[0196] Table 3c Comparison of different magnetic beads for Human p-RIPK1

[0197]

[0198] Table 3d Human p-RIPK1 Linear Formula for Comparison of Different Magnetic Beads

[0199] Linear formula R-squared value <![CDATA[y1=208.77x+159851]]> <![CDATA[R1 2 =0.9929]]> <![CDATA[y2=31.749x+208257]]> <![CDATA[R2 2 =0.9296]]> <![CDATA[y3=201.95x+707438]]> <![CDATA[R3 2 =0.9131]]>

[0200] according to Figure 2 The area results and linear formulas obtained from Tables 3c and 3d show that magnetic bead 1 has the best linearity. Therefore, in subsequent experiments, magnetic bead 1 was selected as the coating of the Human p-RIPK1 antibody with the capture antibody.

[0201] 4. Comparison of different blocking agents

[0202] In the development of in vitro diagnostic reagents, to reduce the interference from proteins in patient samples or other factors during the testing process, and to minimize false positives and false negatives, we need to add blocking agents to the assay. Proper use of blocking agents also helps improve the sensitivity and specificity of the test. These blocking agents effectively reduce non-specific binding, increasing the signal-to-noise ratio and improving the clinical usability of the diagnostic reagents. Therefore, we compared different blocking agents to achieve better experimental results.

[0203] Table 4a): Comparison of different blockers of Human total-RIPK1

[0204]

[0205] Table 4b Linear formula for comparing different blockers of human total RIPK1

[0206] Linear formula R-squared value <![CDATA[y1=18.848x+4116.8]]> <![CDATA[R1 2 =0.9996]]> <![CDATA[y2=14.06x+3274.9]]> <![CDATA[R2 2 =0.9994]]> <![CDATA[y3=14.299x+3224.1]]> <![CDATA[R3 2 =0.9981]]> <![CDATA[y4=14.629x+2869.4]]> <![CDATA[R4 2 =0.9996]]>

[0207] according to Figure 3 The area results and linear formulas obtained in Tables 4a and 4b show that the effect of blocking agent 4 is the best. Therefore, blocking agent 4 will be used in subsequent experiments with the Human total RIPK1 antibody.

[0208] Table 4c Comparison of different human p-RIPK1 blockers

[0209]

[0210] Table 4. Linearity formulas for human p-RIPK1 antibody against different blocking agents.

[0211] Linear formula R-squared value <![CDATA[y1=672.83x+139052]]> <![CDATA[R1 2 =0.9989]]> <![CDATA[y2=175.5x+3657.2]]> <![CDATA[R2 2 =0.9836]]> <![CDATA[y3=547.03x+100462]]> <![CDATA[R3 2 =0.9942]]> <![CDATA[y4=278.21x+48840]]> <![CDATA[R4 2 =0.9998]]> <![CDATA[y5=870.84x+210742]]> <![CDATA[R5 2 =0.9942]]>

[0212] According to Tables 4c, d and Figure 4 Based on the obtained area results and linear formula, the effect of blocker 4 was the best. Therefore, blocker 4 was used in subsequent experiments with the Human p-RIPK1 antibody.

[0213] 5. Comparison of different dyes

[0214] Based on the principle of fluorescence detection in semi-detection, we need to directly label the detection antibody with fluorescence. In order to detect a more stable binding between the antibody and the fluorescence and obtain better detection results, we selected four different fluorescent dyes to label the Humantotal-RIPK1 detection antibody.

[0215] Table 5a Comparison of Human total-RIPK1 antibody against different dyes

[0216]

[0217] Table 5b Linearity formulas for Human total-RIPK1 antibody against different dyes

[0218] Linear formula R-squared value <![CDATA[y1=59.445x+1064.5]]> <![CDATA[R1 2 =0.9983]]> <![CDATA[y2=54.351x+375.68]]> <![CDATA[R2 2 =0.9998]]> <![CDATA[y 1-2 =9.1212x+2597.4]]> <![CDATA[R 1-2 2 =0.9928]]> <![CDATA[y 2-2 =41.955x+191.98]]> <![CDATA[R 2-2 2 =0.9997]]>

[0219] according to Figure 5 As shown in Tables 5a and 5b, the results of labeling with different fluorescent dyes show that dye 2 is more effective than the other fluorescent dyes. Therefore, fluorescent dye 2 was selected for labeling the antibody in subsequent experiments.

[0220] 6. Comparison of different antigen diluents

[0221] Different antigens possess different physical and chemical properties, such as molecular size, charge, and hydrophobicity, which affect the compatibility between the antigen and the diluent. Different experimental purposes also require different diluents. For example, ELISA experiments require diluents containing stabilizers and preservatives; impurities or interfering substances in the sample may need to be neutralized using specific diluent components. Therefore, we selected different antigen diluents for comparison during our experimental testing.

[0222] Table 6a Comparison of different antigen dilutions for Human total-RIPK1

[0223]

[0224]

[0225] Table 6b Comparison of linear formulas for different antigen dilutions of Human total-RIPK1

[0226] Linear formula R-squared value <![CDATA[y1=59.171x+1043.9]]> <![CDATA[R1 2 =0.9974]]> <![CDATA[y2=42.413x+470.88]]> <![CDATA[R2 2 =0.998]]> <![CDATA[y3=41.566x+879.78]]> <![CDATA[R3 2 =0.9966]]> <![CDATA[y4=69.637x-401.09]]> <![CDATA[R4 2 =0.9959]]> <![CDATA[y5=55.38x+2961.8]]> <![CDATA[R5 2 =0.998]]>

[0227] like Figure 6 As shown in Tables 6a and 6b, different antigen diluents were used for standard testing. Based on the conclusions of Tables 6a and 6b, antigen diluent 2 was found to be more effective than the other diluents. Therefore, antigen diluent 2 was selected to dilute Humantotal-RIPK1 standard in subsequent experiments.

[0228] Table 6c Comparison of different antigen dilutions for Human p-RIPK

[0229]

[0230] Table 6. Comparison of linear formulas for different antigen dilutions of Human p-RIPK.

[0231] Linear formula R-squared value <![CDATA[y1=243.94x+100752]]> <![CDATA[R1 2 =0.9762]]> <![CDATA[y2=153.15x+861.22]]> <![CDATA[R2 2 =0.9994]]> <![CDATA[y3=51.098x+51532]]> <![CDATA[R3 2 =0.9494]]> <![CDATA[y4=48.068x+104703]]> <![CDATA[R4 2 =0.6702]]> <![CDATA[y5=295.85x+213234]]> <![CDATA[R5 2 =0.9424]]>

[0232] like Figure 7 As shown in Tables 6c and 6d, different antigen diluents were used for standard testing. Based on the conclusions in Tables 6c and 6d, antigen diluent 2 was found to be more effective than the other diluents. Therefore, antigen diluent 2 was selected to dilute the Human p-RIPK1 standard in subsequent experiments.

[0233] Serum blood sample test

[0234] Based on the results of the experimental optimization, our self-developed antibody pair can correctly recognize our standard and performs well. To verify whether our antibody pair can be applied to clinical samples, we purchased some serum and plasma for testing, and the results are as follows:

[0235] Table 7a Human total RIPK1 plasma test

[0236]

[0237] Table 7b Human p-RIPK1 Plasma Testing

[0238]

[0239] Since the background levels of plasma and serum in this test were relatively low, which is suspected to be related to clinical samples, we chose to add different concentrations of standards to the plasma to verify its reliability.

[0240] Table 7c Human total-RIPK1 plasma antigen-added test

[0241]

[0242]

[0243] Table 7d Human p-RIPK1 plasma antigen-added test

[0244]

[0245] The results in Tables 7c and 7d show that our antibody pair and the developed experimental procedure can detect RIPK1 and phosphorylated RIPK1 in plasma, proving that the antibody pair was successfully screened and can be used in clinical samples.

[0246] in conclusion

[0247] Through systematic research and experimental verification, a method based on single-molecule immunoassay for detecting human RIPK1 and phosphorylated RIPK1 has been successfully developed for the detection of RIPK1 content and activity. This method exhibits excellent sensitivity; through multiple experimental verifications, the detection limit for RIPK1 reaches 5 pg / ml, and the detection limit for phosphorylated p-RIPK1 reaches 1 pg / ml, meeting the needs of clinical and research applications for detecting low-concentration samples. Furthermore, the method shows a cross-reactivity rate of less than 1% with other related proteins, demonstrating good specificity.

[0248] sequence list

[0249]

[0250]

[0251] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A standard product, characterized in that, The standard includes a first standard, which comprises a polypeptide as shown in formula (I): Z1-L1-Z2-Z3(I) In the formula, Z1 is the amino acid sequence that specifically recognizes the pRIPK1 antibody, containing FKMW-{pS}-KLNN; L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue); Z2 is an unlabeled or untagged sequence; Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8.

2. The standard product as described in claim 1, characterized in that, The standard also includes a second standard comprising a polypeptide as shown in formula (II): Z4-L1-Z2-Z3(II) In the formula, Z4 has the amino acid sequence shown in SEQ ID NO:1; L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue); Z2 is an unlabeled or untagged sequence; Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8.

3. The standard product as described in claim 1, characterized in that, Z1 has the amino acid sequence shown in SEQ ID NO:

2.

4. The standard product as described in claim 1 or 2, characterized in that, The tag sequence is selected from the following group: His6 tag, GST tag, MBP tag, FLAG tag, HA tag, and Myc tag.

5. The standard product as described in claim 1, characterized in that, The first standard comprises a polypeptide with the amino acid sequence shown in SEQ ID NO:

6.

6. The standard product as described in claim 2, characterized in that, The second standard contains a polypeptide with the amino acid sequence shown in SEQ ID NO:

5.

7. A reagent kit, characterized in that, The kit contains the standard as described in claim 1.

8. The use of the standard as described in claim 1 or the kit as described in claim 7, characterized in that, Used to detect RIPK1.

9. A polypeptide conjugate, characterized in that, The polypeptide conjugate contains: (a) The polypeptide portion, said polypeptide being as shown in formula (I) or formula (II): Z1-L1-Z2-Z3(I) In the formula, Z1 is the amino acid sequence that specifically recognizes the pRIPK1 antibody, and its sequence contains FKMW-{pS}-KLNN; L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue); Z2 is an unlabeled or untagged sequence; Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8; Z4-L1-Z2-Z3(II) In the formula, Z4 has the amino acid sequence shown in SEQ ID NO:1; L1 has the sequence shown in SEQ ID NO:10 (LPXTGGG, where X is any amino acid residue); Z2 is an unlabeled or untagged sequence; Z3 has an amino acid sequence as shown in SEQ ID NO:3 or 8; and (b) A conjugate selected from the group consisting of: detectable markers, therapeutic agents, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

10. A method for detecting RIPK1, characterized in that, Including the following steps: The sample to be tested is contacted with the peptide in the standard as described in claim 1 or the first standard in the kit as described in claim 7, or the peptide conjugate as described in claim 9, and the formation of the peptide p-RIPK1 complex is detected, wherein the formation of the complex indicates the presence of p-RIPK1 in the sample to be tested.

Images