CN117247449B

CN117247449B - Anti-P24 antibody, and reagent and kit for detecting P24

Info

Publication number: CN117247449B
Application number: CN202310667096.6A
Authority: CN
Inventors: 孟媛; 钟冬梅; 唐丽娜; 游辉; 姜瑢瑢
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2022-06-17
Filing date: 2023-06-06
Publication date: 2024-10-22
Anticipated expiration: 2043-06-06
Also published as: WO2023241416A1; CN117247449A

Abstract

The disclosure provides an anti-P24 antibody, a reagent and a kit for detecting P24, and relates to the technical field of antibodies. The anti-P24 antibodies provided by the present disclosure include a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for the detection of P24 and has improved affinity and activity.

Description

Anti-P24 antibody, and reagent and kit for detecting P24

Cross Reference to Related Applications

The present disclosure claims priority from chinese patent application serial No. 202210684577.3, entitled "anti-P24 antibody, reagents and kits for detecting P24" filed on month 17 2022, the entire contents of which are incorporated herein by reference.

Technical Field

The disclosure relates to the technical field of antibodies, and in particular relates to an anti-P24 antibody, a reagent for detecting P24 and a kit.

Background

Human immunodeficiency virus (Human Immunodeficiency Virus, HIV), an AIDS virus (AIDS), also known as acquired immunodeficiency syndrome virus, is a virus responsible for the defects of the human immune system. It is a Lentivirus (Lentivirus) that infects cells of the human immune system, a retrovirus. HIV is the causative agent of aids, and is transmitted primarily through sexual contact, blood, and mother and infant. In recent years, the number of HIV-infected patients has been on the rise. While the current assay for serum HIV antibodies is a routine assay for diagnosing HIV infection, there are limitations to assaying HIV antibodies: more than 70% of HIV infected individuals will detect antibodies after 6 months of infection, and in the loving population, this figure is more than 80%, and the method of detecting antibodies increases the risk of HIV "window" transmission; in addition, the neonatal generation of antibodies requires 1 year after birth, HIV antibodies from the mother will cause the generation of false positive; because HIV antibodies persist during the course of the disease, they disappear only by the late stage of aids, and cannot be used as a stable indicator for therapeutic monitoring.

P24 is the major structural protein of HIV viral particles, the product of the structural gene GAG, playing an important role in the packaging and maturation process of the virus. The amino acid sequence of the P24 protein is highly conserved among HIV strains, and deletion of P24 results in abnormal viral assembly. The P24 protein has strong specificity and does not have cross reaction with most other retroviruses. The HIV is infected in human body, the first virus marker in the blood of the infected person is virus P24 protein, and a longer window period exists between the virus infection and the detection of HIV antibody, so that the detection of HIV-P24 antigen has played an important role in the early diagnosis of HIV infection, the prognosis of patients, the screening and evaluation of anti-HIV drugs, the discovery of maternal and infant transmission and other aspects.

The detection of HIV-1P24 antigen adopts serological diagnosis method, mainly includes double antibody sandwich ELISA method, immune complex cleavage detection method, ultrasensitive EIA method, enzyme-linked immunofluorescence method, etc. At present, a double-antibody sandwich method is generally used for detecting the P24 antigen of the human immunodeficiency virus, and the acquisition of an anti-P24 antibody is a key for realizing the double-antibody sandwich method detection. Thus, there is a strong need in the art for antibodies that efficiently bind to and detect P24.

Disclosure of Invention

The application provides an anti-P24 antibody with improved affinity and/or activity, so as to improve detection of P24 and provide an important raw material source for detection of P24.

The present disclosure provides an anti-P24 antibody or functional fragment thereof comprising three complementarity determining regions having a heavy chain variable region of amino acid sequence SEQ ID No. 17 and three complementarity determining regions having a light chain variable region of amino acid sequence SEQ ID No. 18.

Optionally, the complementarity determining regions are defined by any one system or combination of systems Kabat, chothia, IMGT, lesk, abM or contacts.

Optionally, the complementarity determining regions are defined by Kabat, chothia, IMGT or Lesk systems.

Optionally, the antibody or functional fragment thereof comprises the following complementarity determining regions:

HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1;

HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;

HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No. 3;

LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4;

LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5;

LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6.

The present disclosure provides an anti-P24 antibody or functional fragment thereof comprising an amino acid sequence such as

HCDR1, HCDR2, HCDR3 shown in SEQ ID NO. 1-3, and LCDR1, LCDR2, LCDR3 shown in SEQ ID NO. 4-6.

The disclosure provides an anti-P24 antibody or a functional fragment thereof, which comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO. 3 in sequence, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO.6 in sequence.

Optionally, the antibody or functional fragment thereof of any one of the above further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

The HFR1 comprises SEQ ID NO 7 or an amino acid sequence having at least 80% homology thereto;

The HFR2 comprises the amino acid sequence of SEQ ID NO 8 or at least 80% homology thereto;

the HFR3 comprises the amino acid sequence of SEQ ID NO 9 or at least 80% homology thereto;

the HFR4 comprises the amino acid sequence of SEQ ID NO 10 or having at least 80% homology thereto;

the LFR1 comprises SEQ ID No. 11 or an amino acid sequence having at least 80% homology thereto;

The LFR2 comprises the amino acid sequence of SEQ ID No. 12 or having at least 80% homology thereto;

The LFR3 comprises SEQ ID No. 13 or an amino acid sequence having at least 80% homology thereto;

The LFR4 comprises the amino acid sequence of SEQ ID No. 14 or having at least 80% homology thereto.

Optionally, the antibody or functional fragment thereof further comprises an HFR1, an HFR2, an HFR3, an HFR4 and an amino acid sequence shown in SEQ ID NO. 7 to SEQ ID NO. 10 in sequence, an LFR1, an LFR2, an LFR3 and an LFR4 shown in SEQ ID NO. 11 to SEQ ID NO. 14 in sequence, or an amino acid sequence having at least 80% identity with each of the sequences.

Alternatively, the antibody or functional fragment thereof of any one of the above binds P24 with an affinity of KD < 8.32 x 10 ^-10 M.

Alternatively, an antibody or functional fragment thereof according to any one of the preceding claims comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 17 and a light chain variable region having the amino acid sequence shown in SEQ ID NO. 18.

The present disclosure provides an anti-P24 antibody or functional fragment thereof comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above, wherein the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, HFR2, LFR3, LFR4 is the amino acid sequence of any one of HFR1, HFR2, LCDR3, LFR4, or any one of the above.

Alternatively, the heavy chain variable region amino acid sequence is shown in SEQ ID NO. 17.

Alternatively, the light chain variable region amino acid sequence is shown as SEQ ID NO. 18.

The present disclosure provides an anti-P24 antibody or functional fragment thereof comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID No. 17 and/or a light chain variable region; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18.

Optionally, the antibody or functional fragment thereof of any one of the above further comprises a constant region.

Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region.

Alternatively, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

Alternatively, the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin.

Alternatively, the constant region is of murine species origin.

Alternatively, the heavy chain constant region sequence is as shown in SEQ ID NO. 15 or at least 80% identical thereto.

Alternatively, the light chain constant region sequence is as shown in SEQ ID NO. 16 or at least 80% identical thereto.

Alternatively, the antibody or functional fragment thereof comprises a heavy chain as shown in SEQ ID NO. 19 and a light chain as shown in SEQ ID NO. 20.

Alternatively, the functional fragment of any one of the above is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.

The present disclosure provides an anti-P24 antibody comprising a heavy chain variable region as described in any one of the preceding claims and/or a light chain comprising a light chain variable region as described in any one of the preceding claims.

Alternatively, the amino acid sequence of the heavy chain is shown in SEQ ID NO. 19.

Alternatively, the amino acid sequence of the light chain is shown in SEQ ID NO. 20.

The disclosure provides an anti-P24 antibody, comprising a heavy chain and/or a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO. 19; the amino acid sequence of the light chain is shown as SEQ ID NO. 20.

The present disclosure provides an antibody conjugate comprising an antibody or functional fragment thereof as described in any one of the above.

Optionally, the antibody is conjugated to biotin or a biotin derivative.

Optionally, the antibody is coupled to a solid phase.

Optionally, the antibody is conjugated to a label.

Optionally, the label is selected from the group consisting of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

Optionally, the label is colloidal gold.

The present disclosure provides a reagent or kit for detecting P24, comprising an antibody or functional fragment thereof as described in any one of the above or an antibody conjugate as described above.

The present disclosure provides a method of detecting P24, comprising: contacting the antibody or functional fragment, antibody conjugate, reagent or kit of any of the above with P24 in the sample to be tested to form an immune complex.

Optionally, the immune complex further comprises a second antibody that binds to the antibody or a functional fragment thereof;

Optionally, the immune complex further comprises a second antibody, which binds to P24. The present disclosure also provides a nucleic acid encoding an antibody or functional fragment thereof as described in any one of the preceding claims.

The present disclosure also provides a vector comprising a nucleic acid as described above.

The present disclosure also provides a cell comprising a nucleic acid or vector as described above.

The present disclosure also provides a method of preparing an antibody or functional fragment thereof according to any one of the above, comprising: the cells described above are cultured.

The disclosure also provides the use of an antibody or functional fragment, antibody conjugate, reagent or kit of any of the above in the detection of P24, the preparation of a product for the detection of P24, the diagnosis of HIV infection.

The present disclosure also provides a method of diagnosing HIV infection in a subject comprising: a) Contacting the antibody or functional fragment, antibody conjugate, reagent or kit of any of the above with P24 in a sample from the subject to form an immune complex; and

B) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of HIV in the subject sample.

Optionally, in step a), a second antibody is also included in the immunocomplex, which second antibody binds to the antibody or a functional fragment thereof.

Optionally, in step a), a second antibody is further included in the immunocomplex, the second antibody binding to the P24.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present disclosure and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.

FIG. 1 shows the results of reducing SDS-PAGE of Anti-P24 5F11 mut.

Detailed Description

In the present disclosure, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.

In the present disclosure, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen binding fragment to an antigen or epitope recognized thereby. In particular embodiments of the present disclosure, CDRs refer to the highly variable regions of the heavy and light chains of an antibody.

In the present disclosure, the heavy chain complementarity determining region is denoted by "HCDR", which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by "LCDR" and include LCDR1, LCDR2 and LCDR3.

Embodiments of the present disclosure provide an anti-P24 antibody or functional fragment thereof comprising three complementarity determining regions having the heavy chain variable region of amino acid sequence SEQ ID No. 17 and three complementarity determining regions having the light chain variable region of amino acid sequence SEQ ID No. 18.

CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. The present disclosure uses Kabat annotation standards to identify CDR regions, but other methods to identify CDR regions are within the scope of the present disclosure.

Methods of CDR definition are well known in the art, and include: kabat definition, chothia definition, IMGT definition, contact definition, and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Still other CDR definition methods may not strictly follow one of the above schemes, but still overlap at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. Exemplary defined CDRs are listed in table 1 below, with slightly different definitions in the different documents. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to table 1 are also within the scope of the disclosure.

Table 1: CDR definition ¹

CDR	Kabat	AbM²	IMGT	Chothia
					HCDR1	H31～H35³	H26～H35³	H26～H33..5⁵	H26～H32..34⁴
HCDR2	H50～H65	H50～H58	H51～H57	H52～H56
					HCDR3	H95～H102	H95～H102	H93～H102	H95～H102
LCDR1	L24～L34	L24～L34	L27～L32	L24～L34
					LCDR2	L50～L56	L50～L56	L50～L51	L50～L56
LCDR3	L89～L97	L89～L97	L89～L97	L89～L97

¹ The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with the amino acid numbers on the heavy chain being indicated by "H+ numbers" and the amino acid numbers on the light chain being indicated by "L+ numbers". The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins of Immunological Interest" (1983).

² The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

³ If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if H35A and H35B are present at the same time, then CDR-H1 ends at position 35B.

⁴ If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if H35A and H35B are present at the same time, then CDR-H1 ends at position 34.

⁵ If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

In alternative embodiments, the complementarity determining regions are defined by any one or a combination of systems Kabat, chothia, IMGT, lesk, abM or contacts.

In an alternative embodiment, the complementarity determining regions are defined by the Kabat, chothia, IMGT, lesk system.

In an alternative embodiment, the complementarity determining regions are defined by the Kabat system.

In alternative embodiments, the complementarity determining regions are defined by the Chothia system.

In an alternative embodiment, the complementarity determining regions are defined by the IMGT system.

In an alternative embodiment, the complementarity determining regions are defined by the AbM system.

In an alternative embodiment, the complementarity determining regions are defined by the Contact system.

In alternative embodiments, the complementarity determining regions are defined by Kabat, chothia, IMGT, lesk, abM or a combination of Contact systems.

In an alternative embodiment, the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, chothia, abM or IMGT system above corresponds to the Kabat numbering positions as follows:

CDR	Kabat	AbM	IMGT	Chothia
					HCDR1	H31～H35	H26～H35	H26～H33	H26～H32
HCDR2	H50～H65	H50～H58	H51～H57	H52～H56
					HCDR3	H95～H102	H95～H102	H93～H102	H95～H102
LCDR1	L24～L34	L24～L34	L27～L32	L24～L34
					LCDR2	L50～L56	L50～L56	L50～L51	L50～L56
LCDR3	L89～L97	L89～L97	L89～L97	L89～L97

in alternative embodiments, the Kabat numbering positions corresponding to the amino acid sequence of HCDR1 are H31-H35, the Kabat numbering positions corresponding to the amino acid sequence of HCDR2 are H50-H65, the Kabat numbering positions corresponding to the amino acid sequence of HCDR3 are 95-H100D, the Kabat numbering positions corresponding to the amino acid sequence of LCDR1 are L24-L34, the Kabat numbering positions corresponding to the amino acid sequence of LCDR2 are L50-L56, and the Kabat numbering positions corresponding to the amino acid sequence of LCDR3 are L89-L95.

In an alternative embodiment, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of heavy chain variable region SEQ ID NO. 17 are shown in sequence as SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the antibody or functional fragment thereof are also shown in sequence as SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3.

In an alternative embodiment, the amino acid sequences of LCDR1, LCDR2, LCDR3 of light chain variable region SEQ ID NO. 18 are shown in sequence as SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, and the amino acid sequences of LCDR1, LCDR2, LCDR3 of the antibody or functional fragment thereof are also shown in sequence as SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6.

In alternative embodiments, the antibody or functional fragment thereof comprises the following complementarity determining regions:

The embodiment of the disclosure provides an anti-P24 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6.

The embodiment of the disclosure provides an anti-P24 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 in sequence, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6 in sequence.

In the present disclosure, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, referring to regions of an antibody heavy chain variable region and a light chain variable region other than CDRs; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

In the present disclosure, the heavy chain variable region is obtained by ligating the CDRs of the following numbering with the FR in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In alternative embodiments, the antibody or functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

HFR1 comprises SEQ ID NO 7 or an amino acid sequence having at least 80% homology thereto;

HFR2 comprises the amino acid sequence of SEQ ID NO 8 or at least 80% homology thereto;

HFR3 comprises SEQ ID NO 9 or an amino acid sequence having at least 80% homology thereto;

HFR4 comprises the amino acid sequence of SEQ ID NO 10 or having at least 80% homology thereto;

LFR1 comprises the amino acid sequence of SEQ ID NO. 11 or having at least 80% homology thereto;

LFR2 comprises the amino acid sequence of SEQ ID NO. 12 or having at least 80% homology thereto;

LFR3 comprises the amino acid sequence of SEQ ID NO. 13 or having at least 80% homology thereto;

LFR4 comprises the amino acid sequence of SEQ ID NO. 14 or having at least 80% homology thereto.

In alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having the amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3, and LFR4 having the amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.

In an alternative embodiment, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 7 to SEQ ID NO. 10 in sequence and having amino acid sequences shown in SEQ ID NO. 11 to SEQ ID NO. 14 in sequence.

In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided by the present disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.

In an alternative embodiment, the antibody or functional fragment thereof binds P24 with an affinity of KD < 8.32 x 10 ^-10 M.

In alternative embodiments, the antibody or functional fragment thereof binds P24 with an affinity of KD.ltoreq.10 10 ^-10 M or KD.ltoreq.10 10 ^-11 M.

In an alternative embodiment, the antibody or functional fragment thereof binds P24 with an affinity of KD.ltoreq.2.61X 10 ^-11 M.

In alternative embodiments, the antibody or functional fragment thereof comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 17 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 18.

The embodiment of the present disclosure also provides an antibody or a functional fragment thereof against P24, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR3, HFR2, HFR4, HFR3, LFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR4, LFR2, LFR3, LFR 4. In an alternative embodiment, the heavy chain variable region amino acid sequence is shown in SEQ ID NO. 17.

In an alternative embodiment, the light chain variable region amino acid sequence is set forth in SEQ ID NO. 18.

The embodiment of the disclosure also provides an anti-P24 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18.

In alternative embodiments, the antibody or functional fragment thereof further comprises a constant region.

In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.

In alternative embodiments, the heavy chain constant region is selected from the group consisting of the heavy chain constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD, and the light chain constant region is selected from the group consisting of the kappa-type or lambda-type light chain constant regions.

In alternative embodiments, the constant region is of species origin of cattle, horses, cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, cocktails or humans.

In an alternative embodiment, the constant region is of murine species origin.

In an alternative embodiment, the sequence (CH) of the heavy chain constant region is shown in SEQ ID NO. 15 and the sequence of the light chain constant region (CL) is shown in SEQ ID NO. 16.

In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the constant region (SEQ ID NO:15 or 16) described above.

In alternative embodiments, the antibody or functional fragment thereof comprises a heavy chain as set forth in SEQ ID NO. 19 and a light chain as set forth in SEQ ID NO. 20.

In alternative embodiments, the functional fragment is selected from any one of the group consisting of F (ab ') 2, fab', fab, fv and scFv of an antibody.

The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The functional fragments described above are readily available to those skilled in the art, given the structure of the present disclosure that provides an intact antibody.

Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

Embodiments of the present disclosure also provide an antibody against P24, comprising a heavy chain comprising the heavy chain variable region described above and the heavy chain constant region described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.

In an alternative embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO. 19.

In an alternative embodiment, the amino acid sequence of the light chain is shown in SEQ ID NO. 20.

The disclosed embodiments also provide an antibody comprising a heavy chain and/or a light chain, the heavy chain having an amino acid sequence as shown in SEQ ID NO. 19; the amino acid sequence of the light chain is shown as SEQ ID NO. 20.

The embodiment of the disclosure also provides an antibody conjugate, which comprises the antibody. Wherein the antibody is directly or indirectly covalently coupled to the conjugate. Or the antibody is coupled to the conjugate to be conjugated in a non-covalent adsorption manner.

In alternative embodiments, the antibody in the antibody conjugate is conjugated to biotin or a biotin derivative.

In alternative embodiments, the antibody in the antibody conjugate is conjugated to a label.

In alternative embodiments, a marker refers to a substance of a type having a property that can be directly observed by the naked eye or detected by an instrument, such as luminescence, color development, radioactivity, etc., by which a qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, a person skilled in the art can select an appropriate marker according to the detection conditions or actual needs, and no matter what marker is used, it is within the protection scope of the present disclosure.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).

In alternative embodiments, enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18F.

In alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In an alternative embodiment, the colloidal metal is colloidal gold.

In alternative embodiments, the antibody in the antibody conjugate is conjugated to a solid phase.

In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.

In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In an alternative embodiment, the solid phase is a nitrocellulose membrane.

The disclosed embodiments also provide a reagent or kit for detecting P24, the reagent or kit comprising the above-described antibody or a functional fragment thereof or the above-described antibody conjugate.

The disclosed embodiments also provide for the use of the above antibodies or functional fragments thereof, antibody conjugates, or reagents or kits described above in the detection of P24.

The disclosed embodiments also provide a method of detecting P24, comprising: contacting the above antibody or a functional fragment, antibody conjugate, reagent or kit thereof with P24 in the sample to be detected to form an immune complex.

In an alternative embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

In an alternative embodiment, the immune complex further comprises a second antibody, which binds to P24.

The disclosed embodiments also provide a nucleic acid molecule encoding the above antibody or functional fragment thereof.

The disclosed embodiments also provide vectors containing the above nucleic acid molecules.

The disclosed embodiments also provide cells comprising the above vectors.

The disclosed embodiments also provide a method of making an antibody or functional fragment thereof, comprising: the cells as described above were cultured.

The disclosed embodiments also provide the use of the above-described antibodies or functional fragments, antibody conjugates, reagents or kits thereof for detecting P24, or for preparing a product for detecting P24, for diagnosing HIV infection.

Embodiments of the present disclosure also provide a method of diagnosing HIV infection in a subject comprising: a) Contacting the antibody or functional fragment thereof, antibody conjugate, reagent or kit described above with a sample from a subject to form an immune complex; and

B) Detecting the presence of an immune complex, the presence of the complex being indicative of the presence of HIV in a sample from the subject.

In an alternative embodiment, in step a) a second antibody is also included in the immune complex, the second antibody being bound to the antibody or a functional fragment thereof.

In an alternative embodiment, in step a), a second antibody is also included in the immunocomplex, the second antibody binding to P24.

The present disclosure provides an anti-P24 antibody, a reagent and a kit for detecting P24, which can specifically bind to P24, have improved activity and/or affinity thereto, and detect P24 with the antibody having improved sensitivity and specificity. The present disclosure provides a richer antibody selection for the detection of P24.

On the basis of the present disclosure that an amino acid sequence of an antibody or a functional fragment thereof is disclosed, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying a culture product of a recombinant cell capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present disclosure to prepare the antibody or the functional fragment thereof by any one of the techniques.

In order to make the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.

The features and capabilities of the present disclosure are described in further detail below in connection with the examples.

EXAMPLE 1 preparation of Anti-P24 5F11 monoclonal antibody

Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART ^TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.

1 Construction of recombinant plasmid

(1) Antibody Gene production

MRNA is extracted from hybridoma cell strains which are prepared in the laboratory and secrete monoclonal antibodies against P24, a DNA product is obtained through an RT-PCR method, the product is inserted into a pMD-18T vector after an A adding reaction is carried out by rTaq DNA polymerase, the product is transformed into DH5 alpha competent cells, heavy chain and light chain gene clones are respectively taken after bacterial colonies grow out, and each 4 clones are sent to a gene sequencing company for sequencing.

(2) Sequence analysis of Anti-P24F 11 antibody variable region Gene

The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI 11.5.5 software is utilized for analysis to determine that the amplified genes of the heavy chain and the light chain primer pair are correct, wherein in the gene fragment amplified by the light chain, the VL (light chain variable region) gene sequence is 342bp, and the front of the VL (light chain variable region) gene sequence is 57bp leader peptide sequence; in the gene fragment amplified by the heavy chain primer pair, the VH (heavy chain variable region) gene sequence is 363bp, belongs to the VH1 gene family, and a leader peptide sequence of 57bp is arranged in front of the VH1 gene family.

(3) Construction of recombinant antibody expression plasmids

pcDNA^TM 3.4The vector (vector) is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced into HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and is named as pcDNA3.4A expression vector, and is hereinafter abbreviated as 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific primers of the antibody are designed, and the two ends of the VL and VH gene specific primers are respectively provided with HindIII, ecoRI enzyme cutting sites and protective bases, and a 0.70kb light chain gene fragment and a 1.42kb heavy chain gene fragment are amplified by a PCR amplification method.

The heavy chain and light chain gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the heavy chain gene and the light chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the heavy chain and the light chain are respectively obtained.

2 Stable cell line selection

(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity

The plasmid was diluted to 40. Mu.g/100. Mu.l with ultrapure water, CHO cells were conditioned to 1.43X10 ⁷ cells/mL in centrifuge tubes, 100. Mu.l of plasmid was mixed with 700. Mu.l of cells, transferred to an electrotransfer cup, electrotransfer, sampling counts were taken on days 3,5, 7, and sampling assays were performed on day 7.

The coating solution (main component NaHCO 3) dilutes the P24 recombinant antigen to 1. Mu.g/mL, 100. Mu.L per well, and overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50 mu L/hole, EDTA.2Na+ concentrated H ₂SO₄); OD was read on the microplate reader at 450nm (reference 630 nm). The results show that the reaction OD after 1000-fold dilution of the cell supernatant is still greater than 1.0, and the reaction OD without cell supernatant hole addition is less than 0.1, which indicates that the antibodies generated after plasmid transient are active on the P24 recombinant antigen.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 (volume ratio) sequentially with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain

Diluting the plasmid to 40 mug/100 uL by using ultrapure water, regulating 1.43X10 ⁷ cells/mL of CHO cells in a centrifuge tube, mixing 100 mu L of plasmid with 700 mu L of cells, transferring into an electrorotating cup, electrorotating, and counting the next day; 25 mu mol/L MSX 96 wells were incubated under pressure for approximately 25 days.

Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; 3 days later, seed preservation and batch culture are carried out, the cell density is regulated to be 0.5X10 ⁶ cells/mL, batch culture is carried out by 2.2mL, the cell density is regulated to be 0.3X10 ⁶ cells/mL, and seed preservation is carried out by 2 mL; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.

3 Recombinant antibody production

(1) Cell expansion culture

After cell recovery, the cells were first cultured in 125mL shake flasks with an inoculation volume of 30mL and a medium of 100% Dynamis, and placed in a shaker at a speed of 120r/min at 37℃and carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/mL, and calculating the expanded culture volume according to the production requirement, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/mL for production.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding is started every day when the culture is carried out in a shake flask for 72 hours, hyCloneTM Cell BoostTM Feed a is fed with 3% of the initial culture volume every day, and the feeding amount of Feed 7b is one thousandth of the initial culture volume every day until the 12 th day (feeding on 12 th day). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 6 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr at 50KD and the other Mr at 28KD.

Example 2 affinity and Activity optimization

The Anti-P24 5F11 monoclonal antibody obtained in example 1 has an ability to bind to P24, but has insufficient affinity and antibody activity, and thus the applicant has performed directed mutation on the light chain CDR and the heavy chain CDR of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. Monoclonal antibodies with remarkably improved affinity and antibody activity are obtained through screening and are named as: anti-P24F 11mut. The heavy and light chain amino acid sequences of Anti-P24F 11mut are shown in SEQ ID NO. 19 and SEQ ID NO. 20, respectively.

Example 3 detection of Performance of antibodies

1. Affinity analysis

Affinity analysis was performed using the instrument Biacore8K+, CM5 chip, purified antibodies were gradient diluted with HBS-EP starting at 200nM and P24 recombinant antigen diluted to 500nM with 10mM acetic acid pH 4.5.

The operation flow is as follows: after premixing EDC with NHS, 10. Mu.L/min 60 s.fwdarw.antigen, 10. Mu.L/min 420 s.blocking solution (composition 10.5mL 1.0M ethanolamine-HCl pH 8.5), 10. Mu.L/min 60 s.fwdarw.antibody, 30. Mu.L/min 120 s.fwdarw.10. Mu.L/min 90s by HBS-EP cleavage.fwdarw.sensor regeneration: 10mM GLY pH2.5, 10. Mu.L/min 30s.

KD represents equilibrium dissociation constant, i.e. affinity; ka represents the binding rate; kd represents the dissociation rate. HBS-EP contains 0.01M HEPES,pH 7.4,0.15M NaCl,3mM EDTA,0.005%v/v Surfactant P20 as main component; an aqueous solution of N-ethyl-N' - (dimethylaminopropyl) -carbodiimide with EDC of 0.2 mol/L; n-hydroxysuccinimide aqueous solution with NHS of 0.05 ml/L).

Table 2 affinity data

Sample name	KD(M)	Ka(1/Ms)	Kd(1/s)
				Control	8.32E-10	5.19E+04	4.32E-05
Anti-P24 5F11mut	2.61E-11	2.68E+05	7.00E-06

2. Activity assay

The coating solution (main component NaHCO 3) dilutes the P24 recombinant antigen to 1. Mu.g/mL, 100. Mu.L per well, and overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding the diluted monoclonal antibody into the mixture at the temperature of 37 ℃ for 30-60 min at the concentration of 100 mu L/hole; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; stop solution (50. Mu.L/well, 0.75 g/EDTA.2Na and 10.2ml/L concentrated H ₂SO₄) was added; OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in the following table.

TABLE 3 Activity data

Sample concentration (ng/mL)	500	250	125	62.5	31.25	0
							Control	1.893	1.356	0.812	0.497	0.299	0.029
Anti-P24 5F11mut	2.107	1.789	1.036	0.651	0.479	0.021

3. Stability assessment

Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 4 below shows the results of the detection of OD after 21 days of antibody examination.

Table 4 stability data

Sample concentration (ng/ml)	250	125	0
				4 ℃,21 Days sample	1.756	0.982	0.011
Sample at-80℃for 21 days	1.755	0.988	0.012
				37 ℃ And 21 days of sample	1.748	0.991	0.011

4. Detection performance assessment

Coating magnetic beads by using the antibody Anti-P24F 11mut of the example as a coating antibody; another antibody (obtained from the Pentium organism) was used as a labeled antibody, acridinium ester was labeled, and the antibody was paired with the Anti-P24F 11mut antibody coated with magnetic beads, which was abbreviated as Anti-P24F 11mut in the following table. The performance of the antibodies was tested on a chemiluminescent platform using a double antibody sandwich method. The instrument is used: yingkai Shine I2910, reaction mode: 1) 20. Mu.L of test sample+100. Mu.L of magnetic bead coated antibody (concentration 0.05%), reacted at 37℃for 10min, washed 3 times with PBST; 2) Adding 100 mu L of acridinium ester-labeled antibody, reacting for 10min at 37 ℃, and washing 3 times by PBST; 3) 200. Mu.L of acridine ester substrate was added and the instrument read. Remarks: NIBSC is a positive sample and LC is a clinically negative serum sample.

The result shows that the Anti-P24 5F11mut paired antibody clinical sample has the average value of 535 and the standard deviation of 100.69, has good specificity, does not detect the suspected false positive sample, and does not detect the suspected false positive sample due to HAMA interference. The Anti-P24 5F11mut paired antibody activity and specificity were advantageous over the control. The specific performance evaluation results are shown in the following table:

Table 5 performance evaluation data

The foregoing description of the preferred embodiments of the present disclosure is provided only and not intended to limit the disclosure so that various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

The partial amino acid sequences according to the present application are shown in the following Table 6:

TABLE 6 amino acid sequence

Claims

1. An anti-P24 antibody or antigen-binding fragment thereof having HCDR1 to HCDR3 of the heavy chain variable region of amino acid sequence shown in SEQ ID No. 17 and LCDR1 to LCDR3 of the light chain variable region of amino acid sequence shown in SEQ ID No. 18, said HCDR1 to HCDR3 and LCDR1 to LCDR3 being defined by the Kabat, chothia, IMGT, lesk, abM or Contact system, the numbering of said systems being according to Kabat numbering.

2. An anti-P24 antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 having amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 3, and LCDR1, LCDR2, LCDR3 having amino acid sequences shown in SEQ ID No. 4 to SEQ ID No. 6.

3. The antibody or antigen-binding fragment thereof of any one of claims 1 to 2, further having at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

4. The antibody or antigen-binding fragment thereof according to claim 3, further comprising an amino acid sequence having the amino acid sequence of HFR1, HFR2, HFR3, HFR4, shown in SEQ ID No. 7 to SEQ ID No. 10, and an amino acid sequence of LFR1, LFR2, LFR3, and LFR4, shown in SEQ ID No. 11 to SEQ ID No. 14, or an amino acid sequence having at least 80% identity to the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 sequences.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 2, wherein the antibody or antigen-binding fragment thereof binds P24 with an affinity of KD < 8.32 x 10 ^-10 M.

6. An anti-P24 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR3, HFR2, HFR4, HFR1, LFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LCDR3, LFR4 of claim 3 or 4.

7. An anti-P24 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 17; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 2, wherein the antibody or antigen-binding fragment thereof further comprises a constant region.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

11. The antibody or antigen-binding fragment thereof of claim 8, wherein the constant region is of a species origin of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the constant region is of murine species origin.

13. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region sequence is as shown in SEQ ID No. 15 or has at least 80% identity thereto; the light chain constant region sequence is shown in SEQ ID NO. 16 or has at least 80% identity thereto.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 2, wherein the antigen-binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.

15. An anti-P24 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain variable region of claim 6 or 7 and the heavy chain constant region of claim 9, 10 or 13; the light chain comprises the light chain variable region of claim 6 or 7 and the light chain constant region of claim 9, 10 or 13.

16. An anti-P24 antibody comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence as shown in SEQ ID No. 19; the amino acid sequence of the light chain is shown as SEQ ID NO. 20.

17. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 14 or the antibody of any one of claims 15 to 16 conjugated to biotin or a label.

18. The antibody conjugate of claim 17, wherein the antibody is conjugated to a solid phase.

19. The antibody conjugate of claim 17, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

20. The antibody conjugate of claim 19, wherein the nanoparticle-based label is colloidal gold.

21. A reagent or kit for detecting P24, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the antibody according to any one of claims 15 to 16 or the antibody conjugate according to any one of claims 17 to 20.

22. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or an antibody according to any one of claims 15 to 16 for the preparation of a test P24 product, comprising:

contacting the antibody or antigen binding fragment thereof with P24 in the sample to be tested to form an immune complex.

23. The use of claim 22, wherein the immune complex further comprises a second antibody that binds to the antibody or antigen binding fragment thereof.

24. The use of claim 22, wherein the immune complex further comprises a second antibody that binds to P24.

25. A nucleic acid encoding the antibody or antigen binding fragment thereof of any one of claims 1 to 14 or the antibody of any one of claims 15 to 16.

26. A vector comprising the nucleic acid of claim 25.

27. A cell comprising the nucleic acid of claim 25 or the vector of claim 26.

28. A method of preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or an antibody according to any one of claims 15 to 16, comprising: culturing the cell of claim 27.

29. Use of the antibody or antigen binding fragment thereof of any one of claims 1 to 14, the antibody of any one of claims 15 to 16, the antibody conjugate of any one of claims 17 to 20, or the reagent or kit of claim 21 in the preparation of a product for detecting P24.