CN112646040B

CN112646040B - Protein specifically binding to human IgG4 and application thereof

Info

Publication number: CN112646040B
Application number: CN202011644854.5A
Authority: CN
Inventors: 蒋会会; 易维京; 吴春荣; 汤定斌; 赵忠灏; 蔡方琴
Original assignee: Zhongyuan Huiji Biotechnology Co Ltd
Current assignee: Zhongyuan Huiji Biotechnology Co Ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-03-25
Anticipated expiration: 2040-12-31
Also published as: CN114920845A; CN112646040A; CN114891112A; CN114891110A; CN114891111A

Abstract

The invention relates to the technical field of biological medicines, in particular to a protein specifically binding to human IgG4 and application thereof, discloses a protein sequence of an anti-human IgG4 antigen, can be well applied to a clinical detection reagent, realizes simple, rapid and high-specificity detection of IgG4 in a sample, breaks through the technical barrier of independent research and development of an IgG4 antibody or an antigen binding fragment in China at present, realizes low-cost and high-efficiency raw material development, and has an important effect on future domestic detection reagents.

Description

Protein specifically binding to human IgG4 and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a protein specifically binding to human IgG4 and application thereof.

Background

The IgG4 related diseases are chronic and systemic diseases closely related to IgG4 lymphocytes, and the diseases are characterized by the rise of serum IgG4 level and the infiltration of IgG4 positive cells into various organs and tissues, and are frequently affected with various organs, so that the accurate measurement of IgG4 can provide reference for clinical research and has high application value.

However, IgG4 has very high homology with other subclasses IgG1, IgG2 or IgG3 in IgG, and IgG4 in normal healthy serum accounts for only about 4%, and is an immunoglobulin G subtype with the lowest content in human body, so that detection of IgG4 is very difficult, and the requirements on sensitivity and specificity of a detection reagent are very high, so that it is necessary to develop an antibody with excellent performance, but at present, domestic development of IgG4 antibody still has a high technical barrier, and there is basically no detection reagent related to IgG4 on the market, and only a biochemical reagent of siemens is widely used, but the detection reagent of siemens does not have universality, and needs to be matched with an expensive special protein instrument, and has a problem of long detection time.

Therefore, it is very important to develop an IgG4 specific antibody with excellent performance, so that the antibody can be widely applied to various IgG4 detection reagents, and finally, the high sensitivity, high specificity and rapid measurement of IgG4 can be realized.

Disclosure of Invention

The invention provides human IgG4 binding protein, which aims to solve the problems of difficult detection and long detection time of IgG4 and realize high sensitivity, high specificity and rapid measurement of trace IgG4 in human blood by using low cost.

In order to achieve the purpose, the invention adopts the following technical means:

a protein that specifically binds human IgG4, said protein being an anti-IgG 4 antibody or antigen-binding fragment comprising:

a) the heavy chain variable region CDR1 as set forth in SEQ ID NO. 1;

b) the heavy chain variable region CDR2 as set forth in SEQ ID NO. 2;

c) the heavy chain variable region CDR3 as set forth in SEQ ID NO. 3;

d) light chain variable region CDR1 as set forth in SEQ ID NO. 4;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 5;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 6;

the anti-human IgG4 antibody or antigen-binding fragment was named Ab16

Or

a) Heavy chain variable region CDR1 as set forth in SEQ ID NO. 7;

b) the heavy chain variable region CDR2 as set forth in SEQ ID NO. 8;

c) the heavy chain variable region CDR3 as set forth in SEQ ID NO. 9;

d) light chain variable region CDR1 as set forth in SEQ ID NO. 10;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 11;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 12;

the anti-human IgG4 antibody or antigen-binding fragment was named Ab18

Or

a) The heavy chain variable region CDR1 as set forth in SEQ ID NO. 13;

b) the heavy chain variable region CDR2 as set forth in SEQ ID NO. 14;

c) the heavy chain variable region CDR3 as set forth in SEQ ID NO. 15;

d) light chain variable region CDR1 as set forth in SEQ ID NO. 16;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 17;

f) 18, light chain variable region CDR3 as set forth in SEQ ID NO;

the anti-human IgG4 antibody or antigen-binding fragment was named Ab20

Or

a) The heavy chain variable region CDR1 as set forth in SEQ ID NO. 19;

b) the heavy chain variable region CDR2 as set forth in SEQ ID NO. 20;

c) heavy chain variable region CDR3 as set forth in SEQ ID NO. 21;

d) light chain variable region CDR1 as set forth in SEQ ID NO. 22;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 23;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 24;

the anti-human IgG4 antibody or antigen-binding fragment was named Ab27

Or

a) Heavy chain variable region CDR1 as set forth in SEQ ID NO. 25;

b) heavy chain variable region CDR2 as set forth in SEQ ID NO. 26;

c) the heavy chain variable region CDR3 as set forth in SEQ ID NO. 27;

d) light chain variable region CDR1 as set forth in SEQ ID NO 28;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 29;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 30;

the anti-human IgG4 antibody or antigen-binding fragment was named Ab28

Preferably, the protein is an anti-IgG 4 monoclonal antibody.

In another aspect, the invention also discloses nucleic acids encoding the variable regions of SEQ ID NOS 1-6, 7-12, 13-18, 19-24, and/or 25-30.

In another aspect, the invention discloses an expression vector comprising the nucleic acid.

In another aspect, the invention discloses a host cell comprising the expression vector.

In another aspect, the present invention also discloses a protein comprising any of the above proteins that specifically binds human IgG4, wherein the protein is preferably: monoclonal antibodies Ab16, Ab18, Ab20, Ab27 or Ab 28.

Preferably, the kit comprises a combination of any two or more of the antibodies, and the antibodies in each combination simultaneously recognize the IgG4 protein, and the concentration of IgG4 protein is determined.

In another aspect, the invention features a conjugate comprising a monoclonal antibody of any of the above covalently linked to a chemical label or a biomarker.

In another aspect, the invention discloses a conjugate formed by coupling the monoclonal antibody, and/or the conjugate with a solid medium or a semi-solid medium.

In addition, the invention also discloses application of the protein and/or the conjugate in preparing products for detecting IGG4 expression.

The invention has the beneficial effects that: the protein specifically binding to human IgG4 and the application thereof disclosed by the invention can realize the specific detection of IgG4, and compared with the detection method widely used in the market, the protein is simple and convenient to operate and short in time.

Drawings

FIG. 1 is a reagent anti-interference experiment prepared from Ab 28;

FIG. 2 is a comparison of correlation between different process measurements;

FIG. 3 shows the correlation between the kit prepared by Ab16 and the Siemens kit detection;

FIG. 4 shows the correlation between the kit prepared by Ab18 and the Siemens kit detection;

FIG. 5 shows the correlation between the kit prepared by Ab20 and the Siemens kit detection;

FIG. 6 shows the correlation between the kit prepared by Ab27 and the Siemens kit detection;

FIG. 7 shows the correlation between the kit prepared by Ab28 and the Siemens kit detection;

fig. 8 is the clinical relevance of the kit prepared by Ab16 to the siemens kit.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The invention provides an anti-IgG 4 monoclonal antibody, which aims to solve the problems of difficulty in detection of IgG4 and long detection time.

Example 1 expression purification of recombinant protein human IgG4

Cloning the human pollen allergen IgG4 gene sequence to a eukaryotic expression vector to construct a eukaryotic expression plasmid, transfecting HEK293 cells with the expression plasmid, culturing for 5 days by suspension, and centrifugally collecting cell supernatant. And purifying by using a G column to obtain the antigen protein.

Example 2 mouse immunization and antibody detection

Freund's complete adjuvant was mixed and emulsified in equal volumes with the antigen protein at a concentration of 2 mg/ml. Emulsified antigen immunized 6-8 week old female BALB/c SPF grade mice. Each mouse was injected 40ug of antigen protein using either plantar injection or dorsal subcutaneous injection. Two weeks after the primary immunization, the antigen protein was mixed and emulsified with Freund's incomplete adjuvant, and 40ug of antigen protein was injected again into each mouse by foot sole injection or back subcutaneous injection. Two weeks later blood was taken via the tail vein, and the supernatant was collected by centrifugation and assayed for serum titer by ELISA. Immunizations were performed every two weeks and serum titers were measured. Screening to 10⁶The mice were lympholyzed to separate lymphocytes for cell fusion.

Example 3 cell fusion and Positive hybridoma cell selection and subcloning

Screening hybridomas obtained by three step screening were screened: 1) confirming the ability to bind IgG 4; 2) the specificity was confirmed.

1) To screen for high affinity anti-human IgG4 antibodies, positive well screens were performed using binding human IgG4 protein. And selecting the hole with the higher ELISA positive value and cell ratio value for multiple subcloning, and selecting the monoclonal hybridoma cell with the highest affinity.

2) Evaluation of specificity: the specificity of the antibodies produced by the clones selected in 1) was evaluated. Myeloma human IgG1(Sigma), myeloma human IgG2(Sigma), myeloma human IgG3(Sigma), myeloma human IgG4(Sigma) were adjusted to 1. mu.g/mL with PBS. The adjusted samples were added to a 96-well plate in 100 μ L portions. The operation after shaking with a shaker was the same as in 1) above. Among the numerous clones, 17 clones were selected which showed a response to myeloma human IgG4 compared to myeloma human IgG1, 2 and 3, of which 5 were more reactive.

EXAMPLE 4 production purification of monoclonal antibodies

BALB/c mice were used for 6-8 weeks, and 500ul paraffin oil was intraperitoneally injected to suppress the immune response in the mice. Injecting about 1X10 into abdominal cavity after one week⁶A quantity of hybridoma cells of interest. Ascites collection began two weeks later. The collected ascites is treated with ammonium sulfate precipitation and affinity purification of protein A to obtain the target antibody.

EXAMPLE 5 subtype identification of monoclonal antibodies and Gene sequence cloning

The subtypes of the heavy and light chains of the monoclonal antibody were identified by the SBA cloning System-HRP kit from southern Biotech according to the protocol of the instruction. The heavy chains of the 17-strain antibody are all IgG1, and the light chains are Kappa. mRNA was prepared by extracting hybridoma cells using a reverse transcriptase PrimeScript from Takara^TMII Reverse Transcriptase Reverse transcription was performed in combination with primers designed for a specific subtype. Finally, standard antibody variable region gene cloning primers are adopted to carry out antibody gene sequence amplification. And obtaining an antibody gene sequence by plasmid sequencing after the amplified product is linked with the T vector.

EXAMPLE 6 Linear assay of monoclonal antibodies

The reagent formula comprises: r1: TRIS buffer solution 100mmo/L, KCl 0.1mol/L, Tween 203g/L, sodium azide 1 g/L; r2: 50mmoL of PBS buffer solution, 500mg/L of KCl 0.3mol/L, IgG4 monoclonal antibody and 40mL/L, BSA 30g/L of latex microspheres.

The detection instrument comprises: full-automatic biochemical analyzer (Hitachi 7180)

The specific operation method comprises the following steps: the Ab12-Ab28 antibody is prepared into a kit, namely the IgG4 monoclonal antibody in the reagent 2 is Ab12-Ab28 respectively,

(1) mixing the serum sample to be detected with the reagent R1 and the reagent R2, and stirring and uniformly mixing the mixture to make the mixture fully react;

(2) measuring absorbance difference (main wavelength is 340nm, and sub wavelength is 700nm) after reaction by using a full-automatic biochemical analyzer (Hitachi 7180);

(3) the concentration of IgG4 in the sample was calculated from the absorbance change values.

(4) Plotting the concentration as X axis and the light absorption value as Y axis to calculate R²And verifying the performances of different antibodies.

The detection principle is as follows:

IgG4 in the sample can be combined with anti-IgG 4 antibody in the reagent to form an antigen-antibody-microsphere complex, certain turbidity is generated, the turbidity is proportional to the content of the antigen when certain antibody exists, the turbidity is measured at certain wavelength, and the IgG4 can be quantitatively measured through a multi-point calibration curve.

Complement IgG4(g/L) ═ CS × Δ AT/Δ AS (g/L) in the sample

(wherein. DELTA.AT represents the absorbance of the sample tube AS compared with the absorbance of the blank tube,. DELTA.AS represents the absorbance of the calibration tube AS compared with the absorbance of the blank tube, and the concentration of IgG4 in the CS calibration solution)

The results of the experiments are shown in the following table

TABLE 1 preparation of monoclonal antibody Linear experiment

The results show that R2 of Ab16, Ab18, Ab20, Ab27 and Ab28 is more than 0.9, which proves that the linearity is better and the accuracy of the detection result can be ensured; and the K values are all larger than 1000, so that the sample detection gradient is better, the antigen-antibody binding capacity is stronger, the Ab28 has the best effect, and the Ab28 is selected as an antibody used in a subsequent test.

Example 7 antibody Titer assay

IgG4 full-length prokaryotic antigen was diluted to 1ug/ml, added to a 96-well plate at 100ul per well, incubated overnight at 4 ℃ and washed. The antibody is diluted to a proper concentration, diluted according to a 3-fold ratio, 100ul is put in each hole, incubated at 37 ℃ for 1h, and plates are washed. Goat anti-mouse HRP enzyme-labeled antibody was expressed as 1: diluting at 3000, incubating at 37 deg.C for 30min, developing for 3min, and detecting absorbance at A450 nm.

The control was the antibody itself incubated at 4 ℃ and the results are shown in tables 2-6.

TABLE 2 Ab16 Indirect potency assay

TABLE 3 Ab18 Indirect potency assay

TABLE 4 Ab20 Indirect potency assay

TABLE 5 Ab27 Indirect potency assay

TABLE 6 Ab28 Indirect potency assay

The results show that: under lower concentration, the detection signal has larger gradient, which indicates that the sensitivity of the four antibodies is higher, and the four antibodies are suitable for subsequent tests.

Example 8 antibody stability assay

The antibodies were thermally accelerated at 37 ℃ for 7 days in a defined buffer, and the accelerated antibodies were evaluated by SDS-PAGE and ELISA indirect methods, and the long-term stability of the antibodies was identified in comparison with 4 ℃. Further, the freeze-thaw stability of the antibody was evaluated by repeating the freeze-thaw at-20 ℃ for 5 times in the same manner, and the results are shown in tables 8 to 12.

TABLE 8 Ab16 stability assay

Concentration ng/ml	Freezing for 3 times	Freezing for 5 times	Acceleration 7d	Acceleration 14d	Acceleration 21d		4℃
								1000	2.437	2.409	2.613	2.657	2.655	2.586
300	2.318	2.305	2.502	2.524	2.533	2.479
							100	2.28	2.172	2.351	2.407	2.359	2.324
33.33	1.658	1.698	1.757	1.695	1.719	1.738
							11.11	1.092	1.181	1.21	1.253	1.271	1.219
3.70	0.509	0.579	0.688	0.731	0.776	0.769
							1.23	0.261	0.275	0.316	0.369	0.351	0.275
0.41	0.125	0.147	0.151	0.169	0.174	0.137
							0.14	0.085	0.093	0.098	0.111	0.106	0.096
0	0.061	0.061	0.063	0.061	0.061	0.062

TABLE 9 Ab18 stability assay

TABLE 10 Ab20 stability assay

Concentration ng/ml	Freezing for 3 times	Freezing for 5 times	Acceleration 7d	Acceleration 14d	Acceleration 21d		4℃
								1000	2.323	2.225	2.387	2.438	2.402	2.339
300	2.223	2.156	2.245	2.256	2.310	2.255
							100	2.132	2.107	2.130	2.284	2.272	2.154
33.33	1.753	1.721	1.792	1.854	1.833	1.801
							11.11	1.052	1.185	1.231	1.334	1.365	1.295
3.70	0.582	0.576	0.657	0.703	0.727	0.608
							1.23	0.223	0.217	0.304	0.328	0.325	0.295
0.41	0.124	0.137	0.152	0.165	0.173	0.140
							0.14	0.082	0.092	0.097	0.112	0.106	0.096
0	0.063	0.048	0.063	0.061	0.061	0.060

TABLE 11 Ab27 stability assay

TABLE 12 Ab28 stability assay

Concentration ng/ml	Freezing for 3 times	Freezing for 5 times	Acceleration 7d	Acceleration 14d	Acceleration 21d		4℃
								1000	2.687	2.563	2.773	2.879	2.885	2.735
300	2.546	2.449	2.643	2.771	2.760	2.535
							100	2.431	2.272	2.416	2.568	2.575	2.433
33.33	1.745	1.925	1.975	1..953	2.091	1.985
							11.11	1.191	1.285	1.513	1.645	1.475	1.415
3.70	0.685	0.629	0.687	0.735	0.755	0.619
							1.23	0.359	0.286	0.356	0.368	0.397	0.324
0.41	0.134	0.127	0.151	0.165	0.173	0.157
							0.14	0.085	0.095	0.097	0.123	0.121	0.093
0	0.061	0.061	0.063	0.061	0.062	0.061

The experimental result shows that all the antibodies (Ab16, Ab18, Ab20, Ab27 and Ab28) can be stably stored at 4 ℃, the property is stable after accelerating for one month at 37 ℃, the performance of the reagent used after the bottle is opened is ensured, the accuracy and the stability of the detection result are ensured, the performance of the antibody after the acceleration is more sensitive, and other additives in the acceleration condition can have a positive effect on the antibody.

In addition, the antibody can be repeatedly frozen and thawed, the performance of the antibody is not influenced, and the antibody is very suitable for practical application scenes.

Example 9 anti-interference experiment

The reagent formula of the embodiment is the same as that of the reagent formula, wherein the IgG4 monoclonal antibody in R2 is Ab 28.

Because there is a high similarity between IgG1, IgG2, IgG3, and IgG4 in serum, this experiment examined whether the presence of IgG1, IgG2, and IgG3 interferes with the antibodies of the invention, and because the ratio of IgG1: IgG2: IgG3: IgG4 in blood is 66:23:7:4, the interfering substances are added to this experiment.

Purified recombinant IgG4 protein at a concentration of 1mg/ml was mixed well with IgG3, 5.75mg/ml IgG2, 16.5mg/ml IgG1 at a concentration of 1.75 mg/ml. Serum without IgG4 was used as a diluent to prepare recombinant IgG4 protein at concentrations of 0g/L, 0.5g/L, 1g/L, 2g/L, and 4g/L, and a mixture. The concentration of each diluted sample was measured using the IgG4 content assay kit prepared as described above, according to the experimental conditions described above. The result shows that the linear regression straight line slope of the purified recombinant IgG4 protein measured by the prepared IgG4 content detection kit and the recombinant IgG4 protein (figure 1) in the mixed solution reaches above 0.99, which indicates that the difference between the simple IgG4 protein and the mixed IgG4 protein measured by the IgG4 detection kit prepared by the monoclonal antibody is small, and the test strip can tolerate the interference of IgG1, IgG2 and IgG3 on the content measurement of IgG 4.

EXAMPLE 10 investigation of the preparation Process

In this example, the process influence of the Ab28 prepared by the present invention on the small-batch production and the large-batch production is explored.

Wherein, the small batch production adopts vortex, and the large batch production adopts stirring.

The results are shown in the following table and FIG. 2

TABLE 13 different Processes

The results show R²The result is 0.998, which shows that the mixing mode does not have a substantial influence on the performance of the antibody, and the mode can be selected in the practical operation.

Example 11 correlation experiment

The reagents and detection methods of the present invention are as shown in example 6.

The control test is a Siemens BN II special protein apparatus for detecting IGG 4.

The specific operation is as follows (refer to the Siemens BN II operation instruction in detail):

(1) the instrument is automatically initialized for about 20 minutes;

(2) inputting a dilution strip, a quality control product, a calibration product and a cleaning solution;

(3) inputting sample information, and selecting a dilution multiple according to the concentration of the sample;

(4) take out and wash the diluted strip, oven dry → start a new round of testing.

The current Siemens BN II detection sample has the following problems: the initialization time is long, and about 20 minutes is required. Secondly, the sampling speed is low, sample information needs to be input firstly, specific requirements on the placement and the state of the sample are met, and the experiment operation is complex. The instrument adopts a dilution method for testing, the concentration exceeding the concentration range needs to be diluted and then tested, the detection time (30 samples need to be tested, more than 2 hours are needed) is greatly increased, once the dilution strip is used up, the shutdown is needed for replacing the dilution strip, all the operations are carried out again, and the time is greatly lost.

Compared with a special protein instrument with complex operation, the operation is greatly simplified by using the biochemical instrument, the time is shortened, the operation of the biochemical instrument does not need to place or replace a dilution strip in the instrument, and only the startup, the parameter setting, the quality control product inputting and the sample collecting are needed for detection, and about 10 minutes is needed for testing 30 samples.

In this example, the kits prepared from Ab16, Ab18, Ab20, Ab27 and Ab28 monoclonal antibodies with better performance in the above examples were selected, and the kits of siemens corporation were compared with the kit of the present invention to detect samples with different concentrations at the same time, thereby verifying the performance of the monoclonal antibody of the present invention in practical applications.

The results are shown in the following table

TABLE 14 correlation of kits of the invention with Siemens kits

As can be seen from the above table and FIGS. 3-7, when the monoclonal antibody against rhenium is applied to the kit, the relative deviation between the detection reagent sample and the Siemens detection result is within 15%, R is²Above 0.99, the kit prepared by the monoclonal antibody is shown to have better correlation with a Siemens kit, the existing Siemens kit is a detection reagent which is commonly used in the market and has excellent performance, and the excellent performance of the monoclonal antibody can be proved through the correlation research of the Siemens kit and the Siemens reagent, but the detection of the existing Siemens kit has the biggest problem that a special protein instrument is needed, the detection time is longer, the requirement of clinical tests on the timeliness and the rapidness of the detection is not facilitated, the detection of the monoclonal antibody is convenient and rapid, other instruments are not needed, and the detection of the IGG4 in a sample can be realized only by utilizing a biochemical instrument which is commonly existing in the market.

EXAMPLE 12 clinical trial

The procedures and reagents were the same as in the above examples

The results are shown in the following table and in FIG. 8:

TABLE 15 clinical relevance of the kits of the invention to the Siemens kit

The result shows that the sample with the content of IGG4 being more than 4g/L is diluted to the detectable concentration for measurement to obtain the result, wherein the correlation between the sample and the Siemens is better in the detection range of less than 4g/L, and the sample with the content of IGG4 being more than 4g/L is diluted during detection.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Sequence listing

<110> Yuanhui Biotechnology Ltd in Chongqing

<120> protein specifically binding to human IgG4 and application thereof

<130> 2020.12.29

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> mouse

<400> 1

Asn Tyr Gly Met Ser

1 5

<210> 2

<211> 17

<212> PRT

<213> mouse

<400> 2

Thr Ile Ser Ser Gly Ser Ser Tyr Thr Tyr Tyr Leu Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 11

<212> PRT

<213> mouse

<400> 3

His Glu Gly Tyr Tyr Lys Tyr Lys Phe Thr Tyr

1 5 10

<210> 4

<211> 15

<212> PRT

<213> mouse

<400> 4

Arg Ala Ser Gln Ser Val Asp Tyr Asn Gly Ile Ser Tyr Met His

1 5 10 15

<210> 5

<211> 7

<212> PRT

<213> mouse

<400> 5

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> mouse

<400> 6

Gln Gln Ser Ile Glu Glu Pro Pro Thr

1 5

<210> 7

<211> 5

<212> PRT

<213> mouse

<400> 7

Ser Tyr Gly Met Ser

1 5

<210> 8

<211> 17

<212> PRT

<213> mouse

<400> 8

Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 9

<211> 11

<212> PRT

<213> mouse

<400> 9

His Glu Asp Tyr Tyr Lys Tyr Lys Phe Ala Tyr

1 5 10

<210> 10

<211> 15

<212> PRT

<213> mouse

<400> 10

Arg Ala Ser Gln Ser Val Asp Tyr Asn Gly Ile Ser Phe Phe His

1 5 10 15

<210> 11

<211> 7

<212> PRT

<213> mouse

<400> 11

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 12

<211> 9

<212> PRT

<213> mouse

<400> 12

Gln Gln Ser Ile Glu Glu Pro Pro Thr

1 5

<210> 13

<211> 5

<212> PRT

<213> mouse

<400> 13

Ser Tyr Gly Met Ser

1 5

<210> 14

<211> 17

<212> PRT

<213> mouse

<400> 14

Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 15

<211> 11

<212> PRT

<213> mouse

<400> 15

His Glu Asp Tyr Tyr Lys Tyr Lys Phe Ala Tyr

1 5 10

<210> 16

<211> 15

<212> PRT

<213> mouse

<400> 16

Arg Ala Ser Gln Ser Val Asp Tyr Asn Gly Ile Ser Tyr Met His

1 5 10 15

<210> 17

<211> 7

<212> PRT

<213> mouse

<400> 17

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 18

<211> 9

<212> PRT

<213> mouse

<400> 18

Gln Gln Ser Ile Glu Glu Pro Pro Thr

1 5

<210> 19

<211> 5

<212> PRT

<213> mouse

<400> 19

Thr Tyr Val Met His

1 5

<210> 20

<211> 17

<212> PRT

<213> mouse

<400> 20

Asn Phe Asn Pro Tyr Asn Asp Gly Thr Leu Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 21

<211> 10

<212> PRT

<213> mouse

<400> 21

Asn Asp Tyr Asp Pro Ala Tyr Phe Asp Tyr

1 5 10

<210> 22

<211> 11

<212> PRT

<213> mouse

<400> 22

Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser

1 5 10

<210> 23

<211> 7

<212> PRT

<213> mouse

<400> 23

Arg Ala Asn Arg Leu Leu Asp

1 5

<210> 24

<211> 9

<212> PRT

<213> mouse

<400> 24

Leu Gln Tyr Asp Glu Phe Pro Pro Thr

1 5

<210> 25

<211> 5

<212> PRT

<213> mouse

<400> 25

Ser Ser Ile Ile His

1 5

<210> 26

<211> 17

<212> PRT

<213> mouse

<400> 26

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe Lys

1 5 10 15

Gly

<210> 27

<211> 10

<212> PRT

<213> mouse

<400> 27

Gly Gly Gly Asn Tyr Gly Trp Phe Val Tyr

1 5 10

<210> 28

<211> 11

<212> PRT

<213> mouse

<400> 28

Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ile

1 5 10

<210> 29

<211> 7

<212> PRT

<213> mouse

<400> 29

Arg Ala Asn Arg Leu Val Asp

1 5

<210> 30

<211> 9

<212> PRT

<213> mouse

<400> 30

Leu His Tyr Asp Asp Phe Pro Pro Thr

1 5

Claims

1. A protein that specifically binds human IgG4, wherein the protein is an anti-IgG 4 antibody or antigen-binding fragment comprising:

a) the heavy chain variable region CDR1 as set forth in SEQ ID NO. 1;

b) the heavy chain variable region CDR2 as set forth in SEQ ID NO. 2;

c) the heavy chain variable region CDR3 as set forth in SEQ ID NO. 3;

d) light chain variable region CDR1 as set forth in SEQ ID NO. 4;

e) light chain variable region CDR2 as set forth in SEQ ID NO. 5;

f) light chain variable region CDR3 as set forth in SEQ ID NO. 6;

the anti-human IgG4 antibody or antigen-binding fragment was designated Ab 16.

2. The protein of claim 1, wherein said antibody or antigen-binding fragment is an anti-IgG 4 monoclonal antibody.

3. Nucleic acid encoding an anti-human IgG4 monoclonal antibody, wherein the nucleic acid encodes an antibody having a variable region as set forth in SEQ ID NO: 1-6.

4. An expression vector comprising the nucleic acid of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. An IgG4 detection kit, comprising a protein of claim 1 or 2 that specifically binds to human IgG 4.

7. A conjugate comprising a protein according to any one of claims 1 or 2 covalently linked to a chemical or biological marker.

8. A conjugate formed by conjugating a protein according to claim 1 or 2, and/or a conjugate according to claim 7 to a solid or semi-solid medium.

9. Use of a protein according to claim 1 or 2 and/or a conjugate according to claim 7 and/or a conjugate according to claim 8 for the preparation of a product for detecting the expression of IgG 4.