CN118294677A

CN118294677A - Method for detecting fibroblast growth factor 21

Info

Publication number: CN118294677A
Application number: CN202410442586.0A
Authority: CN
Inventors: 李剑; 梁庆环
Original assignee: Hangzhou Diag Biotechnology Co ltd
Current assignee: Hangzhou Diag Biotechnology Co ltd
Priority date: 2024-04-12
Filing date: 2024-04-12
Publication date: 2024-07-05

Abstract

The invention discloses a method for detecting fibroblast growth factor 21, which uses a sandwich method Elisa kit prepared by an independently developed rabbit monoclonal antibody pair, and comprises the following steps: a, preparing FGF21 antibody pair raw materials; b, preparation of streptavidin micro-pore plates: dissolving streptavidin with sodium carbonate buffer solution, adding 50-200ul of streptavidin into each hole, and coating at 0-4deg.C overnight; c, preparing a biotin-labeled antibody; d, preparing enzyme-labeled antibody: selecting enzyme for marking FGF21 detection antibody, replacing the marked antibody in PBS buffer solution, and preserving for standby; e, establishing an ELISA one-step method. The method simplifies the steps of determining FGF21 in the prior art, shortens the operation time and improves the detection efficiency. In addition, the streptavidin-biotin system is adopted, so that the signal amplification effect is achieved, the sensitivity of the ELISA reaction can be improved, and a new path is opened for detecting micro-antigen and antibody.

Description

Method for detecting fibroblast growth factor 21

Technical Field

The invention belongs to the field of monoclonal antibody preparation, and particularly relates to a method for detecting fibroblast growth factor 21.

Background

Fibroblast growth factor 21 (fibroblast growth factor, FGF 21) belongs to the FGF family and was found in mouse embryos by Nishimura et al, 2000. FGF21 is a secreted protein consisting of 210 amino acids, of which about 30 constitute the signal peptide. The gene is specifically expressed mainly in the liver, but is also expressed in skeletal muscle, thymus, pancreatic cells, and the like. Studies have shown that FGF21 is a novel glycolipid metabolism regulator, which facilitates fat metabolism, promotes glucose absorption, and promotes insulin secretion. FGF21 has some characteristics necessary for the treatment of diabetes, and does not produce the two side effects of hypoglycemia and edema which are common in the current diabetes treatment, and is expected to become an ideal medicament for treating diabetes. In addition, FGF21 has no obvious mitogenic effect on cells, so that the clinical medication risk can be greatly reduced, and the application prospect for treatment is great. There have been clinical studies to find that FGF21 analogues have great potential in the treatment of diabetes, obesity and non-alcoholic steatohepatitis. Based on the above advantages, FGF21 has become a research hotspot in recent years, and at the same time, higher requirements are also put on the detection of FGF 21. Currently, the immune detection method for FGF21 adopts a traditional ELISA sandwich method, and the detection time of the method is long. The scheme is to adopt a one-step method which is independently researched and developed for detecting the sample, so that the measuring step can be simplified, the operation time is shortened, and the detection efficiency is improved. In addition, due to the limited sensitivity of the conventional detection method, when the antigen content in the sample is small, the detection result is still negative in some cases, thus causing false break. However, the one-step method adopts a streptavidin-biotin system, has a signal amplification effect, can improve the sensitivity of an ELISA reaction, and opens up a new way for detecting trace antigens and antibodies.

Disclosure of Invention

The invention aims at providing a method for detecting fibroblast growth factor 21, which comprises the following steps:

a, preparing FGF21 antibody pair raw materials;

b, preparation of streptavidin micro-pore plates: dissolving streptavidin with sodium carbonate buffer solution, adding 50-200ul of streptavidin into each hole, and coating at 0-4deg.C overnight;

c preparation of biotin-labeled antibody: dissolving biotin by using DMSO, mixing biotin and FGF21 capture antibody according to the volume ratio of 1:10-1:50, standing for 2 hours at room temperature, removing redundant free state biotin, replacing the free state biotin with PBS buffer solution, and preserving for later use;

d, preparing enzyme-labeled antibody: selecting enzyme for marking FGF21 detection antibody, replacing the marked antibody in PBS buffer solution, and preserving for standby;

e, establishing an ELISA one-step method: and adding 20-150ul of each of the biotin-labeled antibody, the sample to be detected and the enzyme-labeled antibody into a microwell plate coated with streptavidin, incubating, washing, beating, adding a substrate, and stopping the reaction after color development.

As a preferred embodiment of the present invention, the step a includes FGF21 antigen preparation:

① The amino acid sequence of FGF21 protein is selected as aa29-209, and the amino acid sequence ：HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVK TSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLP LPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS;

② Constructing FGF21 plasmid;

③ And carrying out transient expression on the constructed FGF21 plasmid in a eukaryotic cell expression system, and collecting the purified FGF21 recombinant protein for antigen preparation.

As a preferred embodiment of the present invention, the step a includes animal immunization:

Selecting an immunized animal;

The FGF21 recombinant protein is emulsified by Freund's incomplete adjuvant and then used as an antigen for immunization;

Primary immunization: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

two weeks later, a second immunization: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

third immunization after one week: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

four immunization after two weeks: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

fifth immunization after two weeks: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

Taking a small amount of whole blood after one week, and centrifuging to prepare antiserum for serum titer test;

After one week, 0.2-0.5mg of FGF21 recombinant protein without adjuvant is injected intravenously for enhancing immunity.

As a preferred embodiment of the present invention, the step a further includes: antiserum detection, single B cell culture, FGF21 positive hole screening, antibody gene plasmid construction of positive holes, antibody expression, chessboard screening antibody pairing and large-scale production of antibody pairing.

As a preferred embodiment of the present invention, the immunized animal is one of a rabbit, a sheep and a guinea pig.

As a preferred embodiment of the present invention, the single B cell culture and FGF21 positive well selection described above comprises:

① Isolating whole blood B lymphocytes;

② Spread isolated B lymphocytes at 3000 cells/well to sterile 96 Kong Biaoban with FGF21 antigen plates, cultured overnight;

③ Washing twice with PBS containing 10% -20% fetal bovine serum;

④ Culturing the rest B lymphocytes after washing with 1640 culture medium;

⑤ After 5 days of culture, supernatants were used to screen positive wells by ELISA indirect method.

The beneficial effects of the invention are as follows: based on a streptavidin-biotin system, each streptavidin can be combined with 4 biotins, so that the multistage amplification effect is achieved, and the FGF21 detection sensitivity is improved; the streptavidin is coated in the micro-pore plate, so that the streptavidin is easy to preserve, and the inactivation phenomenon generated during antibody coating is avoided; the complex formed by the biotin-labeled FGF21 antibody, the antigen in the sample and the enzyme-labeled FGF21 antibody is combined with avidin on the microplate through biotin, and the reaction has high specificity and stability due to the extremely high affinity of the avidin combined with the biotin; meanwhile, the method adopts a one-step method, can simplify operation steps, shortens detection time, and is suitable for wide application.

Drawings

FIG. 1 is a schematic representation of purity of the antibodies of the invention after SDS PAGE analysis and purification.

Detailed Description

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.

EXAMPLE 1FGF21 antibody against drug substance

Preparation of FGF21 antigen

1.1 Selecting FGF21 protein with the amino acid sequence aa29-209, wherein the specific amino acid sequence is as follows ：HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVK TSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLP LPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS;

1.2 Constructing FGF21 plasmid;

1.3 transient expression of the constructed FGF21 plasmid in eukaryotic cell expression system, and obtaining about 20mg after collection and purification

FGF21 recombinant proteins are used for antigen preparation.

2. Immunization of animals

2.1 Selecting four rabbits as immunized animals;

2.2FGF21 the recombinant protein is emulsified with Freund's incomplete adjuvant and then used as an antigen for immunization;

2.3 first immunization: each animal was multi-injected with a total of 0.2mg of emulsified antigen;

2.4 second immunization after two weeks: each animal was multi-injected with a total of 0.2mg of emulsified antigen;

third immunization after 2.5 weeks: each animal was multi-injected with a total of 0.2mg of emulsified antigen;

four immunizations after 2.6 weeks: each animal was multi-injected with a total of 0.2mg of emulsified antigen;

A fifth immunization after 2.7 weeks: each animal was multi-injected with a total of 0.2mg of emulsified antigen;

taking a small amount of whole blood after 2.8 weeks, and centrifuging to prepare antiserum for serum titer test;

2.9 weeks later, 0.2mg of FGF21 recombinant protein without adjuvant was intravenously injected to boost the immunity.

3. Antiserum assay

3.1, Performing ELISA titer test on antisera of four rabbits after immunization by using the prepared FGF21 antigen protein respectively, and the results are shown in Table 1;

3.2 selection of the RB223 rabbit with highest antisera titer for whole blood.

Table 1 results of antiserum titration in four rabbits

4. Single B cell culture and FGF21 positive well selection

4.1 Isolating whole blood B lymphocytes;

4.2 spread isolated B lymphocytes at 3000 cells/well to sterile 96 Kong Biaoban with FGF21 antigen plates, cultured overnight;

4.3 washing twice with PBS containing 10% fetal bovine serum;

4.4 culturing the residual B lymphocytes after washing with 1640 medium;

4.5 after 5 days of culture, the supernatant was taken and positive wells were screened by ELISA indirect method, the results are shown in tables 2.1-2.6;

TABLE 2.1-TABLE 2.6RB223B lymphocyte titration results

TABLE 2.1	1	2	3	4	5	6	7	8	9	10	11	12
													A	0.263	0.182	0.392	0.254	0.298	0.178	0.179	0.309	0.155	0.336	0.368	0.316
B	0.198	0.257	0.242	0.206	0.221	0.383	0.366	0.390	0.391	0.139	0.251	0.192
													C	0.360	0.123	0.125	0.208	0.157	0.370	0.147	0.249	0.399	0.195	0.397	0.262
D	0.112	0.386	0.147	0.595	0.347	0.123	0.295	0.332	0.395	0.330	0.271	0.181
													E	0.233	0.116	0.155	0.359	0.348	0.181	0.199	0.124	0.242	0.214	0.233	0.174
F	0.141	0.279	0.194	0.252	0.154	0.137	0.221	0.231	0.326	0.214	0.388	0.308
													G	0.227	0.201	0.386	0.247	0.337	0.125	0.266	0.249	0.306	0.302	0.168	0.252
H	0.249	0.236	0.337	0.119	0.388	0.132	0.355	0.233	0.231	0.124	0.297	0.279

TABLE 2.2	1	2	3	4	5	6	7	8	9	10	11	12
													A	0.263	0.323	0.277	0.112	0.138	0.189	0.386	0.258	0.145	0.214	0.373	0.217
B	0.204	0.314	0.124	0.384	0.215	0.280	0.228	0.288	0.364	0.210	0.226	0.232
													C	0.235	0.204	0.211	0.135	0.342	0.246	0.295	0.213	0.303	0.267	0.392	0.205
D	0.189	0.222	0.587	0.280	0.251	0.131	0.145	0.208	0.302	0.284	0.371	0.176
													E	0.356	0.384	0.154	0.269	0.176	0.290	0.234	0.177	0.171	0.258	0.302	0.169
F	0.184	0.237	0.352	0.261	0.314	0.172	0.316	0.286	0.190	0.279	0.207	0.396
													G	0.314	0.361	0.351	0.318	0.258	0.150	0.174	0.163	0.251	0.327	0.373	0.297
H	0.291	0.332	0.346	0.134	0.262	0.302	0.336	0.164	0.284	0.233	0.398	0.116

TABLE 2.6	1	2	3	4	5	6	7	8	9	10	11	12
													A	0.327	0.118	0.370	0.164	0.319	0.335	0.392	0.254	0.177	0.332	0.327	0.147
B	0.244	0.287	0.361	0.309	0.293	0.385	0.201	0.313	0.389	0.199	0.136	0.270
													C	0.209	0.703	0.281	0.268	0.295	0.329	0.115	0.329	0.148	0.257	0.282	0.379
D	0.373	0.162	0.311	0.392	0.322	0.347	0.222	0.282	0.377	0.278	0.295	0.234
													E	0.120	0.187	0.398	0.251	0.216	0.283	0.385	0.288	0.243	0.319	0.327	0.320
F	0.395	0.375	0.284	0.379	0.371	0.218	0.204	0.117	0.143	0.345	0.159	0.339
													G	0.214	0.362	0.389	0.354	0.240	0.315	0.201	0.116	0.239	0.383	0.164	0.205
H	0.363	0.378	0.361	0.156	0.316	0.231	0.120	0.248	0.194	0.316	0.357	0.332

5. Construction of antibody Gene plasmid for Positive well and expression of antibody (Small-Scale production)

5.1 In order to construct antibody gene plasmids, 24 Kong Yangxing-hole B cells are firstly selected, the antibody gene plasmids are respectively constructed through a molecular cloning technology, 10 antibody gene plasmids are successfully constructed, and specifically, DNA sequences of antibody genes are extracted from the cells through PCR amplification and enzyme digestion technologies. Then, the DNA fragments are connected with plasmid vectors to form recombinant plasmids, the process involves key steps of restriction enzyme cutting, DNA connection, transformation and the like, each plasmid is precisely designed and verified, and plasmid construction numbers and successful construction numbers are shown in Table 3;

5.2 expression of plasmids constructed successfully by transfecting eukaryotic cells to obtain the corresponding antibody supernatant (miniprep);

5.3 purification of miniprep antibodies, see fig. 1, for monoclonal antibody purification results, wherein Marker: protein molecular mass standard, rear lane: after purification of the antibody, furthermore, bands were clearly visible at a molecular weight of about 20kD as analyzed by SDS PAGE, and no other bands appeared, which experiments indicated that the purified antibody was very pure and unaffected by other impurities. These data demonstrate the effectiveness and reliability of the purification system, providing important technical support for the preparation of high purity antibodies.

TABLE 3 antibody Gene plasmid numbering

Positive hole site numbering

1D4

2D3

3D5

4F4

5C3

6C2

7E4

8E2

9F4

10D2

10G1

11E3

Plasmid construction numbering

#1

#2

#3

#4

#5

#6

#7

#8

#9

#10

#11

#12

Build success number

Failed

#2

Failed

#5

Failed

#7

Failed

#10

#11

Failed

Positive hole site numbering

12E1

13E2

14C1

15E2

15G3

16A2

17A3

18D1

19D2

19F5

20A11

20G2

Plasmid construction numbering

#13

#14

#15

#16

#17

#18

#19

#20

#21

#22

#23

#24

Build success number

#13

Failed

#17

Failed

#19

Failed

#21

#22

Failed

6. Chessboard screening optimal pairing

6.1, Respectively marking half of 10 monoclonal antibodies obtained by small-scale production with biotin, and reserving the other half of the monoclonal antibodies;

6.2ELISA Sandwich method 10 (pairs of) antibodies were subjected to a checkerboard assay with antigen, the results are shown in Table 4;

6.3 selecting the best pairing of 6 pairs, see table 5;

6.4ELISA Sandwich method the best 6 pairs of paired antibodies were tested with known FGF21 positive human serum to obtain 2 pairs of rabbit monoclonal antibodies positive against human serum samples, see Table 6.

Table 4 checkerboard screening data

	#2-Biotin	#5-Biotin	#7-Biotin	#10-Biotin	#11-Biotin	#13-Biotin	#17-Biotin	#19-Biotin	#21-Biotin	#22-Biotin
											#2	0.151	0.862	0.423	0.481	0.571	0.781	0.881	0.812	0.671	0.589
#5	0.734	0.176	0.721	0.211	0.215	0.982	0.131	0.251	0.356	0.431
											#7	0.732	1.334	0.462	0.567	1.658	0.924	0.883	1.239	0.891	0.591
#10	0.799	0.178	0.899	0.143	0.212	0.905	0.776	0.188	0.341	0.881
											#11	0.869	0.348	0.433	0.312	0.302	0.345	0.315	0.313	0.661	0.556
#13	0.794	0.982	0.895	0.334	0.923	0.331	0.335	1.288	0.443	0.944
											#17	0.931	0.173	0.491	0.901	0.303	0.661	0.141	0.211	1.321	0.976
#19	0.799	0.422	0.322	0.561	0.537	0.761	0.478	0.367	0.453	0.63
											#21	0.512	0.766	1.315	0.605	0.521	0.604	0.601	0.825	0.241	0.254
#22	0.541	0.851	0.291	0.623	0.708	0.607	0.722	0.761	0.404	0.318

TABLE 5 optimal 6 pairs of paired antibodies

Optimal pairing table
	#7&#5-Biotin
#7&#11-Biotin
	#7&#19-Biotin
#13&#19-Biotin
	#17&#21-Biotin
#21&#7-Biotin

TABLE 6FGF 21-Positive human serum titration data

7. Mass production of optimal pairing

7.1, Selecting a #7& #5 antibody pair and a #17& #21 antibody pair, which have higher sensitivity in the pair of 2 pairs of human serum sample positive antibodies, and performing a large number of plasmid amplification and extraction;

7.2, transfecting eukaryotic cells with the amplified plasmid for mass production and purifying to obtain an antibody which is the core raw material of the FGF21ELISA reagent.

EXAMPLE 1FGF21 assay kit

1. Preparation of enzyme-linked immunosorbent reagent:

1.1 preparation of biotin-labeled antibodies: dissolving biotin with DMSO, mixing biotin and FGF21 capture antibody according to a molar ratio of 1:20, standing for 2 hours at room temperature, removing redundant free state biotin by ultrafiltration and centrifugation, and replacing the free state biotin with PBS buffer solution for standby;

1.2 horseradish peroxidase (HRP) labeled antibody: FGF21 detection antibody is marked by sodium periodate oxidation method. The marked antibody is ultrafiltered and centrifugally replaced in PBS buffer solution, and the antibody is preserved for standby;

2 sample preparation: taking about 5ml of venous blood, centrifuging in time to obtain a supernatant, and placing the supernatant into a centrifuge tube to serve as a blood plasma sample for detection, and preserving at-20 ℃ for later use;

3 specific experimental steps:

3.1 streptavidin coated microwell plates: dissolving streptavidin with sodium carbonate buffer (pH 9.6), adding 100ul per well, and coating at 4deg.C overnight;

3.2 washing: removing the coating liquid, adding 300ul of 1 XPBST washing plates, repeating for 3 times each for 3min, and finally beating on absorbent paper;

3.3 closing: 200ul of 1% BSA blocking solution is added to each well, and incubated for 1h at room temperature;

3.4 washing: removing the coating liquid, adding 300ul of 1 XPBST washing plates, repeating for 3 times each for 3min, and finally beating on absorbent paper;

3.5 sample addition: adding 50 ul/hole of each of the FGF21 biotin labeled antibody, the standard substance or the sample to be detected and the FGF21 enzyme-labeled antibody into a microplate, and incubating at 37 ℃ for 1h;

3.6 washing: removing the coating liquid, adding 300ul of 1 XPBST washing plates, repeating for 3 times each for 3min, and finally beating on absorbent paper;

3.7 developing: adding 100 ul/hole of TMB substrate, developing for 15min, adding 100 ul/hole of stop solution 1N H2S04, and stopping the reaction;

3.8 reading and calculating the result: and reading at the wavelength of 450nm by using an enzyme-labeled instrument, drawing a standard curve, and calculating the concentration of the antigen in the sample to be detected.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the present invention is not limited to the embodiments described above, but is capable of numerous variations and modifications without departing from the spirit and scope of the invention as hereinafter claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1.A method for detecting fibroblast growth factor 21, comprising the steps of:

a, preparing FGF21 antibody pair raw materials;

c preparation of biotin-labeled antibody: dissolving biotin with DMSO, mixing biotin and FGF21 capture antibody according to a molar ratio of 1:10-1:50, standing at room temperature, removing redundant free state biotin, replacing with PBS buffer solution, and preserving for later use;

2. The method of claim 1, wherein step a further comprises FGF21 antigen preparation:

② Constructing FGF21 plasmid;

3. The method of claim 1, wherein step a further comprises the step of immunizing the animal:

① Selecting an immunized animal;

② The FGF21 recombinant protein is emulsified by Freund's incomplete adjuvant and then used as an antigen for immunization;

③ Primary immunization: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

④ Two weeks later, a second immunization: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

⑤ Third immunization after one week: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

⑥ Four immunization after two weeks: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

⑦ Fifth immunization after two weeks: each animal was multi-injected with a total of 0.2-0.5mg of emulsified antigen;

⑧ Taking a small amount of whole blood after one week, and centrifuging to prepare antiserum for serum titer test;

⑨ After one week, 0.2-0.5mg of FGF21 recombinant protein without adjuvant is injected intravenously for enhancing immunity.

4. The method of detecting fibroblast growth factor 21 according to claim 1, wherein step a further comprises: antiserum detection, single B cell culture, FGF21 positive hole screening, antibody gene plasmid construction of positive holes, antibody expression, chessboard screening antibody pairing and large-scale production of antibody pairing.

5. The method of claim 3, wherein the immunized animal is one of a rabbit, sheep, and guinea pig.

6. The method of detecting fibroblast growth factor 21 of claim 4, wherein the single B cell culture and FGF21 positive well selection comprises:

① Isolating whole blood B lymphocytes;

③ Washing twice with PBS containing 10% -20% fetal bovine serum;

④ Culturing the rest B lymphocytes after washing with 1640 culture medium;