A METHOD OF DISCOVERING SPECIFIC FUNCTIONAL ANTIBODIES

TECHNICAL FIELD OF THE INVENTION

The invention relates to the biotechnology field, in particular to a method of discovering specific functional antibodies, in particular to a method of discovering specific functional antibodies based on the composition and frequency analysis of variable regions of immune host antibodies.

BACKGROUND TECHNOLOGY

A completely identical antibody produced by a single B-cell and only against a specific antigen epitope is called a monoclonal antibody. Since Kohler and Milstein reported that hybridoma techniques were used to obtain monoclonal antibodies against sheep erythrocytes in 1975, more and more monoclonal antibodies have been widely used in the fields of detection of medical diagnosis and separation of biomolecules and others. Because of their strong specificty, high purity and good uniformity, monoclonal antibodies have improved the efficiency and accuracy of medical testing. In recent years, with the development of tumor immunotherapy, monoclonal antibodies against immune checkpoint show a great prospect in tumor immunotherapy.

In addition to hybridoma techniques, many techniques have been applied to the production of monoclonal antibodies, such as vitro display techniques represented by phage display, single cell cloning techniques, EB virus-mediated B cell immortalization techniques, protein spectrum analysis combined with DNA high-throughput sequencing techniques etc. These techniques have their own strong points and weakness, for example, hybridoma techniques are limited to a limited variety such as mice or rabbits, and the subsequent isolation of monoclonal hybridoma is time-consuming and huge work. The production of monoclonal antibodies is unstable and easy to be lost. The vitro display techniques also require a long period and the generated antibodies are the same as hybridoma antibodies which need to be humanized.

The method of antibody discovery based on high-throughput sequencing developed in recent years reveals the variation of specific antibody abundance in antibody gene spectrum under specific immune status by comparing the frequency of CDR3 sequences in host antibody gene spectrum before and after immunization. The full-length sequence of CDR3 sequence with largest frequency variation is selected for functional validation in vitro pairing expression as a candidate sequence of specific antibodies. This method is not limited to species. A large number of candidate pairs can be generated from the antibody gene spectrum in a short period for functional verification. It is possible to obtain antibodies with high affinity.

The characteristics of second generation sequencing, such as reading length, accuracy, throughput, cost performance and son on, are suitable for studying the diversity of antibody library composition, but there are some technical difficulties in antibody sequencing library construction, high throughput antibody sequencing and data analysis etc. As a result, the obtained antibody gene spectrum information is inaccurate, the contained antibody gene information is biased, it is difficult to accurately define the variation of CDR3 frequency, and the verification efficiency of vitro pairing function of the selected full-length genes is low.

Second generation sequencing has been widely used to study the diversity of the composition of antibody gene spectrum. However, how to avoid the introduction of biases in the sequencing library construction is always a difficult problem. As present, RT or RACE methods are frequently used to construct high-throughput antibody sequencing library. RT method designs primers and amplifies the variable regions of antibody according to the conserved sequences of known variable regions of antibody sequences. Because of the very high sequence diversity of antibody genes, multiple primers or merger primers are frequently used to amplify the multiple PCR in the real operation, in order to cover the antibody genes to the maximum extent. However, multiple primer amplification, sample RNA degradation, incomplete database and so on inevitably introduce biases in library construction and data analysis. As a result, the results of sequencing cannot truly reflect the composition and changes of the variable regions of antibody. Another difficulty in constructing RACE library is that the requirements of quality and quantity of sample RNA are high. However, it is difficult to obtain enough samples in practical work; in particular, the number of clinicopathological samples is often difficult to meet the needs of RACE library construction.

SUMMARY

According to the shortcomings of the prior arts and the actual requirements, the invention provides a method of discovering specific functional antibodies. The method can roundly study the composition and distribution of the variable regions of multi-species antibody genes without bias.

For this purpose, the invention adopts the following technical scheme:

The invention provides a method of discovering specific functional antibodies, including the following steps:

(1) Extract the total RNAs of at least one target of immunize host subjects and construct a high-throughput antibody sequencing library;

(2) The high-throughput sequencing library constructed by step (1) is used for high-throughput sequencing of the variable regions of the immunoglobulin gene, and the gene spectrum of variable regions of antibody for the at least one target anitgen is obtained.

(3) Candidate CDR3 and corresponding heavy chain and light chain antibody nucleic acid sequences are selected as candidate pairing sequences from the variable region gene spectrum of antibody.

(4) The light chain and heavy chain genes are selected to pairwise express in vitro to produce candidate recombinant antibodies.

Among them, the selection of candidate CDR3 from the gene spectrum of the variable regions of antibody includes the analysis of the results of high-throughput sequencing of Read1 and/or Read2 separately, select candidate CDR3 homologous clusters, and combine with the results of Read1 and Read2 splicing, the full-length gene of the variable regions of antibody containing this CDR3 homologous cluster is identified.

In the invention, the result of Read1 and/or Read2 and the result of Read1 and Read2 splicing are analyzed.

As the preferably technical scheme, the method of constructing high-throughput antibody sequencing library described in step (1) is RACE library construction and/or RT library construction, such as single RACE library construction single RT library construction or both RACE library construction and RT library construction at the same time.

Preferably, the RACE library construction includes the following steps:

(a) Obtain the cells of the subjects and isolate the total RNA;

(b) Use oligo (dT) as primer and total RNA in step (a) as template, synthesize cDNAs by reverse transcription.

(c) Use cDNA produces in step (b) as template, the high-throughput antibody sequencing library is constructed by using the amplicon library construction method after using the two-step PCR amplification method or the first step PCR method to amplify the antibody genes.

The invention constructs the RACE libraries of the heavy chain, Kappa chain and Lambda chain respectively, uses the Illumina system to sequence the library, to sequence the heavy chain library separately, and to combine then sequence or separately sequence the Kappa and Lambda chain libraries.

In the invention, the optimized two-step PCR amplification is used to construct the library, which can reduce the RNA input volume of the sample to 20 ng, simplify the construction process of the library, and maintain the coverage degree and accuracy of the obtained gene spectrum of the variable regions of antibody.

Preferably, the RT library construction includes the following steps:

(a′) Obtain the cells of the subjects and isolate the total RNA;

(b′) Use oligo(dT) as primer and total RNA in step (a′) as template, synthesize cDNAs by reverse transcription.

(c′) Use cDNA produced in step (b′) as template, specific primers are used to amplify the antibody genes, and then the PCR amplified products are constructed DNA library.

In the invention, the PCR amplified program in the RACE library construction and RT library constructions is as follows:

- {circle around (1)} 95°C. 2 min;
- {circle around (2)} 95°C. 30 sec, Tm 30 sec, 72°C. 30 sec; 15-35 cycles
- {circle around (3)} 72°C. 7 min;
- {circle around (4)} 4°C. store

Preferably, the subjects described in the step (a) of the RACE library construction should be any or at least two combinations of mammals, amphibians, fish or birds;

Preferably, the mammals should be any or at least two combinations of humans, mice, primates, rabbits, amphibians, fish or birds.

Preferably, the cells described in the step (a) of the RACE library construction should be derived from any or at least two combinations of the peripheral blood, lymphoid organs, spleen, bone marrow or liver of the subjects;

Preferably, the cells described in the step (a) of the RACE library construction should include any or at least two combinations of memory B cells, plasma cells, or plasmablast.

Preferably, the primers of PCR used in the first round of two-step PCR amplification described in step (c) of RACE library construction include forward primer and reverse primer. The forward primer contains partial forward joint sequence and sequence as shown by SEQ ID NO.1. The reverse primer contains a partial reverse joint sequence and a sequence as shown in one of the SEQ ID NO.2-4;

SEQ ID NO. 1:

AAGCAGTGGTATCAACGCAGAGTA;

SEQ ID NO. 2:

GGAAGACCGATGGGCCCTTGGTGG;

SEQ ID NO. 3:

GCAGGCACACAACAGAGGCAGTTCCAG;

SEQ ID NO. 4:

CACACCAGTGTGGCCTTGTTGGCTT

The forward primers used in the first round of PCR in the two-step PCR amplification described in the step (c) of the RACE library construction are as follows:

Partial joint sequence —AAGCAGTGGTATCAACGCAGAGTA (The forward primer is from Clontech SMARTer RACE 5′/3′ Kit).

As an example, the reverse primers used in the first round of PCR in the two-step PCR amplification described in the step (c) of the RACE method can adopt below three modes:

Partial joint sequence —GGAAGACCGATGGGCCCTTGGTGG (heavy chain IgG reverse primer); Partial joint sequence —GCAGGCACACAACAGAGGCAGTTCCAG (kappa reverse primer); Partial reverse joint sequence —CACACCAGTGTGGCCTTGTTGGCTT (lambda reverse primer)

Preferably, the partial forward joint sequence includes 5-60 bp of the 3′ end of the Illumina forward joint primer, and the partial reverse joint sequence includes 5-60 bp of the 3′ end of the Illumina reverse joint primer;

Preferably, the sequence of the Illumina forward joint primer is shown by SEQ ID NO.5, and the Illumina reverse joint primer is shown by SEQ ID NO.6.

SEQ ID NO. 5:

AATGATACGGCGACCACCGAGATCT

ACACTATAGCCTACACTCTTTCCCTACAC

GACGCTCTTCCGATCT;

SEQ ID NO. 6:

CAAGCAGAAGACGGCATACGAGATA

GCTTCAGGTGACTGGAGTTCAGACGTGT

GCTCTTCCGATCT.

Preferably, the primers used in the second round of PCR in the two-step PCR amplification described in the step (c) of the RACE library construction are conventional Illumina library construction primers.

Preferably, the high-throughput sequencing is composed by any or at least two combinations of sequencing by synthesis, sequencing by joining, sequencing by hybridization, single molecule DNA sequencing, multiple polymerase community sequencing or nano-pore sequencing, then selected as sequencing by synthesis, and further selected as Illumina platform sequencing.

In the invention, using Illumina Miseq 2x300 sequencing system, although the antibody variable region gene sequencing is long, it can basically reach the upper limit of Illumina sequencing, but compared with other high-throughput sequencing methods such as Roche 454, Illumina Miseq 2x300 system is with high accuracy, high-throughput and low cost; compared with the third generation sequencing method, although it does not have the strong point in reading long, but the strong points of Illumina system in throughput and sequencing cost are obviously.

In the invention, the analysis method adopts the method of two-end sequencing and then selecting, which makes up for the deficiency of the reading length of Illumina sequencing, and the full-length sequence can be obtained more accurately by two-end sequencing.

In the invention, it is difficult for the existing Illumina Miseq 2x300 system to ensure the full length of the antibody variable region. An optimized selected method of full length of the antibody variable region is developed, and the full-length sequence of the antibody variable region is obtained by combining the results of RACE and RT library construction and sequencing, and the bias due to incomplete data can be reduces.

Preferably, the principle of selecting candidate CDR3 is: any or at least two combinations of high frequency sequence after CDR3 clustering, selection CDR3 sequences with significantly higher frequency after immunization or outbreak phase than in pre-immunization or convalescence phase, or the sequence of V gene family corresponding to CDR3 after immunization or outbreak phase significantly different from that in the pre-immunization or convalescence phase.

Preferably, the selection of candidate CDR3 in the step (3) and corresponding heavy chain and light antibody nucleic acid sequences as candidate pairing sequence includes the following steps:

(1′) Select candidate CDR3 homologous clusters;

(2′) Anchor the CDR3 homology cluster, and the full length amino acid sequences of the high frequency antibody heavy chain and the light chain variable region containing the CDR3 homology cluster are selected as the first and second pairing candidate sequences in the gene spectrum of the variable region of antibody;

(3) Determine nucleic acid sequences of first and second pairing candidate sequences; Preferably, the selectin in the step (2′) of the full-length amino acid sequences of the high-frequency antibody heavy chain and the light chain variable region containing this candidate CDR3 homologue cluster includes the following steps: all the Read1 and Read2 of the CDR3 homologous cluster are selected from the gene spectrum of antibody variable region to compare the antibody database and get the most frequent amino acid sequence in CDR region and FR region; at the same time, the sequence results Read1 and Read2 are spliced, and all the spliced sequences are compared with the antibody database to determine the highest frequency amino acid sequences in the CDR region and FR region; compare and combine the amino acid sequences of CDR region and FR region; compare and combine the amino acid sequences of CDR region and FR region obtained from two sources, obtain the full-length amino acid sequence of the variable region of antibody corresponding to the CDR3 homology cluster as the candidate pairing sequence, and select the corresponding full-length nucleotide sequences of the highest frequency variable region in the Read1 and Read2 spliced sequences.

Preferably, the full-length nucleotide sequence of the variable region described in step (3′) is compared with the sequence of the highest frequency antibody variable region containing specific CDR3 homology cluster obtained by RT library construction, and the full-length sequence of the variable region of antibody is obtained as a candidate pairing sequence.

In the invention, for the partial antibody variable region, due to the inclusion of a longer CDR region, FR region or a longer 5′UTR region, the splicing rate is very low, so it is difficult to obtain the full length sequence of the high frequency variable region by the method of sequentially selection with the locking of CDR3. The invention adopts RT method to construct library at the same time to make up for the problem of low splicing rate of RATE library.

In the invention, the method of obtaining full-length antibody by combining RACE library sequencing and RT library sequencing is as follows: parallel use RACE library construction sequencing and RT library construction sequencing for the same sample, compare the consistency of high-frequency CDR3 in the two libraries, when achieve a certain standard under the consistency, lock CDR3 and select the highest frequency DNA sequence containing the CDR3 in the RT library.

Preferably, the method also includes the identification of selected antibodies.

Preferably, the identification includes the identification of the obtained antibody with at least one target antigen;

Preferably, the identification steps include scFv or Fab fragments or IgG of the antibody obtained by expressing in vitro, and the resultant scFv, Fab fragments or IgG with the target antigen;

Preferably, the vitro expression method is to express scFv or Fab fragments through the display method of prokaryotic cells, phages or yeast systems, or to express Fab or IgG through the exogenous gene of mammalian cells;

Compared with the prior arts, the invention has the following beneficial effects:

(1) The analysis method of the invention adopts the method of two-end sequencing and splicing then selecting, which makes up for the deficiency of the reading length of the Illumina sequencing, and the full-length sequence can be obtained more accurately by the two-end sequencing;

(2) The invention adopts the optimized two-step PCR amplification library construction, which can reduce RNA input amount of the sample to 20 ng, simplify the construction process of the library, and maintain the coverage degree and accuracy of the acquired variable region gene spectrum of the antibody.

(3) The invention aims at the existing Illumina Miseq 2x300 system which is difficult to obtain the full length of the variable region of antibody, develops an optimized method of selectin full length of the variable region of antibody, develops an optimized method of selecting the full length of the variable region of antibody, obtain the full-length sequence of the variable region of antibody by combining with the results of RACE and RT library construction sequencing, and reduces bias due to incomplete data;

(4) The discovery method for specific antibodies in the invention is simple, fast and low cost, can detect micro samples, and makes a foundation for the discovery of a large number of antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the optimized discovery method for antibodies in the invention.

FIG. 2 is a schematic diagram of concrete steps of the embodiment 1 of the invention.

FIG. 3 is the correlation analysis of antibody gene CDR3 frequency of different RNA inputs: FIG. 3(a) is the comparison of heavy-chain CDR3 when RNA input is 600 ng and 20 ng; FIG. 3(b) is the comparison of heavy chain CDR3 when RNA input is 600 ng and 100 ng; FIG. 3(c) is the comparison of kappa CDR3 when RNA input is 600 ng and 20 ng; FIG. 3(d) is the comparison of kappa CDR3when RNA input is 600 ng and 100 ng; FIG. 3(e) is the comparison of lambda CDR3 when RNA input is 600 ng and 20 ng; FIG. 3(f) is the comparison of lambda CDR3when RNA input is 600 ng and 100 ng.

FIG. 4 is a pairing expression result of a candidate dengue virus antibody of the embodiment 2 in the invention.

FIG. 5 is a pairing expression result of a candidate flu virus antibody of the embodiment 3 in the invention;

FIG. 6 is the correlation analysis of CDR3 frequency of RT and RACE libraries: FIG. 6(a) is the correlation analysis of the CDR3 frequency of heavy chain in RT and RACE libraries; FIG. 6(b) is the correlation analysis of CDR3frequency Lambda chain in RT and RACE libraries.

SPECIFIC IMPLEMENTATIONS

In order to further elaborate the technical means adopted by the invention and its effect, the technical scheme of the invention is further explained by combining with the drawings and by concrete embodiments, but the invention is not limited to the scope of the embodiment.

Embodiment 1 An Optimized High-Throughput Library Construction Method for Few Samples

The concrete steps to build the library are shown in FIG. 2. Sample preparation and first chain cDNA synthesis: Peripheral blood mononuclear cells (PBMC) are isolated from human or animal peripheral blood by density gradient centrifugation. Total RNAs are isolated from PBMC by Trizol. 20 ng, 100 ng and 600 ng RNAs are used as template and oligo (dT) is used as primer for the synthesis of first-chain cDNA. The concrete operation can be carried out by referring to the manual of SMARTer® RACE 5′/3′ Kit.

The optimized two-step PCR method is used to construct the antibody library. In the first round of PCR amplification, the forward primer is the optimized SMARTer® RACE 5′/3′ Kit UPM with the partial joint primer containing Illumina, the reverse primer containing the sequence of the invariant region of the antibody and the partial join primer containing Illumina. VH, VK and VL are amplified respectively. The reaction system is

Reaction System

Component
Volume

PCR
1 × TransStart ® FastPfu Fly
5 μL

reaction
Buffer

Optimized UPM
5 μL

Reverse primer
0.2 μL

TransStart ® FastPfu Fly DNA
0.5 μL

Polymerase

cDNA
5 μL

dNTPs
0.2 mM

Total volume
50

PCR reaction conditions are as follows:

Reaction program
Cycles number

Amplification
95° C. 2 min
1

program
95° C. 30 s
25

67° C.-62° C. 30 s

72° C. 30 s

72° C. 7 min
1

The first round of PCR products are recovered by magnetic beads.

The primers of the second round of PCR is conventional Illumina library construction primer, and the reaction system is as follows:

Reaction System

Component
Volume

PCR
1 × TransStart ® FastPfu Fly
5 μL

reaction
Forward primer
0.2 μM

Reverse primer
0.2 μM

TransStart ® FastPfu Fly DNA
0.5 μL

Polymerase

First round PCR recovered

product
20 μL

dNTPs
0.2 mM

Total volume
50

PCR reaction conditions are as follows:

Reaction program
Cycles number

Amplification
95° C. 2 min
1

program
95° C. 30 s
6-8

60° C. 30 s

72° C. 30 s

72° C. 7 min
1

The steps of gel cutting and purification of products can be referred to the manual of QIAquick Gel Extraction Kit. After the library is constructed, Bioanalyzer High Sensitivity DNA chip is used for QC, sequencing platform is Illumina Miseq 2x300, the result is shown in FIG. 3(a)-(f).

The results of FIG. 3(a)-(f) show that the frequency correlation R2 values of heavy chain, Kappa chain and Lambda chain CDR3 are 0.83, 0.90 and 0.98 respectively at the 20 ng RNA input, comparing with 600 ng RNA input, it shows a high correlation. And the figure shows the ranking of high frequency CDR3, For example, CDR3, which ranks the top 10 in the gene spectrum of the variable region of antibody, also shows a high consistency at 600 ng, 100 ng and 20 ng RNA inputs. The comparison of CDR3 frequency correlation explains that the RACE library constructed with 20 ng RNA input can meet the requirements of antibody library sequencing in terms of antibody gene coverage and CDR3 region bias.

Embodiment 2 An Optimized Antibody Gene Screening Method for Dengue Virus Monoclonal Antibody

Use the method of RACE antibody library construction described in embodiment 1, total RNAs are isolated form PBMC of dengue patients in acute stage and convalescence stages, and construct the RACE libraries of heavy chain, Kappa chain and Lambda chain respectively. Use Illumina Miseq 2x300 system to sequence the library: the heavy chain library is sequenced alone, and the Kappa and Lambda chain libraries are combined and sequenced. The sequencing results are processed by software Trimmomatic-0.30, the parameters are set as follows: phred33, LEADING:20, TRAILING:20, SLIDINGWINDOW:20:20, MINLEN:200). Remove poor quality data. The following improvements are made to data analysis. Comparing the data amount in the antibody library after removing and preserving Singleton sequence, it is found that the data amount changes greatly after Singleton is removed, so Singleton sequence cannot be removed. And the way of repeated sequence normalization can reduce the occupation of server resources: first, find out each sequence and its repeat times, then analyze each sequence by IgBlast, and obtain each CDR3 frequency by counting the repeat times.

TABLE 1

Comparison of data amount of antibody

library before and after removing Singleton

sequence

Reads amount before
Reads amount after

removing singleton
removing singleton

6H-R1
30179
4573

6H-R2
30179
471

6K-R1
574845
141848

6L-R1

6K-R2
574845
16785

6L-R2

TABLE 2

Comparison of data amount of antibody

library before and after repeated sequence

normalization

Reads amount before
Reads amount after

repeated sequence
repeated sequence

normalization
normalization

6H-R1
30179
27278

6H-R2
30179
29913

6K-R1
574845
463091

6L-R1

6K-R2
574845
563921

6L-R2

TABLE 3

Comparison of library quality of dengue

patients in acute and convalescent stages

Sequence

amount
Q30 index

Sequence
after
after

Library
amount
organization
organization

Convalescent
2365300
812294
81.90

stage H

Convalescent
1569720
647412
82.88

stage K

Convalescent

stage L

Acute stage H
4721106
60358
83.98

Acute stage K
1726272
1149690
82.45

Acute stage L

Because CDR3 sequences are almost unique markers for different antibody genes, the abundance of CDR3 sequences can be evaluated in the gene spectrum of the variable region of antibodies according to the number of obtained CDR2 sequences. The sequences of FR region and CDR region are determined according to IgBlast website, but the sequence of CDR3 region is not determined by IgBlast website, so we use characteristic sequence of FR4 region to determine CDR3 amino acid sequence, and the characteristic sequence of heavy chain FR4 initiation region is WG*G or GQG, characteristic sequences of Kappa and Lambda chains are FG*GT. Run IgBlast respectively for the Read1 and Read2 sequences and merge the results, and select the pairing candidate CDR3 sequence based on one or all of the following principles: 1) High frequency sequence after CDR3 clustering; 2) Select the CDR3 sequences which are significantly higher in after-immunization or outbreak sage than those in the pre-immunization or convalescent stage; 3) The V gene family corresponding to CDR3 is significantly different between that of after-immunization or outbreak stage and that of pre-immunization or convalescent stage.

The CDR3 frequency changes of heavy chain and light chain of dengue patients in acute and convalescent stages are show in tables 4, 5, and 6. The results show that the concentration of CDR3 in acute stage is significantly higher than that in convalescent stage, indicating that it may be related to specific antigen.

TABLE 4

Analysis of the CDR3 frequency of

heavy chain of dengue patients in the acute

and convalescent stages

Frequency

of

Frequency
convalescent
CDR3

Heavy chain
of acute
stage
Cluster

CDR3 sequence
stage (%)
(%)
(%)

ARALSEKSLT
8.53
0.11
9.76

TSYLDC

VKDASTTSIG
5.97
0.03
10.04

AAPFDY

THRRPSLRYP
5.45
0.15
5.62

DV

ASLGGTVTDA
3.13
0.03
5.55

FDL

AKDASTTSKG
2.49
0
3.14

AAPFDY

ARGFTYGHYF
2.23
0
2.24

DY

VKDASTTSIG
2.05
0
10.04

AAPFDN

AKTVVTAPGV
1.86
0
2.15

FDY

ASDILDAFDV
1.84
0
1.85

VKDASTTSIG
1.72
0
10.04

AAPFDS

ASLGGTVTDA
1.21
0
5.55

FDV

ASLGGTVTDA
1.15
0
5.55

FDI

ATGYTYGYYF
1.00
0
1.01

DY

TABLE 5

Analysis of CDR3 frequency of Kappa

chain of dengue patients in the acute and

convalescent stages

Frequency

Frequency
of
CDR3

Kappa chain
of acute
convalescent
Cluster

CDR3 sequence
stage (%)
stage (%)
(%)

QQANSFPWT
5.94
0.06
6.31

HQYSSWPPG
4.87
0.18
5.22

GT

HQYNSWPPG
2.83
0
3.04

GT

HQYNAWPPG
2.81
0
3.78

GT

MQGTHWRS
2.63
0
2.68

QQYSFWPWT
2.45
0
2.56

QQYDSFPLT
1.68
0
1.75

QQANNFPWT
1.57
0
1.66

HQYTSWPPG
1.28
0
1.37

GT

QQRSNWPRT
1.24
0.01
1.3

QQISNFPIT
1.17
0
1.2

QQSFSPPWT
1.09
0.07
1.17

QQSGSSLH
1.02
0
1.27

QQRSTWPYT
1
0.07
1.09

TABLE 6

Analysis of CDR3 frequency of

Lambda chain of dengue patients in

the acute and convalescent stages

Frequency of

Frequency
convalescent
CDR3

Lambda chain
of acute
stage
Cluster

CDR3 sequence
stage (%)
(%)
(%)

CSYAASYYDT
10.08
1.06
10.96

GV

SSYRSISPFYV
5.2
0.64
5.45

SSYRSSSPFYV
5.06
0.52
5.18

QVWDRSTNHR
4.49
0.97
4.53

V

QVWDRSSDHR
3.69
0.61
3.82

L

QSYDTSLRAG
3.58
0.65
3.67

V

SSYTGSSTV
2.06
0.19
2.06

LLSYGGAPCV
1.5
0.27
1.58

CSYAGRSTWV
1.48
0.04
1.53

QSFDDSLSGW
1.38
0.24
1.4

V

QVWDRSTNHR
1.26
0.15
1.28

L

CSYAGSNTWI
1.24
0.21
2.55

CSYAGSNTWV
1.16
0.17
2.55

Anchor candidate CDR3 sequences, take the heavy-chain CDR3 sequence THRRPSLRYPDV as an example, run all Read2 and corresponding Read1 containing the CDR3 homology cluster respectively by IgBlast to obtain the amino acid sequences of CDR region and FR region, such as CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4. The results of IgBlast analysis are combined into the R12 file. In parallel, the sequencing results of R1 and R2 are spliced, and the amino acid and nucelotide sequences of each domain are determined by running IgBlast with the spliced sequences, and the Contig file is obtained.

The selection of full length sequence of the variable region follows the following steps: taking the heavy chain. CDR3-THRRPSLRYPDV homologous cluster as an example, the highest frequency variable region of the antibody gene is screened and obtained from the R12 file. The amino acid sequence of each domain are as follows, as shown in Table 7:

FR1(QITLKESGPMLVKPTQTLTLTCTFS)-

CDR1(GFSLSTSGVG)-FR2(VGWIRQPPGEAL

EWLAI)-CDR2(IYWDDDK)-

FR3(RYSPSLRSRLTISKDTSKNQVVLTMTN

LDPVDTATYFC)FR4(WGQG).

Further, the corresponding nucleotide sequences are selected in turn from Contig file according to the sequence of nnFR4-nnCDR3-nnFR3-nnCDR2-nnFR2-nnCDR1-nnFR1-contig. The corresponding full-length nucleotide sequence of the antibody variable region of the highest frequency nucleotide sequence is obtained, which should contain the complete FR and and CDR regions and the signal peptide sequence.

TABLE 7

Heavy chain CDR3 THRRPSLRYPDV

homologous clusters corresponding to

combinations of the highest frequency

FR and CDR regions

Heavy chain-THRRPSLRYPDV-R12

highest frequency amino acid sequence

CDR1
GFSLSTSGVG

FR1
QITLKESGPMLVKPTQTLTLTCTFS

FR3
RYSPSLRSRLTISKDTSKNQVVLTMTNLD

PVDTATYFC

FR4
WGQG

FR2
VGWIRQPPGEALEWLAI

CDR2
IYWDDDK

Take Kappa chain —HQYSSWPPGGT homologous cluster as an example, the most abundant sequence combinations of FR and CDR regions are screened and obtained from the R12 file. The amino acid sequence of each region is as follows: FR1(EIVMTQSPATLSVSPGERATLSCRAS)—FR2(LAWYQHKPGQAPRLLLY)—FR3(TRAAGIPDRFSGSGSGTEFTLTISSLQSEDFAVYFC)—CDR2(GAS) —CDR1(QSVSSN)—FR4(FGQGT), refer to FIG. 8. The corresponding highest frequency nucleotide sequence is selected in turn from the Contig file according to the sequence of nnFR4-nnCDR3-nnFR3-nnCDR2-nnFR2-nnCDR1-nnFR1-contig, the corresponding highest frequency full-length DNA sequence of the antibody variable region is obtained.

TABLE 8

Kappa chain CDR3 HQYSSWPPGGT

homologous clusters corresponding to

combinations of the highest frequency

FR and CDR regions

Kappa-HQYSSWPPGGT-R12 highest

frequency amino acid sequence

CDR1
QSVSSN

FR1
EIVMTQSPATLSVSPGERATLSCRAS

FR3
TRAAGIPDRFSGSGSGTEFTLTISSLQSED

FAVYFC

FR4
FGQGT

FR2
LAWYQHKPGQAPRLLLY

CDR2
GAS

This selecting method of sequence of antibody variable region has the following benefits: while sequencing data are processed, Singleton reads are retained; the splicing rates of variable regions of antibody genes corresponding to different high frequency CDR3 are significantly different, the full length splicing rate of heavy chain high frequency CDR3 sequences in the following table ranges from 34% to 91% (Table 9). If the splicing sequence analysis is used directly, in some cases the data loss may be huge and the CDR3 frequency analysis may even be biased. For current process, R1 and R2 sequences are used for CDR3 composition analysis at first, which avoids the risk of effective data loss and also helpful to obtain accurate frequency of CDR3 sequences; for current process, the highest frequency amino acid sequences information of FR and CDR regions are obtained from R1 and R2 files, and the highest frequency nucelotide sequences of variable to region of antibody gene are selected from Contig sequence file, it can effectively guarantee the sequences of variable regions are the highest frequency sequences, and guarantee the integrity and accuracy of the sequence information. A separate selection of sequences FR and CDR region in the R12 file also verifies the accuracy of the current process as show in FIG. 4.

TABLE 9

Analysis of sequencing and splicing of

variable region of antibody

Splicing

Heavy chain
Reads
Reads
Splicing

CDR3_Seq
amount
amount
rate(%)

ARALSEKSL
2394
810
33.8

TTSYLDC

VKDASTTSI
1676
730
43.6

GAAPFDY

THRRPSLRY
1531
1387
90.6

PDV

ARGFTYGH
628
415
66.1

YFDY

Analysis of antibody gene pairing and expressing: an expression frame is constructed by overlapping PCR, and the expression frame includes promoter, variable region and antibody constant region. The selected antibody heavy chain gene and light chain gene are combined and paired, then transfected 293FT cells for expression. The supernatant is obtained for Elisa assay and tested its affinity with antigen. Concrete implementation steps are as follows: The variable regions of candidate heavy chain and light chain are constructed expression frame by using the method of bridging PCR. The expression frame includes promoter, variable region and constant region of human antibody. The plasmids containing heavy chain and light chain expression frames are paired and transfected 293FT cells for vitro expression and antibody analysis. 293T cells for vitro expression and antibody analysis. 293T cells are collected, adjusted cell density and inoculated with 28-hole plate 1.2×10⁵/hole; after cultivation in an incubator at 37° C. and 5% Co2 for overnight, the cells are transfected when the cell density reaches 60-80%. The plasmids of 0.25 ug heavy chain and 0.75 ug light chain are incubated at room temperature for 5 minutes, mixed with the transfection reagent then cultivated at room temperature for 20 min to form the transfection complex. The transfection complex is added into the cell pore and mixed softly, then cultivated in an incubator at 37° C. and 5% CO2 for 72 hours. The supernatant is collected and detected activity by Elisa. First, an anti-human IgG(Fc) and a detection antigen are diluted with pH 9.6 carbonic acid coating solution to 10 μg/mL, 96-well microtiter plate coated with 100 μL at 4° C. for overnight; or at 37° C, 2 hours; then sealed off by 4% skim milk powder-PBS and 300 μL/well, treated for 1˜2 h at 37° C. The liquid in microtiter plate is discarded, the rest is washed with PBST for three times, then added transfection and cultivated for 48 hours, the supernatant of 100 μl/well is treated at 37° C. for 1 h. The culture medium and PBS control are set up. The liquid in the microtiter plate is discarded and the rest is washed with PBST for three times, and HRP goat anti-human IgG(Fc) 1: 2000 and HRP goat anti-human IgG 1: 5000 are added respectively. then 100 μl/well is treated at 37° C. for 1 h. The liquid in the microtiter plate is discarded, the rest is washed with PBST for five times, and the OPD chromogenic solution is added, 100 μL/well, avoid light for coloration; The absorption value of OD490 wavelength is read by Microplate Reader.

Four heavy chains are paired with five Kappa chains and four Lambda chains respectively. Elisa results (FIG. 4) show that there is no positive result in the pairing of kappa chains and heavy chains, and five positive clones are obtained from the pairing of lambda chains and heavy chains. The positive rate is 5/36.

Embodiment 3 An Optimized Antibody Gene Screening Method for Flu Virus Monoclonal Antibody

Use the method of RACE antibody library construction described in embodiment 1, total RNAs are isolated from PBMC of volunteers before and 7 days after the injection of influenza vaccine, and construct the RACE libraries of heavy chain, Kappa chain and Lambda chain respectively according to the method described in embodiment 2. Use Illumina Miseq 2x250 system to sequence high-throughput. The methods of data processing, the determination of FR and CDR regions, and the selection of candidate CDR3 amino acid sequences are the same as those described in embodiment 1.

TABLE 10

CDR3 frequency analysis of heavy

chain before and after immunization

of influenza vaccine

Frequency
Frequency

Heavy
of 7^th day
before

Frequency

chain
after
immuni-

change of

CDR3_
immuniza-
zation
V
V gene

Seq
tion (%)
(%)
gene
(%)

MSW
4.84
0.07
IGHV2-
100%

NDRV

70*13

VAP

SVVS
4.17
0.05
IGHV3-
75%

FPPY

74*01

EVGG
2.88
0.06
IGHV3-
25%

ERAY

7*01

DRDA
1.95
0.03
IGHV1-
0%

SGDF

3*01

DI

APGG
1.21
0.01
IGHV3-
25%

QWA

7*01

Y

SSVV
0.97
0.03
IGHV3-
75%

SFPP

74*01

Y

DRVT
0.78
0.02
IGHV3-
0%

GDNF

23*01

YYY

MGV

GSTI
0.76
0.01
IGHV3-
100%

MVTL

53*01

LQFF
0.67
0
IGHV2-
100%

EGRH

70*13

MDV

DRVA
0.47
0.02
IGHV3-
0%

SGDF

23*01

DI

LLSG
0.4
0.01
IGHV2-
100%

GENP

70*01

SYYY

HMD

V

KTYG
0.35
0.01
IGHV4-
40%

SGSF

59*01

DYFD

Y

GATV
0.33
0
IGHV3-
100%

VNDL

53*01

EY

GGTI
0.33
0.01
IGHV3-
100%

RVTL

53*01

MNW
0.33
0
IGHV2-

NDRV

70

VDP

GGTI
0.32
0
IGHV3-
100%

MVTL

53*01

DYLS
0.32
0
IGHV1-
100%

GTYT

24*01

PPLY

ERGE
0.28
0
IGHV4-
40%

VGDS

59*01

VDNF

YYY

MDV

PMTY
0.28
0.01
IGHV4-
0

YYDI

4*02

SDAG

AYYF

DT

PPTA
0.28
0.01
IGHV4-
22%

HHFN

39*01

AFYI

TABLE 12

CDR3 frequency analysis of Lambda

chain before and after immunization of

influenza vaccine

Frequency

Frequency

Lambda
of 7^th
Frequency

change

chain-
day after
before

of V

CDR3_
immuniza-
immuniza-
V
gene

Seq
tion (%)
tion (%)
gene
(%)

AAW
16.83722
0.23
IGLV1-
100%

DDDL

47*01

SGPV

AAW
2.558374
0.03
IGLV1-
20%

DDSQ

44*01

NGPL

QT
1.711464
3.48
IGLV4-
−100%

69*01

ATWD
1.589042
0.02
IGLV1-
100%

DNLS

47*01

GPV

QV
1.483721
0
IGLV3-
100%

21*01

AAW
1.269279
0.01
IGLV1-
100%

DDNF

47*01

SGAE

V

SSYTS
1.175902
0.05
IGLV2-
0%

SSTP

14*

WV

01/03

AVW
1.161244
0.01
IGLV1-
20%

DNSL

44*01

NGFY

V

AAW
1.099626
0.01
IGLV1-
100%

DDSL

47*

SPPE

01/02

V

ATWD
1.037193
0.01
IGLV1-
100%

DDLS

47*

GPV

01/02

TABLE 11

CDR3 frequency analysis of Kappa

chain before and after immunization of

influenza vaccine

Frequency

Kappa
of 7^th day
Frequency

Frequency

chain
after
before

change

CDR3_
immuniza-
immuniza-
V
of V

Seq
tion (%)
tion (%)
gene
gene (%)

QQRS
2.993274
0.01
IGKV3-
−9%

DWP

11*01

YT

QQY
2.178924
0.01
IGKV1-
100%

DA

33*01

HQSS
1.560191
0.01
IGKV1-
21%

IRSW

39*01

T

LQHN
1.399475
0.01
IGKV1D-
100%

TYPQ

17*02

T

QQSS
1.274091
0
IGKV1-
21%

TSSW

39*01

T

QQY
1.19955
0.48
IGKV3-
−31%

NNW

15*01

PPYT

MQG
1.103035
0.01
IGKV2-
100%

KYW

30*01

PT

QQY
1.060809
0
IGKV3-
50%

RGSS

20*01

CT

QQY
1.020738
0.07
IGKV3-
0.5

GSSP

20*01

PWT

QQY
0.998763
0
IGKV3-
0.5

DYSS

20*01

ST

The expression frame is constructed by fusion of PCR; the frame includes promoter, variable region and antibody constant region.

The selected antibody heavy chain gene and light chain gene are randomly paired and transfected 293FT cells for expression and the supernatant is obtained for Elisa assay, its affinity to antigens is tested.

The concrete methods for expression in vitro can be referred to dengue virus cases. Elisa results show that one functional antibody per pair is obtained form the pairing of candidate Kappa chain and Lambda chain with heavy chain respectively. From FIG. 5, we can see that the success rate of pairing is 2/15.

Embodiment 4 Full-Length Sequences of Variable Region of Antibody Obtained by Combining the Sequencing Results of RACE and RT libraries.

The optimized full-length sequence selection method of the variable region described in embodiment 2 may partially make up for the insufficient reading length of the sequence. But for partial antibody variable region, such as containing long length of CDR and FR regions of 5′ UTR may lead to low splicing rate. It is difficult to obtain the full length sequence of the high frequency variable region by the described selection in turn method with locking CDR3. For example, in four sequences of highest-frequency CDR3 in Table 9, the corresponding splicing rate of two CDR3 is about 40%.

A method is developed to obtain the full-length sequence of variable region of antibody by combining the results of RACE and RT libraries sequencing. The RACE and RT libraries sequencing. The RACE and RT methods are used to construct library and sequence for the same sample in parallel, and the consistency of high-frequency CDR3in the two libraries is compared. The CDR3 homology cluster is locked and the maximum frequency variable region full length DNA sequence containing the CDR3homology cluster is selected from the RT library.

The method of concrete implementation is as follows:

The RT method and the RACE method described in embodiment 1 are used to separate RNA from PBMC of blood sample of dengue patient at acute stage; construct heavy chain and light chain libraries by RT method at the same time.

The steps of RT library construction are as follows:

According to the method described in the embodiment 1, the first chain of cDNA is obtained, and the specific primers are used to amplify variable regions of heavy chain (VH), Kappa chain (VK) and Lambda chain (VL). The forward primer is designed in the conserved region of the variable region, and the reverse primer is designed in the constant region. The primer sequences are shown in Table 13, in which heavy chain primers are quoted from REF1.

Adopt the 25 μL PCR amplification system consisting of 2.2 μM VH or 3.6 μM VK or 2.2 μM VL forward primers, 1 μM reverse primers, 2.5 μL cDNA and 0.75 μL AccuPrime Taq Polymerase.

PCR program: 95° C. min; 25 cycles (95° C. 30 sec, 56° C. 30 sec, 72° C. 1 min); 72° C. 7 min; 4° C. The operation steps of gel cutting and purification of PCR products can be referred to the manual of QIAquick Gel Extraction Kit (Qiagen).

The construction of high-throughput sequencing library can be referred to the manual of NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB). After the library construction is finished, Bioanalyzer High Sensitivity DNA chip (Agilent) is used for QC and the sequencing platform is Illumina Miseq 2x300.

TABLE 13

List of primers used in RT

Library Construction

VH chain
IGHV_

CGCAGACCCTCTCA

forward
LR1

CTCAC

primers
IGHV_
TGGAGCTGAGGTGAA

LR2
GAAGC

IGHV_
TGCAATCTGGGTCTG

LR3
AGTTG

IGHV_
GGCTCAGGACTGGTG

LR4
AAGC

IGHV_
TGGAGCAGAGGTGAA

LR5
AAAGC

IGHV_
GGTGCAGCTGTTGGA

LR6
GTCT

IGHV_
ACTGTTGAAGCCTTC

LR7
GGAGA

IGHV_
AAACCCACACAGACC

LR8
CTCAC

IGHV_
AGTCTGGGGCTGAGG

LR9
TGAAG

IGHV_
GGCCCAGGACTGGTG

LR10
AAG

IGHV_
GGTGCAGCTGGTGGA

LR11
GTC

VH chain
HGR
AAGACCGATGGGCCC

reverse primers

TTG

VK chain
kvf1
TGACCCAGTCTCCAT

forward

CCTCC

primers
kvf2
TGACCCAGTCTCCAT

CCTCA

kvf3
TGACCCAGTCTCCAT

CCTTCC

kvf4
TGACCCAGTCTCCAT

CCTTACT

kvf5
TGACCCAGTCTCCAT

CTGCC

kvf6
TGACCCAGTCTCCAT

CTTCC

kvf7
TGACACAGTCTCCAG

CCACC

kvf8
TGACACAGTCTCCAG

GCACC

kvf9
TGACGCAGTCTCCAG

GCAC

kvf10
TGACCCAGTCTCCAG

ACTCC

kvf11
TGACTCAGTCTCCAC

TCTCCC

kvf12
TGACCCAGTCTCCAT

TCTCCC

kvf13
TGACCCAGACTCCAC

TCTCTC

kvf14
TGACCCAGACTCCAC

TCTCC

kvf15
TGACCCAGTCTCCTTC

CACC

kvf16
TCACGCAGTCTCCAG

CATTC

kvf17
TGACGCAGTCTCCAG

CCAC

kvf18
TGACCCAGTCTCCAT

CTTCTG

VK chain
KCR
ACACAACAGAGGCAG

reverse

TTCCAG

primers

VL chain
lvf1
GGTCCTGGGCCCAGT

forward

CTGTCG

primers
lvf2
GGTCCTGGGCCCAGT

CTGCC

lvf3
GTCCTGGGCCCAGTC

TGTGCT

lvf4
TGTCAGTGGTCCAGG

CAGGGC

lvf5
GATCCTGGGCTCAGT

CTGCCCTG

lvf6
GCTCTGAGGCCTCCT

ATGAGCTG

lvf7
GATCCGTGGCCTCCT

ATGAGCTG

lvf8
TCTCTGAGGCCTCCT

ATGAGCTG

lvf9
GCTCTGCGACCTCCT

ATGAGCTG

lvf10
GTTCTGTGGTTTCTTC

TGAGCTGAC

lvf11
GCTCTGTGACCTCCT

ATGTGCTG

lvf12
TCTCTGTGGCCTCCTA

TGAGCTG

lvf13
GTTCTGTGGCCTCCTA

TGAGCTG

lvf14
GTCTCTGTGCTCTGCC

TGTGCTG

lvf15
GGTCTCTCTCCCAGC

CTGTGCTG

lvf16
GGTCTCTCTCCCAGCT

TGTGCTG

lvf17
GTTCCCTCTCGCAGC

CTGTGCT

lvf18
GTTCCCTCTCGCAGG

CTGTGCT

lvf19
GGTCCAATTCTCAGA

CTGTGGTGAC

lvf20
GGTCCAATTCCCAGG

CTGTGGTG

lvf21
GAGTGGATTCTCAGA

CTGTGGTGAC

lvf22
GGTCCCTCTCCCAGC

CTGTGC

VL chain
LCR
AGTGTGGCCTTGTTG

reverse primers

GCTTG

The library sequencing and analysis are carried out according to the method described in embodiment 1, the correlation of high frequency CDR3 region between RACE library and RT library is compared, see FIG 6(a) and 6(b) for the analysis of CDR3 frequency correlation between RACE library and RT library.

The results show that the correlation R²value of heavy chain and Lambda chain CDR3 in the two libraries is about 0.7; as show in FIG. 6(a), the consistency of the highest frequency heavy chain and light chain CDR3is very good, the high frequency heavy chain and light chain CDR3 in the top 10 of antibody spectrum also show some consistency, indicating that there is no significant deviation in most of the high frequency CDR3 rankings in the two libraries. For example, the highest frequency CDR3 (ARETDGMDV) of heavy chain of the sample 11-2 in FIG. 6(b) is the highest frequency CDR3 in both RACE and RT libraries. The amino acid sequences of the most abundant FR and CDR regions of the CDR3-ARETDGMDV homology cluster selected by using the RACE R12 file are FR1:QVQLVQSGAEVKRPGASVKVSCKAS, FR2: MHWVRQAPGQRLEWMGW, FR3: KYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYC,CDR1:GYTFTTYA,FR4:WGQG, CDR2:INAGNGNT. The amino acid sequence of the most abundant FR region and CDR region of CDR2-ARETDGMDV homology cluster selected by RT file are also consistent, which indicates that this method can be used to splice the variable region of high frequency CDR3 gene.

Embodiment 5 An Optimized Antibody Gene Screening Method for HA Monoclonal Antibody

Use the method of RACE antibody library construction described in embodiment 1, total RNAs are isolated from spleen cells of mice immunized with HA antigen, and construct the RACE libraries of heavy chain and light chain respectively. Use Illumina Miseq 2x250 system for high-throughput sequencing. The methods of data processing, the determination of FR and CDR regions, and the selection of candidate CDR3 amino acid sequences are the same as those described in embodiment 1.

The expression frame is constructed by fusion of PCR; the frame includes promoter, variable region and antibody constant region. The selected antibody heavy chain gene and light chain gene are randomly paired and transfected 293FT cells for expression and the supernatant is obtained for Elisa assay, its affinity to antigens is tested.

The concrete methods for expression in vitro can be referred to dengue virus cases. Elisa results show that one affinity antibody is obtained from the pairing of candidate heavy chain and light chain.

TABLE 14

Analysis of heavy chain CDR3

frequency after HA immunization in mice

Frequency after

Heavy chain CDR3_Seq
immunization (%)

TRGDY
5.94

ARHTIPPYVMDY
1.56

ARDEGIYGY
1.48

ARGVYNYGRVWYFDV
1.36

ARRDYDNYVPFAY
1.25

TGDYEFGLFDY
1.24

ARLSGTFAY
1.01

ASLKGSAY
0.92

AREGGYYFDY
0.91

ARDNGHDWFAY
0.87

ARRDYGNYVPFAY
0.76

VLDYYGYAPFAY
0.75

ARDLYYSHGGFAY
0.70

ARVDGYLQGYYFGY
0.69

ARGREGNGAMDY
0.65

ARQEFYYGNYDAMDY
0.61

ASGILNVMDY
0.58

ARATVPAEIAY
0.57

ARWTGTGDYAMDY
0.56

ARSGLIYDGYYAWFAY
0.53

TABLE 15

Analysis of Kappa chain CDR3

frequency after HA immunization in mice

Frequency after

Kappa chain
immunization (%)

WQGTHFPWT
4.62

QNGHSFPYT
4.11

QQYYSYPRT
3.86

QQYYRYPWT
1.97

QQYYNYRT
1.92

MQHLEYPFT
1.83

QQYYSYPWT
1.49

KQSYNLLT
1.31

LHYDNLWT
1.22

QQYYSYRT
1.11

LQYDNLLT
1.10

MQHLEYPYT
1.04

QNDHSFPLT
0.96

SQSTHVPPT
0.89

FQGSHVPWT
0.85

WQGTHFPQT
0.84

QQWSSNPFT
0.79

QQWSSNPPT
0.79

HQWSSYRT
0.78

KQSYNLWT
0.76

The applicant declares that the detailed method of the invention is explained by the above embodiments, but the invention is not limited to the above detailed methods, that is, it does not mean that the invention should rely on the above detailed methods for implementation. Technicians in the technical field should understand that any improvement in the invention, the equivalent replacement of the raw materials of the products of the invention and the addition of auxiliary components, and the selection of concrete ways, etc., fall within the protection scope and the public scope of the invention.

A METHOD OF DISCOVERING SPECIFIC FUNCTIONAL ANTIBODIES

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