CN117304327B - Anti-goat IgG rabbit monoclonal antibody and application thereof - Google Patents
Anti-goat IgG rabbit monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to a rabbit monoclonal antibody of anti-goat IgG and application thereof, belonging to the technical field of biology. The antibody comprises six CDRs of a heavy chain CDR-H1, a CDR-H2, a CDR-H3 and a light chain CDR-L1, a CDR-L2 and a CDR-L3. The rabbit monoclonal antibody of the anti-goat IgG provided by the invention has higher affinity with goat IgG, and can recognize and detect goat IgG with high specificity and high sensitivity. The method can be applied to ELISA detection, is favorable for obtaining more accurate detection and evaluation results, and can reduce detection cost and reduce or avoid interference of background signals.
Description
Technical Field
The invention relates to a rabbit monoclonal antibody of anti-goat IgG and application thereof, belonging to the technical field of biology.
Technical Field
Immune response is an important factor in human resistance to disease. Antibodies can be produced when an animal is stimulated with an antigen, the specificity of which depends on the determinants of the antigen molecule, and each antigen molecule has many antigenic determinants, so that the antibodies produced by immunizing an animal are actually a mixture of antibodies. The traditional method has low antibody preparation efficiency and limited yield, and animal antibodies can generate serious allergic reaction when injected into human bodies. In addition, it is extremely difficult to separate these different antibodies, and thus the advent of monoclonal technology has addressed this problem.
A conventional antibody molecule (IgG) is a protein molecule consisting of two identical heavy chains and two identical light chains that is fairly conserved in structure. The light chain of an antibody comprises 1 variable region and 1 constant region, namely VL and CL. While the heavy chain has 1 variable region and 3 constant regions, namely VH, CH1, CH2 and CH3. The VH and VL regions together constitute the smallest unit of antigen recognition by a conventional antibody, and the sequence differences in the antibody variable regions determine the ability of the antibody to specifically recognize different antigens. Whereas the CL and CH regions are relatively conserved, known as the constant region of the antibody, with the CH2 and CH3 regions of the CH region being important for antibody recruitment to the immune cell for ADCC and CDC functions.
Monoclonal antibody preparation techniques include lymphocyte technique for immunizing mice, phage display technique, humanized antibody transgenic mice and single B cell antibody technique.
The principle of phage display technology is that a section of exogenous gene is inserted into a proper position of a phage coat protein structural gene, and under the condition that the reading frame is normal and the normal function of the coat protein is not influenced, the exogenous gene can be expressed along with the expression of the coat protein, so that polypeptide or protein is displayed on the surface of phage in the form of fusion protein, the displayed protein can keep relatively independent space structure and biological activity, the combination of target protein is facilitated, and therefore, the screening of phage display antibody libraries can be rapidly carried out by using the target protein. After the construction of the display library, the target protein is taken as a stationary phase, incubated with the display library for a period of time, unbound phage are washed away, and adsorbed phage are eluted by a competitive receptor. The phage infection host bacteria obtained by elution are propagated and amplified, and then the next round of elution is carried out. After 3-5 rounds of "adsorption-elution-amplification" (more rounds of elution are required for certain weak affinity antibodies), a high enrichment of phage that specifically bind to the target protein can be obtained.
The application of the rabbit monoclonal antibody mainly comprises the following steps:
1) Use in biomarker development: rabbit monoclonal antibodies are widely used in various fields of life sciences research, and have particular advantages in immunohistochemistry and in detecting post-translational modifications of proteins. Over 100 rabbit monoclonal antibodies have been developed to date for IHC clinical diagnosis and study of cancer. In the field of protein modification research, such as protein phosphorylation, plays a vital role in protein signaling pathways associated with diseases, and the use of rabbit monoclonal antibodies that phosphorylate proteins can clearly show the extent of protein phosphorylation. More than 200 phosphorylation-specific rabbit monoclonal antibodies are currently available.
2) Use in therapeutic antibody drug development: most therapeutic antibodies used clinically require in vitro affinity maturation to increase the affinity of the antibody, which usually takes 8-12 months, with a final affinity of 10 -9 mol/L. Rabbit monoclonal antibodies can reach 10 without in vitro affinity maturation -11 To 10 -13 mol/L. Such high affinity not only reduces the clinical use of antibodies, but also reduces side effects due to the use of large amounts of antibodies. In addition, compared with a mouse monoclonal antibody, the rabbit monoclonal antibody is easier to humanize, the rabbit monoclonal antibody mainly comprises a heavy chain gene and a light chain gene, and a great number of rabbit monoclonal antibody sequences are analyzed to find that the rabbit and human antibodies have higher homology of about 60-76 percent, and the mouse and human antibodies have slightly lower homology of 57-72 percent, so that the development of medicaments by using the rabbit monoclonal antibodies has wider prospect.
3) The rabbit anti-goat IgG marked by horseradish peroxidase is a rabbit monoclonal antibody marked by HRP goat horseradish peroxidase with goat IgG as an antigen, and can be specifically combined with the goat IgG. The horseradish peroxidase labeled rabbit anti-sheep IgG is mainly used for detection experiments of sheep IgG such as ICC/IF, dotblot, ELISA, IHC-P, IHC-Fr, immunomicroscope, WB and the like.
Based on the prior art, there is a need to provide a novel anti-goat IgG rabbit monoclonal antibody which has a higher affinity with goat IgG and can recognize and detect goat IgG with high specificity and high sensitivity.
Disclosure of Invention
The main purpose of the invention is as follows: provides a novel rabbit monoclonal antibody of anti-goat IgG and application thereof. The rabbit monoclonal antibody of the anti-goat IgG can specifically recognize the goat IgG.
The technical scheme for solving the technical problems is as follows:
in a first aspect of the invention there is provided an anti-goat IgG rabbit monoclonal antibody or antigen-binding portion thereof capable of specifically binding goat IgG, said antibody or antigen-binding portion comprising a heavy chain variable region VH comprising heavy chain CDR-H1, CDR-H2 and CDR-H3 and a light chain variable region VL comprising light chain CDR-L1, CDR-L2 and CDR-L3,
the amino acid sequences of the six CDRs of the monoclonal antibody or antigen binding portion thereof are selected from any one of the following four groups:
(1) CDR-H1 with the amino acid sequence shown as SEQ ID NO. 3, CDR-H2 with the amino acid sequence shown as SEQ ID NO. 4, CDR-H3 with the amino acid sequence shown as SEQ ID NO. 5, CDR-L1 with the amino acid sequence shown as SEQ ID NO. 6, CDR-L2 with the amino acid sequence shown as SEQ ID NO. 7 and CDR-L3 with the amino acid sequence shown as SEQ ID NO. 8;
(2) CDR-H1 with the amino acid sequence shown as SEQ ID NO. 11, CDR-H2 with the amino acid sequence shown as SEQ ID NO. 12, CDR-H3 with the amino acid sequence shown as SEQ ID NO. 13, CDR-L1 with the amino acid sequence shown as SEQ ID NO. 14, CDR-L2 with the amino acid sequence shown as SEQ ID NO. 15 and CDR-L3 with the amino acid sequence shown as SEQ ID NO. 16;
(3) CDR-H1 with the amino acid sequence shown in SEQ ID NO. 19, CDR-H2 with the amino acid sequence shown in SEQ ID NO. 20, CDR-H3 with the amino acid sequence shown in SEQ ID NO. 21, CDR-L1 with the amino acid sequence shown in SEQ ID NO. 22, CDR-L2 with the amino acid sequence shown in SEQ ID NO. 23 and CDR-L3 with the amino acid sequence shown in SEQ ID NO. 24;
(4) CDR-H1 with the amino acid sequence shown in SEQ ID NO. 27, CDR-H2 with the amino acid sequence shown in SEQ ID NO. 28, CDR-H3 with the amino acid sequence shown in SEQ ID NO. 29, CDR-L1 with the amino acid sequence shown in SEQ ID NO. 30, CDR-L2 with the amino acid sequence shown in SEQ ID NO. 31 and CDR-L3 with the amino acid sequence shown in SEQ ID NO. 32.
Further, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen binding portion thereof are selected from any one of the following four groups:
(1) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;
(2) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10;
(3) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18;
(4) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 25, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 26.
Preferably, the amino acid sequence of the monoclonal antibody or antigen binding portion thereof is selected from any one of the amino acid sequences shown in SEQ ID NOS.33-36.
In a second aspect of the invention there is provided a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof according to the first aspect.
In a third aspect of the invention there is provided a mammalian system expression vector comprising a nucleic acid as described in the second aspect.
Preferably, the mammalian cell expression vector is pcdna3.4.
In a fourth aspect of the invention there is provided a host cell comprising an expression vector according to the third aspect.
Preferably, the host cell is HEK293.
In a fifth aspect of the invention there is provided the use of an antibody or antigen binding fragment thereof as described in the first aspect as a secondary antibody for detecting goat IgG serum titers.
The invention also provides an application of the double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) for quantitatively detecting goat IgG in the culture supernatant of Vero cells by using the rabbit monoclonal antibody against goat IgG.
The invention also provides application of the double-antibody sandwich immune colloidal gold chromatography technology of the rabbit monoclonal antibody against goat IgG in the first aspect or the second aspect to qualitative detection of goat IgG in the supernatant of Vero cell culture.
The terms referred to in the present invention are defined as follows:
"heavy chain CDR-H1, CDR-H2, CDR-H3" means, in order: CDR1, CDR2 and CDR3 of the heavy chain complementarity determining regions.
"light chain CDR-L1, CDR-L2, CDR-L3" means, in order: CDR1, CDR2 and CDR3 of the light chain complementarity determining regions.
The "single chain antibody (single chain antibody fragment, scFv)" is formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide (linker) of 15 to 20 amino acids.
The invention has the following technical effects:
1) According to the invention, the rabbit monoclonal antibody targeting goat IgG is obtained through phage display technology, and the antibody affinity KD (M) is 2.29E-9-6.57E-9, so that goat IgG can be specifically identified with high affinity.
2) The antibody of the invention can specifically bind goat IgG and can be used as a secondary antibody for detecting goat IgG serum titer.
Drawings
FIG. 1 is a graph showing the binding of goat IgG to enriched phage detected by ELISA in example 1 for each round of panning.
FIG. 2 is a schematic representation of the expression vector of the mammalian system of example 2.
FIG. 3 is an electrophoresis detection diagram of rabbit monoclonal antibody expressed by the lactation system of example 2. Wherein, (a) antibody B619101-B619104 electrophoreses; (B) an electrophoresis detection panel of antibodies B619105-B619114; (c) electrophoresis detection pattern of antibody B619115-B619118.
FIG. 4 is a graph showing ELISA results of rabbit monoclonal antibodies expressed by the lactation system of example 3 as secondary antibodies for detecting goat IgG serum titers.
FIG. 5 is a graph showing the SPR binding detection results of rabbit monoclonal antibody and goat IgG expressed by the lactation system of example 3. Wherein (a) antibody B619101SPR assay; (B) an antibody B619114SPR assay; (c) an antibody B619118SPR assay; (d) antibody B619113SPR assay.
Detailed Description
The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the reagents and materials used are commercially available products unless otherwise specified.
The raw material purchasing sources and the product numbers in the specific embodiments are shown in the following table:
| material name | Manufacturer' s | Goods number |
| Bovine serum albumin | Radix Arenariae | 9048-46-8 |
| ELISA plate | Corning | 3590 |
| Anti-M13Antibody(HRP) | sino biological | 11973-MM05T-H |
| Goat Anti-rabbit IgG-HRP | Jackson | 111-035-008 |
| Protein A chip | Cytiva | 291275551 |
| Goat IgG | Biyun Tian (a kind of Chinese character) | A7007 |
EXAMPLE 1 screening of anti-goat IgG rabbit monoclonal antibodies
Immunization of Japanese white rabbits with goat IgG as antigen, construction of reservoir Capacity 4.05x10 9 Is used for screening single-chain antibodies of anti-goat IgG by phage display technology.
10ug/mL goat IgG was coated on ELISA strips using solid phase panning, overnight at 4 ℃. PBST was washed three times, 2% protein-free blocking solution was added to each well, and blocking was performed at 37℃for 1 hour. After three PBST washes, phage display library (approximately 2x 10) 12 CFU), 37 ℃ for 1 hour. Unbound phage were aspirated and PBST washed 10 times. glycine-HCl (ph=2.2) solution was added to each well, reacted at 37 ℃ for 5 minutes, the adsorbed phage was eluted by gently blowing the wells, and then Tris-HCl (ph=8.8) solution was added to neutralize to neutrality. The eluted phage infected TG1 cells in logarithmic growth phase, and the recovered phage was amplified for the next round of panning.
After three rounds of panning, a Phage-ELISA was used to verify whether the specificity was enriched. 2ug/mL goat IgG was coated on ELISA strips overnight at 4 ℃. After three PBST washes, blocking was performed with 2% protein-free blocking solution at 37 ℃ for 1 hour. After 5 passes of PBST, three rounds of panning phage display library were added, with a first well of about 1X10 12 CFU, 4-fold gradient dilution, final Kong Kongbai, 37 ℃ for 1 hour. After 5 washes of PBST, HRP-labeled secondary antibodies against M13 were added and incubated for 1 hour at 37 ℃. After PBST is washed for 5 times, TMB developing solution is added, the color development is carried out for 5-10 minutes at room temperature in a dark place, finally, 2M sulfuric acid is used for stopping the color development, and an enzyme label instrument is used for reading the light absorption value at the wavelength of 450nm and making a Phage-ELISA binding curve.
ELISA detection results are shown in FIG. 1, in which helper phage is used as a negative control, and after three rounds of enrichment, the affinity of phage population to goat IgG is increased round by round.
Antigen binding assays were performed on each round of enriched phage monoclonal, with the following specific procedures:
TG1 cells were infected with the enriched phage library of each round, 88 were randomly picked in the first round, 352 were randomly picked in the second round, 792 were randomly picked in the third round, and phages were amplified and recovered. 2ug/mL goat IgG was coated on ELISA strips overnight at 4 ℃. After three PBST washes, blocking was performed with 2% protein-free blocking solution at 37 ℃ for 1 hour. The 1232 amplified monoclonal phages and negative control helper phages were incubated with a 2% protein-free blocking solution in PBST solution at a ratio of 1:1 for 1 hour at room temperature, and the incubated phages were added to the blocked elisa plate and incubated at 37 ℃ for 1 hour. After 5 washes of PBST, HRP-labeled secondary antibodies against M13 were added and incubated for 1 hour at 37 ℃. And (3) after PBST is washed for 5 times, TMB is added for color development at room temperature and in a dark place for 5-10 minutes, finally, 2M sulfuric acid is used for stopping color development, an enzyme label instrument is used for reading the light absorption value at the wavelength of 450nm, and the light absorption value is more than twice of that of a negative control, and OD450 is more than 0.5, so that the positive clone is obtained. The results showed that 45 positive clones recognizing goat IgG were found in 1232 monoclonal phages, and sequencing analysis was performed on these 45 positive clones to yield 18 Unique sequences, numbered B619101-B619118.
Example 2: expression and purification of anti-goat IgG rabbit monoclonal antibody in a mammalian system
The expression vector pCDNA3.4 of the mammalian system for the rabbit monoclonal antibody of example 1 was constructed as shown in FIG. 2, and then a plasmid was prepared therefrom. The preparation method comprises the following steps: the positive ScFv clones obtained by phage display technique screening in experimental example 1 were used as templates, and the heavy chain variable region and the light chain variable region of ScFv antibody were respectively linked to the constant region of Human IgG1 by PCR means, and cloned into pcdna3 4 vector.
The light chain and the heavy chain are combined and then co-transfected into a mammalian cell, the mammalian cell is placed on a shaking table for expression for one week, the supernatant is collected and purified, and a NanoDrop instrument is used for reading the absorbance value of 280nm, wherein the antibody expression level of the number B619112 is extremely low, and no protein is collected. The remaining 17 high concentration proteins were pipetted into a dialysis bag and dialyzed in a 1XPBS beaker. Purified antibodies were collected and SDS-PAGE under reducing conditions gave the results shown in FIGS. 3 (a) - (c).
As can be seen from fig. 3 (a) - (c); the molecular weight of the expressed 17 antibodies is about 50-80kDa, and the purity is more than 80%.
EXAMPLE 3 binding ELISA and SPR detection of mammalian System-expressed rabbit monoclonal antibodies
The rabbit monoclonal antibodies were validated by ELISA. 2ug/mL Goat IgG was coated on ELISA strips, blocked at 4℃overnight with 2% protein-free blocking solution at 37℃for 1 hour, anti-Goat IgG Rabbit monoclonal antibody of example 2 was diluted to 10ug/mL as the initial well concentration, 3-fold gradient diluted, the final well as blank, incubated at 37℃for 1 hour, PBST washed plates for 5 times and then pat dried, goat anti-Rabbit IgG, HRP as secondary antibody, incubated at 37℃for 1 hour, PBST washed plates for 5 times and then pat dried. 100uL of TMB is added to each hole to react for 5-10 minutes at room temperature in a dark place, the color development is stopped by using 2M sulfuric acid, and the absorbance at the wavelength of 450nm is read by using an enzyme-labeled instrument.
17 anti-goat IgG rabbit monoclonal antibodies can be combined with goat IgG, and the EC50 is shown in Table 1. OD450 of 17 anti-goat IgG is shown in table 2.
TABLE 1 EC50 s of 17 expressed antibodies
TABLE 2 OD450 of 17 expressed antibodies
And (3) screening antibodies with better goat IgG binding activity and weaker BSA and Human IgG binding from the 17 anti-goat IgG rabbit monoclonal antibodies, and screening four antibodies. See in particular table 3:
TABLE 3 Table 3
The four antibodies obtained were screened and numbered B619101, B619113, B619114 and B619118. The amino acid sequence information for each antibody is as follows:
the antibody numbered B619101,
the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO. 1.
The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 2.
The amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID NO. 3.
The amino acid sequence of CDR-H2 of the heavy chain variable region is shown in SEQ ID NO. 4.
The amino acid sequence of CDR-H3 of the heavy chain variable region is shown in SEQ ID NO. 5.
The amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID NO. 6.
The amino acid sequence of CDR-L2 of the light chain variable region is shown in SEQ ID NO. 7.
The amino acid sequence of CDR-L3 of the light chain variable region is shown in SEQ ID NO. 8.
The antibody numbered B619113,
the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO. 9.
The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 10.
The amino acid sequence of CDR-H1 of heavy chain variable region is shown as SEQ ID NO. 11
The amino acid sequence of CDR-H2 of the heavy chain variable region is shown in SEQ ID NO. 12.
The amino acid sequence of CDR-H3 of the heavy chain variable region is shown in SEQ ID NO. 13.
The amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID NO. 14.
The amino acid sequence of CDR-L2 of the light chain variable region is shown in SEQ ID NO. 15.
The amino acid sequence of CDR-L3 of the light chain variable region is shown in SEQ ID NO. 16.
The antibody numbered B619114,
the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO. 17.
The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 18.
The amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID NO. 19.
The amino acid sequence of CDR-H2 of the heavy chain variable region is shown in SEQ ID NO. 20.
The amino acid sequence of CDR-H3 of the heavy chain variable region is shown in SEQ ID NO. 21.
The amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID NO. 22.
The amino acid sequence of CDR-L2 of the light chain variable region is shown in SEQ ID NO. 23.
The amino acid sequence of CDR-L3 of the light chain variable region is shown in SEQ ID NO. 24.
The antibody numbered B619118,
the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO. 25.
The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 26.
The amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID NO. 27.
The amino acid sequence of CDR-H2 of the heavy chain variable region is shown in SEQ ID NO. 28.
The amino acid sequence of CDR-H3 of the heavy chain variable region is shown in SEQ ID NO. 29.
The amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID NO. 30.
The amino acid sequence of CDR-L2 of the light chain variable region is shown in SEQ ID NO. 31.
The amino acid sequence of CDR-L3 of the light chain variable region is shown in SEQ ID NO. 32.
FIG. 4 shows ELISA results of rabbit monoclonal antibody B619118 expressed by the lactation system of example 3 as a secondary antibody for detecting the serum titer of the Goat IgG. As can be seen from fig. 4: b619118 binds significantly specifically to the coat IgG.
EXAMPLE 4 detection of bound SPR of mammalian System-expressed rabbit monoclonal antibodies
2. Mu.g/mL of the four antibodies screened in example 3 were each injected into the experimental channel at a flow rate of 10. Mu.L/min for 60s with a capture of approximately 300-400RU. The diluted Goat IgG is sequentially injected into the experimental channel and the reference channel at a flow rate of 30 mu L/min, and combined and dissociated for corresponding time. The binding dissociation steps were all performed in running buffer. After each concentration analysis, the chip was regenerated with 10mM pH1.5 Gly-HCl at a flow rate of 30. Mu.L/min for 30s, washing away undissociated analytes. The experimental channel needs to recapture the same amount of ligand for the next concentration analysis. KD values were calculated for each sample using Biacore 8K analysis software Biacore Insight Evaluation Software. The reference channel (Fc 1) was used for background subtraction.
Conclusion: the affinity values for each of the four antibodies for the coat IgG are shown in table 4.
TABLE 4 Table 4
| Antibody numbering | Antigens | Antigen concentration | ka | kd | KD(M) |
| B619101 | Goat IgG | 200nM | 1.39E+05 | 9.16E-04 | 6.59E-09 |
| B619113 | Goat IgG | 200nM | 2.42E+04 | 5.86E-05 | 2.42E-09 |
| B619114 | Goat IgG | 200nM | 2.25E+04 | 5.15E-05 | 2.29E-09 |
| B619118 | Goat IgG | 200nM | 1.13E+04 | 3.85E-05 | 3.41E-09 |
FIGS. 5 (a) - (d) are graphs showing the SPR binding detection results of rabbit monoclonal antibodies B619101, B619114, B619118 and B619113, respectively, with goat IgG, as can be seen from FIGS. 5 (a) - (d): the above 4 antibodies all have affinity with the Goat IgG on nM scale.
The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (7)
1. An anti-goat IgG rabbit monoclonal antibody or antigen-binding portion thereof capable of specifically binding goat IgG, comprising a heavy chain variable region VH comprising heavy chain CDR-H1, CDR-H2 and CDR-H3 and a light chain variable region VL comprising light chain CDR-L1, CDR-L2 and CDR-L3,
the method is characterized in that: the amino acid sequences of the six CDRs of the monoclonal antibody or antigen binding portion thereof are selected from any one of the following four groups:
(1) CDR-H1 with the amino acid sequence shown as SEQ ID NO. 3, CDR-H2 with the amino acid sequence shown as SEQ ID NO. 4, CDR-H3 with the amino acid sequence shown as SEQ ID NO. 5, CDR-L1 with the amino acid sequence shown as SEQ ID NO. 6, CDR-L2 with the amino acid sequence shown as SEQ ID NO. 7 and CDR-L3 with the amino acid sequence shown as SEQ ID NO. 8;
(2) CDR-H1 with the amino acid sequence shown as SEQ ID NO. 11, CDR-H2 with the amino acid sequence shown as SEQ ID NO. 12, CDR-H3 with the amino acid sequence shown as SEQ ID NO. 13, CDR-L1 with the amino acid sequence shown as SEQ ID NO. 14, CDR-L2 with the amino acid sequence shown as SEQ ID NO. 15 and CDR-L3 with the amino acid sequence shown as SEQ ID NO. 16;
(3) CDR-H1 with the amino acid sequence shown in SEQ ID NO. 19, CDR-H2 with the amino acid sequence shown in SEQ ID NO. 20, CDR-H3 with the amino acid sequence shown in SEQ ID NO. 21, CDR-L1 with the amino acid sequence shown in SEQ ID NO. 22, CDR-L2 with the amino acid sequence shown in SEQ ID NO. 23 and CDR-L3 with the amino acid sequence shown in SEQ ID NO. 24;
(4) CDR-H1 with the amino acid sequence shown in SEQ ID NO. 27, CDR-H2 with the amino acid sequence shown in SEQ ID NO. 28, CDR-H3 with the amino acid sequence shown in SEQ ID NO. 29, CDR-L1 with the amino acid sequence shown in SEQ ID NO. 30, CDR-L2 with the amino acid sequence shown in SEQ ID NO. 31 and CDR-L3 with the amino acid sequence shown in SEQ ID NO. 32.
2. The rabbit monoclonal antibody or antigen-binding portion thereof of anti-goat IgG of claim 1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen-binding portion thereof are selected from any one of the following four groups:
(1) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;
(2) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10;
(3) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18;
(4) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 25, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 26.
3. The rabbit monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the amino acid sequence of said monoclonal antibody or antigen-binding portion thereof is selected from any one of the amino acid sequences shown in SEQ ID NOs 33-36.
4. A nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-3.
5. A mammalian expression vector comprising the nucleic acid of claim 4.
6. A host cell comprising the vector of claim 5.
7. Use of a monoclonal antibody or antigen binding portion thereof according to any one of claims 1-3 for the preparation of a secondary antibody for detecting goat IgG serum titers.
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