CN110317270A - Antitoxin snake PLA2Protein antibodies and its application - Google Patents
Antitoxin snake PLA2Protein antibodies and its application Download PDFInfo
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- CN110317270A CN110317270A CN201910384138.9A CN201910384138A CN110317270A CN 110317270 A CN110317270 A CN 110317270A CN 201910384138 A CN201910384138 A CN 201910384138A CN 110317270 A CN110317270 A CN 110317270A
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Abstract
The present invention relates to antitoxin snake PLA2Protein antibodies and its application, antitoxin snake PLA2Protein antibodies include anti-green bamboo snake, solder horn, adder, cobra or pallas pit viper PLA2The monoclonal antibody of albumen has high-affinity and specificity.Monoclonal antibody passes through the PLA of above five kinds of poisonous snakes2Albumen is prepared as mice immunized with antigen and hybridoma fusion techniques, and passes through PLA2Two hypervariable region amino acid sequences in albumen are that antigen carries out antibody screening, finally obtain anti-green bamboo snake, solder horn, adder, cobra and pallas pit viper PLA2Totally ten kinds of monoclonal antibodies of albumen.Any one or more combination all can serve as snakebite diagnosis detection reagent use in ten kinds of monoclonal antibodies.Antitoxin snake PLA provided in an embodiment of the present invention2Protein antibodies can prepare snakebite diagnostic reagent or kit, improve snakebite diagnosis efficiency.
Description
[technical field]
The present invention relates to antibody and field of immunodetection more particularly to antitoxin snake PLA2Protein antibodies and its application.
[background technique]
There are 5,000,000 venomous snake bites in the whole world every year to root according to the statistics of the World Health Organization, and wherein the death rate is 2%, leaves tight
Weight sequelae accounts for 8%.It endangers the summation for being much larger than other tropical diseases.Above data is only to have the data of medical records, real
Venomous snake bite number on border is much bigger.For the annual venomous snake bite in China up to 200,000 people, the snakebite death rate is 5%-10%, the death rate
It is significantly larger than global average value with disability rate.
Contain a large amount of lps molecule in snake venom, each system core albumen of human body, such as blood system can be acted on
System coagulation factor, complement system and ion channel etc. cause the toxic reactions such as blood coagulation disorders, respiratory paralysis, cardiac arrest, finally
Lead to the serious sequelae such as tissue necrosis or death.
Antivenin type in China's is less at present, and antiserum kind can not cover all hypertoxic snake kinds.In addition, at present also
There is no the fast detecting method of effective venomous snake bite.Existing methods for clinical diagnosis relies primarily on family members or patient to the finger of poisonous snake atlas
Recognize, the main methods such as clinical manifestation and snake dental impression identify, such method Error Diagnostics is very big.There are a large amount of cases to identify because failing
It bites out snake kind, delays valuable treatment window time, and it is dead.
Therefore, there is an urgent need to prepare the protein antibodies for being able to detect venomous snake bite.
[summary of the invention]
In view of this, the embodiment of the invention provides antitoxin snake PLA2Protein antibodies and its application can be used in preparing snake
Hurt diagnostic reagent or kit, improves snakebite diagnosis efficiency.
To achieve the goals above, the present invention provides antitoxin snake PLA2Protein antibodies, the antibody can be with the PLA of poisonous snake2
Protein-specific combines, and the antibody is by the PLA of the poisonous snake2The hypervariable region amino acid sequence of albumen is prepared.
Optionally, from the PLA of green bamboo snake, solder horn, adder, cobra and/or pallas pit viper2The hypervariable region amino of albumen
Acid sequence includes SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
At least one of NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
Optionally, the antibody includes: anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 1, the anti-leaf of bamboo
Green PLA2Protein monoclonal antibody Tritoxomab-PLA2- 2, anti-solder horn PLA2Protein monoclonal antibody Protoxomab-
PLA2- 1, anti-solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 2, anti-adder PLA2Protein monoclonal antibody
Viptoxomab-PLA2- 1, anti-adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 2, anti-cobra PLA2Albumen list
Clonal antibody Najatoxomab-PLA2- 1, anti-cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 2, anti-pallas pit viper
PLA2Protein monoclonal antibody Glotoxomab-PLA2- 1, anti-pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-PLA2-2
At least one of.
Optionally, the anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 1 with have such as SEQ ID
The PLA of amino acid sequence shown in NO:62Protein binding, the anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-
PLA2- the 2 and PLA with the amino acid sequence as shown in SEQ ID NO:72Protein binding；The anti-solder horn PLA2Albumen list
Clonal antibody Protoxomab-PLA2- the 1 and PLA with the amino acid sequence as shown in SEQ ID NO:82Protein binding, institute
State anti-solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 2 with have the amino acid sequence as shown in SEQ ID NO:9
The PLA of column2Protein binding；The anti-adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 1 with have such as SEQ ID
The PLA of amino acid sequence shown in NO:102Protein binding, the anti-adder PLA2Protein monoclonal antibody Viptoxomab-
PLA2- the 2 and PLA with the amino acid sequence as shown in SEQ ID NO:112Protein binding；The anti-cobra PLA2Albumen
Monoclonal antibody Najatoxomab-PLA2- the 1 and PLA with the amino acid sequence as shown in SEQ ID NO:122Albumen knot
It closes, the anti-cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 2 with have as shown in SEQ ID NO:13
The PLA of amino acid sequence2Protein binding；The anti-pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-PLA2- 1 with have such as
The PLA of amino acid sequence shown in SEQ ID NO:142Protein binding, the anti-pallas pit viper PLA2Protein monoclonal antibody
Glotoxomab-PLA2- the 2 and PLA with the amino acid sequence as shown in SEQ ID NO:152Protein binding.
Optionally, the antibody is whole antibody or its antigen-binding fragment.
To achieve the goals above, the present invention also provides snakebite diagnostic reagents, including the antibody.
To achieve the goals above, the present invention also provides snakebite diagnostic kit, the snakebite diagnostic kit includes institute
State antibody and positive criteria product.
Optionally, the positive criteria product is the PLA of the poisonous snake of naturally isolated purifying2Albumen or artificial synthesized change containing height
The PLA of region amino acid sequence2Albumen.
Antitoxin snake PLA provided by the invention2Protein antibodies, by the PLA of poisonous snake2It is prepared by the hypervariable region amino acid sequence of albumen
, can be with the PLA in poisonous snake venom2Albumen has high-affinity and specificity, by PLA2Snakebite made of protein antibodies is examined
Which kind of venomous snake bite disconnected reagent or kit can be used in detection by, and using antibody antigen immune response as checkout and diagnosis base
Plinth improves snakebite diagnosis efficiency.
[Detailed description of the invention]
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for this field
For those of ordinary skill, without any creative labor, it can also be obtained according to these attached drawings other attached
Figure.
Fig. 1 is green bamboo snake provided in an embodiment of the present invention, solder horn, adder, cobra and pallas pit viper PLA2The amino of albumen
Acid sequence comparison result, wherein it is PLA that box, which marks area,2Albumen hypervariable region, respectively region one, region two and region three.
Fig. 2 is solder horn difference subspecies PLA provided in an embodiment of the present invention2The amino acid alignment result of albumen.
Fig. 3 is cobra PLA provided in an embodiment of the present invention2The space structure figure of albumen.
Fig. 4 is green bamboo snake provided in an embodiment of the present invention, solder horn, adder, cobra and pallas pit viper natural toxin SDS-
PAGE electrophoresis simultaneously carries out coomassie brilliant blue staining result.
[specific embodiment]
It is clear in order to be more clear the purpose of the present invention, technical solution and advantageous effects, below in conjunction with attached drawing and
Specific embodiment, the present invention will be described in further detail.It should be understood that specific implementation described in this specification
Mode is not intended to limit the present invention just for the sake of explaining the present invention.
In the present invention, term " antitoxin snake PLA2Protein antibodies ", " PLA2Protein antibodies ", " antibody " are used interchangeably, and are all
It refers to and PLA2The antibody that protein-specific combines.
" antigen-binding fragment " of term antibody refers to the polypeptide of the segment comprising full length antibody, keeps specific binding
The ability for the same antigen that full length antibody is combined, and/or the specific binding to antigen is competed with full length antibody, also claimed
For " antigen-binding portion thereof ".
Term " separation " refers to that substance is separated from its primal environment, and if it is crude, primal environment is
It is natural surroundings.As under the native state in active somatic cell polynucleotide and protein do not isolate and purify, will be more
Polynucleotide or protein are separated from native state with other existing substances, then to isolate and purify.
Term " hybridoma " refers to during preparing monoclonal antibody, is melted with myeloma cell and bone-marrow-derived lymphocyte
Cell made of conjunction.
Term " ELISA " refers to that soluble antigen or antibody are integrated on the solid phase carriers such as polystyrene, utilizes antigen
The qualitative and quantitative detecting method that antibody binding specificity is immunoreacted.
The present invention provides a kind of antitoxin snake PLA2Protein antibodies, the antibody can be with the PLA of poisonous snake2Protein-specific knot
It closes, and the antibody is by the PLA of the poisonous snake2The hypervariable region amino acid sequence of albumen is prepared.
Specifically, phospholipase A2(phospholipase A2, PLA2) albumen is generally stored in hypertoxic snake venom, in poisonous snake
Each puberty and Various Seasonal express, only expression quantity is variant, and is high-abundance proteins in poisonous snake venom (see figure
4).Box marks as PLA2Albumen present position, Protein Marker product molecular weight unit are KDa, wherein Marker is not
With Protein Marker product.It can be seen that PLA2Albumen is prevalent in snake venom.
As shown in Figures 1 and 2, by measurement green bamboo snake, solder horn, adder, cobra and pallas pit viper mature PLA2Albumen
Amino acid sequence (as shown in table 1), it is found that its sequence can be divided into conserved region and hypervariable region.Hypervariable region includes region one, region
Two and region three, region one and region two is essentially the same in subspecies sequence and three sequence of region is big in subspecies difference.Example
Property, solder horn difference subspecies PLA2The amino acid sequence very high homology of albumen, and region one and region diamino acid sequence
It is consistent and region triamido sequence difference is big.
As shown in figure 3, passing through analysis PLA2Protein steric structural finds that region one and region two are in PLA2Protein steric
The both ends of structure.Therefore, the PLA of poisonous snake is chosen2The polypeptide sequence (totally 10 in the region one of the amino acid sequence of albumen and region two
Short peptide sequence, as shown in table 1) it is that antigen carries out artificial synthesized and prepares polyclonal antibody, 10 polypeptides can as the result is shown
Successfully prepare specific antibody and no cross reaction.
Antitoxin snake PLA provided by the invention2Protein antibodies, by the PLA of poisonous snake2It is prepared by the hypervariable region amino acid sequence of albumen
, can be with the PLA in poisonous snake venom2Albumen has high-affinity and specificity, by PLA2Snakebite made of protein antibodies is examined
Which kind of venomous snake bite disconnected reagent or kit can be used in detection by, and using antibody antigen immune response as checkout and diagnosis base
Plinth improves snakebite diagnosis efficiency.
The antibody is whole antibody or its antigen-binding fragment.Antigen-binding fragment is Fab segment, Fab ' segment, F
(ab’)2Segment or single-chain antibody.Specifically, antigen-binding fragment can be obtained by hydrolyzing complete antibody molecule, can also be with
It is made using other other technologies for preparing antigen-binding fragment, it is not limited here.
In the table 1 that the information of partial sequence of the present invention is provided below.
Table 1: the description of sequence
Green bamboo snake PLA2Maturation protein amino acid sequence (SEQ ID NO:1) are as follows:
VIELGKMIFQETGKNPATSYGLYGCNCGPGGRRKPKDATDRCCYVHKCCYKKLTDCDPIKDRYSYSWV
NKAIVCGEDNPCLKEMCECDKAVAICFRENLDTYDKKKKINLKLFCKKTSEQC。
Solder horn PLA2Maturation protein amino acid sequence (SEQ ID NO:2) are as follows:
NLFQFGEMILEKTGKEVVHSYAIYGCYCGWGGQGRAQDATDRCCFVHDCCYGTVNDCNPKTATYSYSF
ENGDIVCGDNDLCLRTVCECDRAAAICLGQNVNTYDKNYEYYSISHCTEESEQC。
Adder PLA2 maturation protein amino acid sequence (SEQ ID NO:3) are as follows:
NLIQFGHIIEHLTGRRPLIYNGYGCYCGLGGSRQPVDATDWCCQVHDCCYQALSRRHCKPKMEKYFYS
VRKDTVTCGGETECRRETCECDKAAALCFRHSKFQGQYIHYRNCLCEGPTPPCQGVCPRWAPTKGG。
Cobra PLA2Maturation protein amino acid sequence (SEQ ID NO:4) are as follows:
RPMPLNLYQFKNMIQCTVPSRSWWDFADYGCYCGRGGSGTPVDDLDRCCQVHDHCYNEAEKISGCWPY SKTYSYECSQGTLTCKGGNNACAAAVCDCDRLAAICFAGAPYNNNNYNIDLKARCQ。
Pallas pit viper PLA2Maturation protein amino acid sequence (SEQ ID NO:5) are as follows:
SLLQFETLIMKVAKKSGMVWCFVHDCCYGKVTGCDPKMDVYSFYSNYGCYCGWGGQGRPQDATDRCSE
ENGDIVCGGDDPCKKEICECDRAAAICFRDNLNTYNDKKYWAFGAKNCPQEESEPC。
Green bamboo snake PLA21 protein amino acid sequence of region (SEQ ID NO:6) are as follows: FQETGKNPATSYGL.
Green bamboo snake PLA22 protein amino acid sequence of region (SEQ ID NO:7) are as follows: KKLTDCDPIKDR.
Solder horn PLA21 protein amino acid sequence of region (SEQ ID NO:8) are as follows: LEKTGKEVVHSYAI.
Solder horn PLA22 protein amino acid sequence of region (SEQ ID NO:9) are as follows: GTVNDCNPKTAT.
Adder PLA21 protein amino acid sequence of region (SEQ ID NO:10) is EHLTGRRPLIYNG.Adder PLA2Region 2
Protein amino acid sequence (SEQ ID NO:11) is QALSRRHCKPKMEK.
Cobra PLA21 protein amino acid sequence of region (SEQ ID NO:12) are as follows: QCTVPSRSWWDFAD.
Cobra PLA22 protein amino acid sequence of region (SEQ ID NO:13) are as follows: NEAEKISGCWPYSKT.
Pallas pit viper PLA21 protein amino acid sequence of region (SEQ ID NO:14) are as follows: MKVAKKSGMVWCFV.
Pallas pit viper PLA22 protein amino acid sequence of region (SEQ ID NO:15) are as follows: CGWGGQGRPQDAT.
From the PLA of green bamboo snake, solder horn, adder, cobra and/or pallas pit viper2The hypervariable region amino acid sequence of albumen
Including SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:
11, at least one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
Correspondingly, the antibody includes anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 1, the anti-leaf of bamboo
Green PLA2Protein monoclonal antibody Tritoxomab-PLA2- 2, anti-solder horn PLA2Protein monoclonal antibody Protoxomab-
PLA2- 1, anti-solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 2, anti-adder PLA2Protein monoclonal antibody
Viptoxomab-PLA2- 1, anti-adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 2, anti-cobra PLA2Albumen list
Clonal antibody Najatoxomab-PLA2- 1, anti-cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 2, anti-pallas pit viper
PLA2Protein monoclonal antibody Glotoxomab-PLA2- 1, anti-pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-PLA2-2
At least one of.And it is also possible that the PLA of other poisonous snakes2Protein monoclonal antibody, herein with no restrictions.
Wherein, anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 1 by green bamboo snake PLA2Albumen
Hypervariable region amino acid sequence SEQ ID NO:6 is prepared；Anti- green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2-
2 by green bamboo snake PLA2The hypervariable region amino acid sequence SEQ ID NO:7 of albumen is prepared.Anti- solder horn PLA2Albumen list
Clonal antibody Protoxomab-PLA2- 1 by solder horn PLA2The hypervariable region amino acid sequence SEQ ID NO:8 of albumen prepare and
Come；Anti- solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 2 by solder horn PLA2The hypervariable region amino acid of albumen
Sequence SEQ ID NO:9 is prepared.Anti- adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 1 by adder PLA2
The hypervariable region amino acid sequence SEQ ID NO:10 of albumen is prepared；Anti- adder PLA2Protein monoclonal antibody
Viptoxomab-PLA2- 2 by adder PLA2The hypervariable region amino acid sequence SEQ ID NO:11 of albumen is prepared.Anti- glasses
Snake PLA2Protein monoclonal antibody Najatoxomab-PLA2- 1 by cobra PLA2The hypervariable region amino acid sequence SEQ of albumen
ID NO:12 is prepared；Anti- cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 2 by cobra PLA2Egg
White hypervariable region amino acid sequence SEQ ID NO:13 is prepared.Anti- pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-
PLA2- 1 by pallas pit viper PLA2The hypervariable region amino acid sequence SEQ ID NO:14 of albumen is prepared；Anti- pallas pit viper PLA2Albumen list
Clonal antibody Glotoxomab-PLA2- 2 by pallas pit viper PLA2The hypervariable region amino acid sequence SEQ ID NO:15 of albumen prepare and
Come.
Further, anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 1 with have such as SEQ ID NO:
The PLA of amino acid sequence shown in 62Protein-specific combines.Anti- green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-
PLA2- the 2 and PLA with the amino acid sequence as shown in SEQ ID NO:72Protein-specific combines.Anti- solder horn PLA2Albumen
Monoclonal antibody Protoxomab-PLA2- the 1 and PLA with the amino acid sequence as shown in SEQ ID NO:82Protein-specific
In conjunction with.Anti- solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 2 with have the amino as shown in SEQ ID NO:9
The PLA of acid sequence2Protein-specific combines.Anti- adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 1 with have such as
The PLA of amino acid sequence shown in SEQ ID NO:102Protein-specific combines.Anti- adder PLA2Protein monoclonal antibody
Viptoxomab-PLA2- the 2 and PLA with the amino acid sequence as shown in SEQ ID NO:112Protein-specific combines.Anti- eye
Mirror snake PLA2Protein monoclonal antibody Najatoxomab-PLA2- 1 with the amino acid sequence as shown in SEQ ID NO:12
PLA2Protein-specific combines.Anti- cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 2 with have such as SEQ ID
The PLA of amino acid sequence shown in NO:132Protein-specific combines.Anti- pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-
PLA2- the 1 and PLA with the amino acid sequence as shown in SEQ ID NO:142Protein-specific combines.Anti- pallas pit viper PLA2Albumen
Monoclonal antibody Glotoxomab-PLA2- the 2 and PLA with the amino acid sequence as shown in SEQ ID NO:152Albumen is special
Property combine.
The present invention also provides a kind of snakebite diagnostic reagent, snakebite diagnostic reagent includes the antitoxin of embodiment of the present invention offer
Snake PLA2 protein antibodies.
The present invention also provides a kind of snakebite diagnostic kit, which includes that embodiment of the present invention provides
Antitoxin snake PLA2Protein antibodies and positive criteria product.Antibody in kit includes anti-green bamboo snake PLA2Protein monoclonal antibody
Tritoxomab-PLA2- 1, anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 2, anti-solder horn PLA2Albumen
Monoclonal antibody Protoxomab-PLA2- 1, anti-solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 2, anti-adder
PLA2Protein monoclonal antibody Viptoxomab-PLA2- 1, anti-adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 2,
Anti- cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 1, anti-cobra PLA2Protein monoclonal antibody
Najatoxomab-PLA2- 2, anti-pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-PLA2- 1, anti-pallas pit viper PLA2Albumen list
Clonal antibody Glotoxomab-PLA2At least one of -2.
In a preferred embodiment, snakebite diagnostic kit is double crush syndrome detection kit.Kit
PLA including at least one poisonous snake2Protein monoclonal antibody group, for example, anti-green bamboo snake PLA2Protein monoclonal antibody group
(Tritoxomab-PLA2- 1 and Tritoxomab-PLA2- 2), wherein Tritoxomab-PLA2- 1 and Tritoxomab-
PLA2- 2 respectively as coated antibody and enzyme labelled antibody, Tritoxomab-PLA2- 2 include detectable label, thus using double
Which kind of poisonous snake the toxin that is snapped in the serum of position of antibody sandwich ELISA method detection human body belongs to, fast and easy diagnose to obtain as a result,
And it is treated with corresponding antitoxin snake serum.
In other embodiments, snakebite diagnostic kit includes the PLA of 5 kinds of poisonous snakes2Protein monoclonal antibody group, respectively
For anti-green bamboo snake PLA2Protein monoclonal antibody group (Tritoxomab-PLA2- 1 and Tritoxomab-PLA2- 2), anti-solder horn
PLA2Protein monoclonal antibody group (Protoxomab-PLA2- 1 and Protoxomab-PLA2- 2), anti-adder PLA2Albumen Dan Ke
Grand antibody group (Viptoxomab-PLA2- 1 and Viptoxomab-PLA2- 2), anti-cobra PLA2Protein monoclonal antibody group
(Najatoxomab-PLA2- 1 and Najatoxomab-PLA2- 2), anti-pallas pit viper PLA2Protein monoclonal antibody group
(Glotoxomab-PLA2- 1 and Glotoxomab-PLA2-2).To realize to green bamboo snake, solder horn, adder, cobra or Pallas pit viper
Snake bite carries out checkout and diagnosis.
Optionally, the positive criteria product of the snakebite diagnostic kit is the PLA of the poisonous snake of naturally isolated purifying2Albumen,
The specific detailed in Example seven of preparation method.
Present invention will be further explained below with reference to specific examples.
Embodiment one:
The present invention provides the preparation method of antitoxin snake PLA2 protein antibodies, comprising:
Step S01 prepares PLA2Proteantigen.
Specifically, PLA is synthesized by solid-phase synthesis by gill biochemistry (Shanghai) Co., Ltd.2Polypeptide antigen.Its
In, the amino acid sequence of ten small peptide antigen as shown in NO:6~15 SEQ ID, adds respectively in the C-terminal of each small peptide antigen
6 histidines, for example in the C-terminal of SEQ ID NO:6 it is added to 6 histidines；Each short peptide antigens synthetic polypeptide antigen
50mg and purity are greater than 95%.
Step S02, uses PLA2Mouse, the splenocyte of the mouse after adaptive immune is immunized in proteantigen.
Specifically, the PLA prepared using step S012Proteantigen is respectively immunized 5 Balb/c mouse.Wherein,
Balb/c mouse is albino lab mouse, every average complete at least 4 times it is immune with 3 blood samplings detection, through first,
Two, the target mouse for filtering out potency higher (being screened with the polypeptide antigen of above-mentioned synthesis) after blood sampling detection three times, at this
In embodiment, 2 mouse are filtered out from 5 mouse, obtain the splenocyte of this 2 mouse.
Step S03 merges splenocyte and murine myeloma cell, and filtering out can be with PLA2The hybridization that proteantigen combines
Tumor cell strain.
It specifically, will be thin from the SP2/0 murine myeloma cell of Balb/c mouse and the spleen of 2 mouse filtered out
Born of the same parents carry out cell fusion, and through culture, observation, detection and yin and yang attribute check experiment after fusion, acquisition can generate antitoxin snake PLA2
The hybridoma cell strain of protein antibodies, and continue culture and selection to it.
Hybridoma is seeded in mouse peritoneal and induces culture, obtains cell culture fluid after culture by step S04, from
Obtain can be with PLA for purifying in the cell culture fluid2The antitoxin snake PLA that proteantigen is immunoreacted2Protein antibodies.
Specifically, before being inoculated with hybridoma, first only to mouse peritoneal injecting fluid paraffin 0.5mL/, after a week small
The hybridoma suspension 2 × 10 that mouse intraperitoneal injection is prepared using the above method6/ mL, every mouse injection 0.5mL hybridoma are thin
Born of the same parents' suspension.
After being inoculated with hybridoma after about 7~10 days, the ascites for the mouse that abdomen obviously expands is acquired under aseptic condition
10000g, centrifugal treating 10min remove the impurity such as lipid, cell, the supernatant for taking centrifugal treating to obtain and 56 DEG C of inactivation 30min,
Obtain PLA2Protein antibodies.With indirect ELISA, (Enzyme Linked Immunosorbent Assay, Enzyme-linked Immunosorbent Assay are surveyed
It is fixed) method detection PLA2Protein antibodies potency (polypeptide antigen that envelope antigen is synthesis).It is anti-by indirect ELISA method verifying
Body and antigen binding situation, and the higher antibody of potency ratio is filtered out, the specificity of the polypeptide antigen of antibody and synthesis is verified, most
Optimal pairing antibody is obtained by matching screening eventually.
It is screened by optimal pairing, the results show that anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2-1
And Tritoxomab-PLA2-2；Anti- solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 1 and Protoxomab-
PLA2-2；Anti- adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 1 and Viptoxomab-PLA2-2；Anti- cobra
PLA2Protein monoclonal antibody Najatoxomab-PLA2- 1 and Najatoxomab-PLA2-2；Anti- pallas pit viper PLA2Protein monoclonal
Antibody Glotoxomab-PLA2- 1 and Glotoxomab-PLA2- 2, this ten kinds of antibody and corresponding poisonous snake PLA2Protein-specific
Binding ability is strong.
The present embodiment is melted into using method preparation monoclonal antibody is induced in intraperitoneal inoculation animal body in hybridoma cell clone
After function, hybridoma, which is inoculated in mouse peritoneal, can induce a large amount of ascites of generation, while secrete lot of antibodies in ascites,
Concentration is higher, conveniently filters out the higher PLA of potency2Protein antibodies.
In other embodiments, monoclonal antibody can also be prepared using extracorporeal culture-ing, this will not be detailed here.
Embodiment two
Anti- green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2-1、Tritoxomab-PLA2- 2 and green bamboo snake
PLA2Protein Epitopes binding test, specific steps:
1) it is coated with: green bamboo snake PLA2Albumen (5 μ g/ml) diluted with coating buffer after according to every 100 μ l of hole amount be added 96 holes
Plate and blank well add 100 μ l coating buffers, and 4 DEG C of coatings are overnight.
2) it washing: outwelling within second day plate hole endoperidium liquid, washed three times with cleaning solution, 250 μ l of every hole is 3-5 minutes each,
It pats dry as far as possible.
3) it closes: confining liquid every hole 200 μ l, 37 DEG C of 1h.It washs three times, pats dry later.
4) 100 μ l antibody Tritoxomab-PLA are added in positive hole2- 1 or antibody Tritoxomab-PLA2- 2 (5 μ g/ml),
100 μ l confining liquids, 37 DEG C of 1h are then added in negative hole and blank well.It washs three times, pats dry later.
5) positive hole, negative hole and blank well are added 100 μ l horseradish peroxidases (HRP) and mark Tritoxomab-
PLA2- 2 or Tritoxomab-PLA2- 1 (5 μ g/ml), 37 DEG C are protected from light incubation 1h.It washs three times, pats dry later.
6) develop the color: developing solution is added in every 100 μ l of hole after washing, and 37 DEG C are protected from light incubation 15-30 minutes.
7) terminate: the every 100 μ l of hole of terminate liquid carries out 450/630nm dual wavelength readings with microplate reader.
When in coating green bamboo snake PLA2Antibody Tritoxomab-PLA is added in the hole of albumen2- 1 adds the anti-of HRP label
Body Tritoxomab-PLA2- 2 or addition antibody Tritoxomab-PLA2- 2 add the antibody Tritoxomab- of HRP label
PLA2In the case where -1, positive hole OD value is obviously higher than negative hole.The result illustrates antibody Tritoxomab-PLA2- 1 and
Tritoxomab-PLA2- 2 and green bamboo snake PLA2Albumen difference epitope combines.
Wherein, above-mentioned used solution formula is as follows:
1, coating buffer: the 0.05M carbonate buffer solution of pH 9.6；Sodium carbonate 1.59g+ sodium bicarbonate 2.93g, is dissolved to 1L
In deionized water.
2, cleaning solution: the Tween-20 of cell phosphate buffer (PBS)+0.1%.Note: cell PBS: potassium chloride,
0.2g；Potassium dihydrogen phosphate, 0.2g；Sodium chloride, 8g；12 water sodium dihydrogen phosphates, 2.16g, deionized water 1L.
3, confining liquid (antibody diluent): every 100ml+ bovine serum albumin(BSA) (BSA) 1g of cleaning solution.
4, developing solution: 3,3', 5,5'- tetramethyl benzidine (TMB) one-component developing solutions (Solarbio, Beijing).
5, terminate liquid: the sulfuric acid of 2mol/L, the 178.3ml water+21.7ml concentrated sulfuric acid slowly stir and evenly mix.
Embodiment three
The present embodiment provides a kind of double crush syndrome detection kits, including are coated with PLA2The solid phase of protein antibodies
Carrier, enzyme mark PLA2Protein antibodies, dilution, chromogenic substrate, terminate liquid, sealer and positive criteria product.Wherein, PLA2Albumen
Antibody includes anti-green bamboo snake PLA2Protein monoclonal antibody group, anti-solder horn PLA2Protein monoclonal antibody group, anti-adder PLA2Egg
White monoclonal antibody group, anti-cobra PLA2Protein monoclonal antibody group, anti-pallas pit viper PLA2Protein monoclonal antibody group.
Anti- green bamboo snake PLA2Protein monoclonal antibody group (Tritoxomab-PLA2- 1 and Tritoxomab-PLA2- 2),
Tritoxomab-PLA2- 1 and Tritoxomab-PLA2- 2 respectively as coated antibody and enzyme labelled antibody, wherein
Tritoxomab-PLA2- 2 include detectable label, and solid phase carrier is microwell plate, magnetic-particle or plastic tube, enzyme marker
It is horseradish peroxidase.
In the present embodiment, the PLA of above-mentioned 5 kinds of poisonous snakes is equipped in kit2Protein monoclonal antibody group, every kind of PLA2Egg
White monoclonal antibody group is independently arranged, to be snapped in the serum of position using double antibody sandwich ELISA detection human body
Which kind of poisonous snake toxin belongs to, and fast and easy diagnoses to obtain as a result, and being treated with corresponding antitoxin snake serum.
In other embodiments, kit may include any 4 kinds, 3 kinds, 2 kinds in above-mentioned 5 kinds of poisonous snakes or a kind of PLA2
Protein monoclonal antibody group.
Further, the present invention also provides a kind of positive criteria product of snakebite diagnostic kit, positive criteria product is natural
The PLA of the poisonous snake isolated and purified2The PLA of albumen or the artificial synthesized amino acid sequence containing hypervariable region2Albumen.In present embodiment
In, positive criteria product is the PLA of the poisonous snake of naturally isolated purifying2Albumen, in other embodiments, positive criteria product can also be with
It is the synthesis PLA of the hypervariable region amino acid sequence containing certain poisonous snake2Albumen, such as two hypervariable region amino acid sequences by green bamboo snake
Arrange the polypeptide of SEQ ID NO:6 and SEQ ID NO:7 synthesis.
Specifically, natural PLA2The preparation method of protein standard substance, comprising:
1, the thick malicious solution of preparation specifically takes out green bamboo snake, the solder horn, adder, eye that suitable laboratory saves respectively
The freeze-dried powder of mirror snake and pallas pit viper is dissolved to obtain the thick malicious solution of 5 kinds of poisonous snakes with the ultrapure water of appropriate volume.
2, molecule is pressed by Sephadex G-75 chromatographic column (buffer: the Tris-HCl solution of 20mmol/L, pH8.0)
Measure the thick malicious solution example of above-mentioned five kinds of size separation purifying.
3, the thick malicious solution example phosphate buffer after first isolating and purifying dilutes 5 times, wherein phosphate buffer
By 0.2g KCl, 0.2g KH2PO4With 3.47g NaHPO4·12H2O is dissolved in 1L water and is prepared, pH 7.0.It uses again
0.22 μm of membrane filtration, and it is splined on AKTA Resource Q or S column, it is carried out with the NaCl solution linear gradient of 0~1mol/L
Elution, flow velocity 1ml/min.Then, each eluting peak is detected and collected at a wavelength of 280 nm, is lyophilized, it is true with lecithin flat band method
Peak activity is determined, until obtaining the natural PLA that purity is greater than 95%2Until protein standard substance.
In the present embodiment, the natural PLA for 5 kinds of poisonous snakes being prepared2Protein standard substance passes through SDS-PAGE
(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, lauryl sodium sulfate poly- third
Acrylamide gel electrophoresis) electrophoresis and dying method with coomassie brilliant blue carry out purity detecting, and purity is greater than 95%.
It is to be appreciated that standard items are correspondingly arranged with antibody group, such as kit includes green bamboo snake PLA2Protein monoclonal
Antibody, solder horn PLA2Protein monoclonal antibody, pallas pit viper PLA2Protein monoclonal antibody, then standard items should include these three poison
The natural PLA of snake2Protein standard substance.
The above is merely preferred embodiments of the present invention, be not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent substitution, improvement and etc. done be should be included within the scope of the present invention.
SEQUENCE LISTING
<110>Kunming Institute of Zoology, Chinese Academy of Sciences
<120>antitoxin snake PLA2 protein antibodies and its application
<130> DD190748I
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 121
<212> PRT
<213>green bamboo snake
<400> 1
Val Ile Glu Leu Gly Lys Met Ile Phe Gln Glu Thr Gly Lys Asn Pro
1 5 10 15
Ala Thr Ser Tyr Gly Leu Tyr Gly Cys Asn Cys Gly Pro Gly Gly Arg
20 25 30
Arg Lys Pro Lys Asp Ala Thr Asp Arg Cys Cys Tyr Val His Lys Cys
35 40 45
Cys Tyr Lys Lys Leu Thr Asp Cys Asp Pro Ile Lys Asp Arg Tyr Ser
50 55 60
Tyr Ser Trp Val Asn Lys Ala Ile Val Cys Gly Glu Asp Asn Pro Cys
65 70 75 80
Leu Lys Glu Met Cys Glu Cys Asp Lys Ala Val Ala Ile Cys Phe Arg
85 90 95
Glu Asn Leu Asp Thr Tyr Asp Lys Lys Lys Lys Ile Asn Leu Lys Leu
100 105 110
Phe Cys Lys Lys Thr Ser Glu Gln Cys
115 120
<210> 2
<211> 122
<212> PRT
<213>solder horn
<400> 2
Asn Leu Phe Gln Phe Gly Glu Met Ile Leu Glu Lys Thr Gly Lys Glu
1 5 10 15
Val Val His Ser Tyr Ala Ile Tyr Gly Cys Tyr Cys Gly Trp Gly Gly
20 25 30
Gln Gly Arg Ala Gln Asp Ala Thr Asp Arg Cys Cys Phe Val His Asp
35 40 45
Cys Cys Tyr Gly Thr Val Asn Asp Cys Asn Pro Lys Thr Ala Thr Tyr
50 55 60
Ser Tyr Ser Phe Glu Asn Gly Asp Ile Val Cys Gly Asp Asn Asp Leu
65 70 75 80
Cys Leu Arg Thr Val Cys Glu Cys Asp Arg Ala Ala Ala Ile Cys Leu
85 90 95
Gly Gln Asn Val Asn Thr Tyr Asp Lys Asn Tyr Glu Tyr Tyr Ser Ile
100 105 110
Ser His Cys Thr Glu Glu Ser Glu Gln Cys
115 120
<210> 3
<211> 134
<212> PRT
<213>adder
<400> 3
Asn Leu Ile Gln Phe Gly His Ile Ile Glu His Leu Thr Gly Arg Arg
1 5 10 15
Pro Leu Ile Tyr Asn Gly Tyr Gly Cys Tyr Cys Gly Leu Gly Gly Ser
20 25 30
Arg Gln Pro Val Asp Ala Thr Asp Trp Cys Cys Gln Val His Asp Cys
35 40 45
Cys Tyr Gln Ala Leu Ser Arg Arg His Cys Lys Pro Lys Met Glu Lys
50 55 60
Tyr Phe Tyr Ser Val Arg Lys Asp Thr Val Thr Cys Gly Gly Glu Thr
65 70 75 80
Glu Cys Arg Arg Glu Thr Cys Glu Cys Asp Lys Ala Ala Ala Leu Cys
85 90 95
Phe Arg His Ser Lys Phe Gln Gly Gln Tyr Ile His Tyr Arg Asn Cys
100 105 110
Leu Cys Glu Gly Pro Thr Pro Pro Cys Gln Gly Val Cys Pro Arg Trp
115 120 125
Ala Pro Thr Lys Gly Gly
130
<210> 4
<211> 124
<212> PRT
<213>cobra
<400> 4
Arg Pro Met Pro Leu Asn Leu Tyr Gln Phe Lys Asn Met Ile Gln Cys
1 5 10 15
Thr Val Pro Ser Arg Ser Trp Trp Asp Phe Ala Asp Tyr Gly Cys Tyr
20 25 30
Cys Gly Arg Gly Gly Ser Gly Thr Pro Val Asp Asp Leu Asp Arg Cys
35 40 45
Cys Gln Val His Asp His Cys Tyr Asn Glu Ala Glu Lys Ile Ser Gly
50 55 60
Cys Trp Pro Tyr Ser Lys Thr Tyr Ser Tyr Glu Cys Ser Gln Gly Thr
65 70 75 80
Leu Thr Cys Lys Gly Gly Asn Asn Ala Cys Ala Ala Ala Val Cys Asp
85 90 95
Cys Asp Arg Leu Ala Ala Ile Cys Phe Ala Gly Ala Pro Tyr Asn Asn
100 105 110
Asn Asn Tyr Asn Ile Asp Leu Lys Ala Arg Cys Gln
115 120
<210> 5
<211> 124
<212> PRT
<213>pallas pit viper
<400> 5
Ser Leu Leu Gln Phe Glu Thr Leu Ile Met Lys Val Ala Lys Lys Ser
1 5 10 15
Gly Met Val Trp Cys Phe Val His Asp Cys Cys Tyr Gly Lys Val Thr
20 25 30
Gly Cys Asp Pro Lys Met Asp Val Tyr Ser Phe Tyr Ser Asn Tyr Gly
35 40 45
Cys Tyr Cys Gly Trp Gly Gly Gln Gly Arg Pro Gln Asp Ala Thr Asp
50 55 60
Arg Cys Ser Glu Glu Asn Gly Asp Ile Val Cys Gly Gly Asp Asp Pro
65 70 75 80
Cys Lys Lys Glu Ile Cys Glu Cys Asp Arg Ala Ala Ala Ile Cys Phe
85 90 95
Arg Asp Asn Leu Asn Thr Tyr Asn Asp Lys Lys Tyr Trp Ala Phe Gly
100 105 110
Ala Lys Asn Cys Pro Gln Glu Glu Ser Glu Pro Cys
115 120
<210> 6
<211> 14
<212> PRT
<213>green bamboo snake
<400> 6
Phe Gln Glu Thr Gly Lys Asn Pro Ala Thr Ser Tyr Gly Leu
1 5 10
<210> 7
<211> 12
<212> PRT
<213>green bamboo snake
<400> 7
Lys Lys Leu Thr Asp Cys Asp Pro Ile Lys Asp Arg
1 5 10
<210> 8
<211> 14
<212> PRT
<213>solder horn
<400> 8
Leu Glu Lys Thr Gly Lys Glu Val Val His Ser Tyr Ala Ile
1 5 10
<210> 9
<211> 12
<212> PRT
<213>solder horn
<400> 9
Gly Thr Val Asn Asp Cys Asn Pro Lys Thr Ala Thr
1 5 10
<210> 10
<211> 13
<212> PRT
<213>adder
<400> 10
Glu His Leu Thr Gly Arg Arg Pro Leu Ile Tyr Asn Gly
1 5 10
<210> 11
<211> 14
<212> PRT
<213>adder
<400> 11
Gln Ala Leu Ser Arg Arg His Cys Lys Pro Lys Met Glu Lys
1 5 10
<210> 12
<211> 14
<212> PRT
<213>cobra
<400> 12
Gln Cys Thr Val Pro Ser Arg Ser Trp Trp Asp Phe Ala Asp
1 5 10
<210> 13
<211> 15
<212> PRT
<213>cobra
<400> 13
Asn Glu Ala Glu Lys Ile Ser Gly Cys Trp Pro Tyr Ser Lys Thr
1 5 10 15
<210> 14
<211> 14
<212> PRT
<213>pallas pit viper
<400> 14
Met Lys Val Ala Lys Lys Ser Gly Met Val Trp Cys Phe Val
1 5 10
<210> 15
<211> 13
<212> PRT
<213>pallas pit viper
<400> 15
Cys Gly Trp Gly Gly Gln Gly Arg Pro Gln Asp Ala Thr
1 5 10
Claims (8)
1. antitoxin snake PLA2Protein antibodies, which is characterized in that the antibody can be with the PLA of poisonous snake2Protein-specific combines, and
The antibody by the poisonous snake PLA2The hypervariable region amino acid sequence of albumen is prepared.
2. antitoxin snake PLA as described in claim 12Protein antibodies, which is characterized in that from green bamboo snake, solder horn, adder,
The PLA of cobra and/or pallas pit viper2The hypervariable region amino acid sequence of albumen includes SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID
At least one of NO:14 and SEQ ID NO:15.
3. antitoxin snake PLA as described in claim 12Protein antibodies, which is characterized in that the antibody includes:
Anti- green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 1, anti-green bamboo snake PLA2Protein monoclonal antibody
Tritoxomab-PLA2- 2, anti-solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 1, anti-solder horn PLA2Albumen
Monoclonal antibody Protoxomab-PLA2- 2, anti-adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 1, anti-adder
PLA2Protein monoclonal antibody Viptoxomab-PLA2- 2, anti-cobra PLA2Protein monoclonal antibody Najatoxomab-
PLA2- 1, anti-cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 2, anti-pallas pit viper PLA2Protein monoclonal antibody
Glotoxomab-PLA2- 1, anti-pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-PLA2At least one of -2.
4. anti-snake venom PLA as claimed in claim 32Protein antibodies, which is characterized in that
The anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 1 with have the ammonia as shown in SEQ ID NO:6
The PLA of base acid sequence2Protein binding, the anti-green bamboo snake PLA2Protein monoclonal antibody Tritoxomab-PLA2- 2 with have such as
The PLA of amino acid sequence shown in SEQ ID NO:72Protein binding；
The anti-solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 1 with have the ammonia as shown in SEQ ID NO:8
The PLA of base acid sequence2Protein binding, the anti-solder horn PLA2Protein monoclonal antibody Protoxomab-PLA2- 2 with have such as
The PLA of amino acid sequence shown in SEQ ID NO:92Protein binding；
The anti-adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 1 with have the ammonia as shown in SEQ ID NO:10
The PLA of base acid sequence2Protein binding, the anti-adder PLA2Protein monoclonal antibody Viptoxomab-PLA2- 2 with have such as
The PLA of amino acid sequence shown in SEQ ID NO:112Protein binding；
The anti-cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 1 with have as shown in SEQ ID NO:12
The PLA of amino acid sequence2Protein binding, the anti-cobra PLA2Protein monoclonal antibody Najatoxomab-PLA2- 2 with tool
Just like the PLA of amino acid sequence shown in SEQ ID NO:132Protein binding；
The anti-pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-PLA2- 1 with have the ammonia as shown in SEQ ID NO:14
The PLA of base acid sequence2Protein binding, the anti-pallas pit viper PLA2Protein monoclonal antibody Glotoxomab-PLA2- 2 with have such as
The PLA of amino acid sequence shown in SEQ ID NO:152Protein binding.
5. antitoxin snake PLA as described in claim 12Protein antibodies, which is characterized in that the antibody is whole antibody or its antigen
Binding fragment.
6. snakebite diagnostic reagent, which is characterized in that the snakebite diagnostic reagent includes described in Claims 1 to 5 any one
Antibody.
7. snakebite diagnostic kit, which is characterized in that the snakebite diagnostic kit includes Claims 1 to 5 any one institute
The antibody and positive criteria product stated.
8. snakebite diagnostic kit as claimed in claim 7, which is characterized in that the positive criteria product is naturally isolated purifying
Poisonous snake PLA2The PLA of albumen or the artificial synthesized amino acid sequence containing hypervariable region2Albumen.
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Cited By (3)
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| CN113832132A (en) * | 2021-10-13 | 2021-12-24 | 中国科学院昆明动物研究所 | Solder tip SVMP protein specific short peptide, solder tip SVMP protein antibody and snake bite detection kit |
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| CN114716567A (en) * | 2022-03-01 | 2022-07-08 | 上海赛伦生物技术股份有限公司 | Preparation method and application of chimeric antigen and anti-bungarus fasciatus snake venom preparation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113832132A (en) * | 2021-10-13 | 2021-12-24 | 中国科学院昆明动物研究所 | Solder tip SVMP protein specific short peptide, solder tip SVMP protein antibody and snake bite detection kit |
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| CN113930408B (en) * | 2021-10-13 | 2023-04-28 | 中国科学院昆明动物研究所 | Bamboo leaf green PLA2 protein specific short peptide, anti-bamboo leaf green PLA2 protein antibody and snake bite detection kit |
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