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Antagene and its collaborator have developed a rapid antibody screen platform (Box “Process timeline”) which compiles comprehensive information for each antibody into a large database called the Antibody AtlasTM. Our precision engineered instrument (Box “Assay instrument”) accurately aligns and captures biomolecules from antibody producing cells onto multiple slides for consistent registration of phenotypic data (antibody affinity & specificity) and genotypic data (full length VH & VL). This novel and patented dual-assay antibody screen is unparalleled in the industry. If you are interested in finding those long sought-after antibodies, you will want to use our platform.

Project details;

Cost:  The pricing is $80,000 per run. 

Note:  Because of the expensive consumable used, we require $40,000 payment upon signing of a contract.  The remaining balance will be paid upon submission of final report with a successful dataset. 

A run includes the following:
·         A pilot run of ELISPOT assay to assess the number of antigen-specific antibody producing cells from splenocytes of a mouse from a cohort of mice.  It is hope that the titer is somewhat correlated with the number of antigen-specific antibody cells.  ELISPOT will determine if immunization was optimal.
·         20,000 CD138+ cells from a high titer mouse are deposited on a picoliter device.
·         A single cell antibody array (SCAA) is generated from the picoliter device.  The SCAA is interrogated with a fluorescently labeled antigen and the antigen-specific spots are identified and affinity (KD) for each antibody is measured.
·         An Agilent chip is used to capture mRNA from all cells on the picoliter device.
·         Tagged cDNA will be generated on the Agilent chip and eluted.
·         Eluted and tagged cDNA will be PCR amplified.
·         A single 2 x 250 bp Illumina MiSeq run is performed.  If the cDNA sequence is not long enough to cover full length V domain, a second run will be performed.

Deliverable:
1.      A summary plot showing the distribution of all measured koff and kon values of each of the captured antibody.
2.      A spreadsheet listing the cDNA and amino acid sequences covering the VH and VL from all antigen-specific IgGs with each kinetic measurement (KD and koff).
3.      A dendrogram showing concatenated HCDR3-LCDR3 sequences clustered into similar families, referred to as paratope clusters, with a kinetic value listed for each antibody member.

II  Antibody Cloning , Expression and Purification

Phase 1:    Cloning of scFv antibody from gene synthesis and production of antibody scFv fusion protein
6-8 weeks $20,000

1- Description: Clone the Vh and Vk sequence from gene synthesis into the mammalian expression vector
Deliverable:  Gene synthesis product                

2- Expression & purification of 10 mg of antibody scFv fragment protein
 * Construction of mammalian expression vector
 * Transient expression and purification of the fusion protein from mammalian culture supernatant
Note: Although scFv could be expressed in bacterial periplasm, solubility and folding is usually an issue and that formation of mixed forms is found quite often, even with a flexible GS linker between the variable domain. Secretary expression of the scFv fusion from mammalian cells in serum-free medium is therefore recommended for this project.
3- The reconstructed scFv antibodies will be assayed by ANTAGENE using ELISA to assess binding activities.
4-  Depending on the nature of the antigen, ANTAGENE will perform appropriate functional assays to assess the scFv antibody’s therapeutic potentials.

Deliverable: 10 mg of the ScFV protein

Phase 2:  Full length antibody expression
                  12-15 weeks     $60,000

ACTIVITIES
5- Subcloning of  antibody Vh and Vk variable regions gene into Antagene’s proprietary vector for full length antibody expression
6- Sequence confirmation of expression vector DNA
- Results will be delivered to Client via email
7-  Transfection of host cell line (CHO-based) with expression vector DNA
8-  Stable cell line selection and stabilization
9-  Scale-up culture
10-  Purification of supernatant from stable pools
11-  Endotoxin and DNA/RNA removal
12-  Characterization of antibody
-Concentration by UV280
- Purity by SDS‐PAGE
- Endotoxin, DNA/RNA concentration analyses          

Phase 3:  1-2 weeks        $10,000

DELIVERABLES
􀂃 Project report
􀂃 Sequence confirmation of expression vector DNA
􀂃 50mg purified full length humanized anti-human mAb antibody
􀂃 Results from Characterization of antibody

-  Concentration by UV280
-  Purity by SDS‐PAGE
-  Endotoxin, DNA/RNA concentration analyses

III  Antigen related Technology:
 
a ) Antigen Preparation: Recombinant Protein Expression in Bacteria                 

1- Cloning of the genes into expression vector (tagged with 6xHis)
Total Time: 3-4 weeks   Price: US $ 2,800

Protein Expression Service combines a variety of expression systems (bacterial and balcuvirus) with a full range of purification options, providing you with the material that you require for your application. The service spans vector construction through to purification of ready to use protein and can be tailored to meet your exact requirements.
A), Construct Generation:
•              Clone coding sequence into one of a wide range of expression vectors
•              The expression construct will be fully sequence verified
•              Preparation of plasmid construct DNA
B), Assessment and Optimization:
•              Introduce plasmid construct DNA into bacterial
•              Selection of over-expressing cells
•              Small scale expression and purification study assessed by SDS-PAGE and 
C), Western blot (anti-tag or specific antiserum) analysis
•              Estimation of protein yield
2-, Scale-up and Purification:
Total Time: 1-2 weeks,  Price: US $ 1,200
•              Large-scale growth of over-expressing cells
•              Cells are harvested and lysed Protein typically purified from cleared lysate via 　　　　　　
purification tag
•              Further purification to remove any contaminants e.g. gel filtration
•              SDS-PAGE and Western blot analysis of purified protein
•              Removal of purification tag and further SDS-PAGE analysis
•              Determination of protein concentration
•              Delivery of at least 10mg of purified protein ready for use in your application

b ) Antigen Preparation: Recombinant Protein Expression in Baculovirus
 
Total Cost:                           $6,300 + Cost of Gene Synthesis                       9 weeks

1.  Gene Synthesis              Gene Synthesis at $0.70/b       
1 weeks         

2. Clone the gene of interest into the suitable baculovirus transfer vector.  
2 weeks at $1,000      
        -PCR/restriction enzyme digestion will be used to clone the gene of interest into a baculovirus transfer vector.
        -If necessary, the sub-clone vector will be confirmed using restriction enzyme digestion and DNA sequence.
        -The recombinant transfer vector will be prepared for further experimentation.              

3.  Generate the recombinant baculovirus for the target gene.  
3 weeks at $2,500
        -Transfect the insect cells (sf9 cells) with recombinant transfer vector and baculovirus DNA.
        -The recombinant virus will be screened by plaque assay.
        -The recombinant virus will be amplified to a 30mL high-titer stock. 
       
4. Amplification of recombinant baculovirus for titer stock. 
2 weeks at $850        
        -A 250mL high-titer virus stock will be generated from a low-titer stock of a  recombinant virus.
        -Several rounds of amplification may be required in order to increase the recombinant virus titer stock.
     
5.   Protein Expression  
1 week          at $950
        -One liter of insect cell culture will be generated.
        -Cell pellet will be harvested and delivered.               
       
6.   Protein Purification   
1 week at $1,000
       -The protein will be purified using affinity column of His-tag and GST-tag         recombinant proteins.
      Total                9 weeks               $6,300 + Cost of Gene Synthesis

Note:
-Purification development is not included and protein yield is variable under the baculovirus vector expression system.
-We offer and provide optimization for protein expression, which is based on M.O.I. and different cell lines. Optimization is optional.
-Price may vary depending on project.
C ),  Antigen Immunization Protocol
                   Cost: $1,000
Mouse Immunization Protocol:

Subject:	Balb/c  mice
Adjuvant:	Complete Freund's Adjuvant (CFA) initially, followed by incomplete Freund's Adjuvant (IFA) for all subsequent injections.
Immunogen:	Typically, proteins and conjugated peptides. Amount of immunogen used for each immunization is 0.1 mg /per mouse /per injection.
Procedure:	The immunogen is diluted to one mL with sterile saline and combined with one mL of the appropriate adjuvant. The antigen and adjuvant are mixed thoroughly to form a stable emulsion which is injected subcutaneously and provides enhanced immune response from the sustained presence of the immunogen.
Bleeds:	Blood is collected from the tail and allowed to clot and retract at 37°C overnight. The clotted blood is then refrigerated for 24 hours before the serum is decanted and clarified by centrifugation at 2500 rpm for 20 minutes.
Schedule:	Day 1 Bleed 0.1-0.2 ml. 1st Immunize with antigen in CFA. Day 20 2nd Immunize with antigen in IFA. Day 40 3rd Immunize with antigen in IFA. Day 50 Test Bleed 0.1-0.2 mL for ELISA screen(internal quality control only, not provide to client). Day 60 4th  Immunize with antigen in IFA. Day 70 Final Total Bleed 0.2-0.5 mL from Each mouse.

 We have protocol available for rabbit, chicken, goat immunization and other animals of your choice