iGEM 2023 — Team yangzhounofls

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Experiment timeline

As
can be seen in the introduction our product is an ELISA kit with a special kind of antibody. So we first
design the plasmids for the protein by homologous recombination. After a series of steps such as
transfection, we successfully obtained the desired antibody. To test it, we coated the  antibody on a
96-well plate as a tool antibody for ELISA detection. Fortunately, our antibody is successful and we
attained a standard curve for our ELISA kit.

**Week 1**

Did
the ice breaking activities and determined our team name and logo. Learned about our project and allocated
jobs to different teamates.

Week
1 Visited the lab. Prepared the LB broth and agarose electrophoresis gel. Prepared the gel for SDS-PAGE,
did the cell resuscitation.

**Week
2**

Digested
the plasmid base.Did the polymerase chain reaction. Constructed the plasmids by homologous recombination.
Tested the plasmid through agaros electrophoresis gel then purified the gel.

**Week
2**

Incited
the monoclonal culture and waited for results for experiment we did before. Communicated with dry lab
about the future collavoration.Picked up monoclone from petri dish

**Week
3**

Reanimate
the cell that we need for the transfection. Transfected the plasmid into cells to make them combine inside
cells. Transfered the cells into a erlenmeyer flask to make them expess the antibody we want.

**Week
3**

Purified
the protein that expressed from the cell which is introduced with plasmids. Analyzed the SDS-PAGE
gel.

**Week
4**

Did
the ELISA test to make sure that our antibody is effective. Make the standard curve for our
product.

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INTRODUCTION

Cancer,
a disease that the world is concerned about. Hundreds of millions of people died of it, and tens of
thousands of families were destroyed by it.

Human
struggle against cancer has been going on for thousands of years, and early people used plants and
minerals to treat cancer, but it didn't work until surgery opened the door to cancer
treatment.Anesthesiology and sterilization have greatly contributed to the development of cancer surgical
treatment, which has continued to be used and improved for decades, until radiation and chemotherapy led
to the first revolution in cancer treatment. In
1943, Alfred Gilman and Louis Goodman, pharmacologists at Yale University in the United States,
successfully conducted a clinical trial to treat lymphoma with nitrogen mustard, which opened the way for
modern cancer chemotherapy.

However,
since chemotherapy drugs or therapies are usually non-targeted, they can also attack normal tissues and
cells while killing cancer cells, causing numerous side effects.Later
on, the
discovery of DNA's double-helix structure revolutionized our understanding of many diseases, pinpointing
mutations at a genetic level, including in cancer. This breakthrough paved the way for targeted drug
therapies. These drugs, purposefully designed, hone in on specific cancer-causing agents, obliterating
tumor cells while sparing surrounding healthy tissues.

To
optimize the application of these targeted drugs, it's imperative to ensure their stability and
specificity. Our innovation lies in an ELISA-based detection method to assess both the stability and
targeting capacity of an ADC drug named "52B8G1-PTX", which is conjugated with paclitaxel (abbreviated as
PTX).

DESIGN

Our
product features a small molecular antibody termed "anti-ptx-mAb". This antibody ensures precise detection
of the stability and targeting abilities of "52B8G1-PTX" in plasma. The "anti-ptx-mAb" is composed of two
main parts: a light chain, referred to as "pEE6.4", and a heavy chain, designated "pEE12.4". These chains
are conjoined using the vector pcDNA3.1 and target fragments "33D9VH" and "33D9VL" through a process of
homologous recombination

Our
entire experimental process, in fact, revolves around the anti-ptx molecular antibody, which is the most
important and unique part of the whole reactor box. Through it we can test the stability and targeting of
52B8G1-PTX.

To
produce the desired antibodies, we began by amplifying the DNA fragments, 33D9VH and 33D9VL, using PCR.
These fragments were then integrated into the pEE12.4 and pEE6.4 vectors through an enzyme-mediated
digestion process. After undergoing homogenization and sequencing tests, we successfully generated the
constructs pEE12.4-33D9H and pEE6.4-33D9L.

Subsequently,
we introduced both constructs into 293F cells via co-transfection, prompting the cells to express the
desired antibody. The expressed antibody was then purified using protein A beads. With our anti-ptx
molecular antibody in hand, we conducted a final validation step using the enzyme-linked immunosorbent
assay (ELISA). This concluded our experimental workflow.

# ![](https://static.igem.wiki/teams/4961/wiki/engineering/enginnering-01.png)

Figure1.
Work flow of our design

#

BUILD

Since
the anti-ptx molecular antibody are composed of heavy chain and light chain, so, we need to construct two
expression plasmids pEE12.4-33D9H, pEE6.4-33D9L that can express antibody heavy chain and antibody light
chain respectively. In order to construct pEE6.4-33D9L and pEE12.4-33D9H, we first carried out an enzyme
cut to obtain a carrier – in the two PCR tubes, the corresponding enzymes, water, templates, and
rapid enzym cut buffers were added, respectively. Finally, 50 microliters of solution were obtained in two
tubes. Place them in the 37°C enzyme cut in the PCR and remove them for 3 hours.

The
second step is to extend the target fragments 33D9VH and 33D9VL.Similarly, two PCR tubes are prepared, and
the corresponding template, water, upstream and reverse downstream and prime STAR MAX premix are inserted.
Configure into 50 microliters of
solution, then put them in the PCR instrument for 1 hour and remove.

The
third step is the purification of the carrier and target fragments, where we have chosen to configure the
liposuction gel to purify, adding the GDP buffer, the DW2 buffer,
the Elution buffer and the HiPure DNA Mini Columns.

When
the experiment reached this stage, the phased testing was introduced.We're going to test the concentration
of purified DNA to see if it's too much lost and determine the optimal input. After the test, we combined
the target fragments with the carrier using the co-source reconfiguration method and mixed them together
in a 37°C connection in the PCR
for 30 minutes.

Finally,
we'll sequence the pEE12.4-33D9H and pEE6.4-33D9L plasmids. We
successfully obtained a monoclonal clone of the recombinant plasmid.

![](https://static.igem.wiki/teams/4961/wiki/engineering/engineering-2.jpg)

Figure 2. The monoclonal
clone of the recombinant plasmid

![](https://static.igem.wiki/teams/4961/wiki/engineering/engineering-3.png)

Figure 3. Heavy chain
sequencing results

![](https://static.igem.wiki/teams/4961/wiki/engineering/engineering-4.png)

Figure 4. Light chain
sequencing results

TEST

We
recovered HEK293F cells for 5 minutes in a 37°C bain-marie.
Raise 293F cells to the appropriate density. Expression plasmids PEE 12.4-33D9H and pEE 6.4-33D9L were
transfected into 293F cells and cultured in 25 ml shake flasks for 5 days. From the culture medium
supernatant of 293F cells, we can purify our antibody using protein A affinity chromatography columns and
test the purity of the antibody we obtained by SDS-PAGE.

The
specific experimental steps were as follows: collect the cell supernatant, centrifuge at 4000 rpm for 45
minutes, and collect the clarified liquid. After the protein A chromatography column was equilibrated with
phosphate buffer salt, the clarified liquid from the previous step was injected, and then equilibrated
with phosphate buffer salt, and finally eluted with glycine at pH=3, to get the antibody we want and test
its protein concentration. Its concentration is:1.754mg/ml。

Next,
we verified its purity using SDS-PAGE, due to the fact that the antibody is composed of light and heavy
chains. Therefore with the addition of the reducing agent, we can see two bands, a 25kD one for the light
chain and a 50kD one for the heavy chain. Non-reduced, the bands of the antibody should be around 150kD.

![](https://static.igem.wiki/teams/4961/wiki/engineering/engineering-5.png)

Figure 5  SDS-PAGE gel

Then
we draw the standard curve for our ELISA kit. We diluted the anti-PTX small molecule antibody with 1
× PBS solution at a concentration of 3 μg / mL, and then coated it on a 96-well plate, added 50
μL to each well, and incubated overnight at 4 ° C.The sample with a concentration of 10 μg / mL
was prepared with 52B8G1-PTX and added to 12 wells with a gradient dilution of 5 times. After incubation,
the pre-made enzyme-labeled secondary antibody working solution was added to continue incubation.Finally,
50 μL TMB color developing solution was added to each well to avoid light, and then 50 μL
concentrated sulfuric acid was added to each well. The detection wavelength was set at 450 nm in the
microplate reader. The detected curve is as follows, and the detection range is about 0.125-8 ng /
ml.

![](https://static.igem.wiki/teams/4961/wiki/engineering/engineering-6.png)

Figure 6.  Standard
Curve of 52B8G1PTX of the ADC
drug "52B8G1-PTX"

LEARN

After
completing the experiment, we found that we could not rule out the non-specific binding of ADC and
antibody during the detection process, and could not determine whether it was only about PTX small
molecule. Therefore, in order to eliminate the interference of ADC, we try to add 52B8G1-PTX antibody and
20 % ethanol dissolved PTX small molecule drug to compete with it, and use competitive ELISA to eliminate
non-specific immunity. Under this method, the color depth of the sample was negatively correlated with the
concentration of PTX small molecule, and the standard curve was also opposite to the first method, showing
a downward trend. Finally, the curve of the following figure was obtained, and the detection range was
0.01 ng / ml-1μg / ml.

![](https://static.igem.wiki/teams/4961/wiki/engineering/engineering-7.png)

Figure 7. Competitive ELISA to eliminate non-specific
immunity

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PART I PREPARATION

**Culture Medium
preparation**

A. **Apparatus**

1. Electronic
balance

2. Measuring
Cylinder

3. Pure
Water Equipment

4. Volumetric
Flask

5. High-pressure
sterilizer

B. **Materials**

1. LB
Broth

2. ddH2O

C. **Procedure**

1. Wash
the volumetric flask;

2. Add
25g LB Broth and 1L ddH2O;

3. Sterilization
in the High-pressure sterilizer for one hour.

**Agarose Gel
Electrophoresis Preparation**

A. **Apparatus**

1. Electronic
balance

2. Erlenmeyer
Flask

3. Microwave
Oven

4. Measuring
Cylinder

B. **Materials**

1. Agarose
Powder

2. TAE
Buffer

C. **Procedure**

1. Add
4.5g Agarose Powder into erlenmeyer flask;

2. Add
300ml TAE Buffer into the erlenmeyer flask;

3. Heat
the solution in the microwave oven until it is clear;

4. Pour
the liquid into the mold.

**Cell
resuscitation**

A. **Apparatus**

1. Liquid
Nitrogen Container

2. Centrifuge
Tube

3. Centrifuge

4. Shaking
Flask

5. Shaker

6. pipetting
gun

B. **Materials**

1. Cell:
HEK293F

2. OPM-293
Culture Medium

C. **Procedure**

1. Take
the cell HEK293F out from liquid nitrogen container;

2. Put
the cell into the centrifuge tube;

3. Add
10ml OPM-293 into the tube;

4. Centrifuge
for 5min, 1000rmp;

5. Pour
the supernatant fluid, and add another 25ml OPM-293;

6. Pour
the mixture into the shaking flask;

7. Shake
for 5 to 6 days.

PART II EXPERIMENT

**Enzyme
Digestion**

A. **Apparatus**

1. Tube

2. pipetting
gun

3. PCR
machine

B. **Materials**

1. Heavy
chain carrier model: PEE12.4-33D9H

2. Light
chain carrier model: PEE6.4-33D9L

3. Enzyme
Pfl 23 II

4. Enzyme
Eco 72 I

5. Enzyme
Cpol

6. ddH2O

7. Fast
Digest Buffer

C. **Procedure**

1. Add
the model of carriers into the tube, 5μL each;

2. Add
Fast Digest Buffer, 5μL each;

3. Add
Pfl 23 II 2.5 μL and Eco 72 I 2.5 μL into the tube containing PEE6.4-33D9L;

4. Add
Cpol 2.5 μL and Eco 72 I 2.5 μL into the tube containing PEE12.4-32D9H;

5. Add
ddH2O
to the tube until the total volume reaches 50 μL;

6. Put
the mixture into the PCR machine, 37 degree Celsius, 3 hours;

7. When
reaches 3 hours, shift the temperature to 4 degree Celsius.

PCR

A. **Apparatus**

1. PCR
machine

2. Tube

3. Pipetting
gun

B. **Materials**

1. Model

2. Forward
primer of light chain

3. Forward
primer of heavy chain

4. Reverse
primer of light chain

5. Reverse
primer of heavy chain

6. PrimeSTAR
MAX premix

7. ddH2O

C. **Procedure**

1. Add
25 μL PrimeSTAR MAX premix, 5 μL forward primer, 5 μL reverse primer, 1 μL model, and 14 μL
ddH2O
to the two system separately;

2. Set
the procedure: 98°C 5 minutes, (98 °C 10 seconds, 54°C 15 seconds, 72 °C 45 seconds,
72°C 5 minutes)\* 30 times, 4 °C for the remaining time.

Agarose Gel Electrophoresis

A. **Apparatus**

1. Gel
bed

2. Electrophoresis
tank

3. Comb

4. Pipetting
gun

B. **Materials**

1. Gel
red

2. DNA
Marker

3. ddH2O

4. Electrophoresis
buffer

5. Nucleic
acid dye

C. **Procedure**

1. Add
Dye: when agarose gel is cooled, add nucleic acid dye,and mix well;

2. Preparation
of gel board: waterproof tape is pasted on both ends of the glue bed to form an 8mm high retaining wall,
press the tape, and place it on the horizontal table. Insert the comb, and the lower end of the comb is
about 1mm away from the bottom of the plate. Pour the gel into the glue bed continuously, 3-4mm high,
avoid bubbles, and solidify at room temperature. After complete solidification, tear off the adhesive
tape, place it in the electrophoresis tank, add the electrophoresis buffer, which is 1-2mm higher than the
glue surface, gently pull out the comb obliquely from one end to remove the bubbles generated;

3. Prepare
the solution: Add 1.8 μL plasmid, 2 μL gel red, and 16.2 μL ddH2O;

4. Sampling:
add DNA maker first, then add DNA sample;

5. Electrophoresis:
cover the electrophoresis tank, the sampling hole is located at the negative pole of the electric field,
and connect the power supply, 140 V, 30 min. When the front edge of the dye moves to the bottom edge for
1-2mm, the electrophoresis ends;

6. Observation
results: turn off the power, take out the gel bed, under the UV detector in the gel of plasmid DNA.

Gel Purification

A. **Apparatus**

1. Centrifugal
tube

2. Centrifuge

3. Knife

4. Bain-marie

5. Column

6. Centrifuge
tube

7. Stopwatch

B. **Materials**

1. Gel

2. Buffer
GDP

3. Buffer
BW2

4. Elution
Buffer

C. **Procedure**

1. When
the DNA fragment is separated, place the gel under ultraviolet light, quickly cut off the gel containing
the target DNA fragment and try to remove the excess gel as much as possible;

2. Take
the weight of the gel and transfer it to the centrifugal tube. Add 1 to 3 times the volume Buffer GDP.
50°C Water bath 10 to 15 minutes, let the gel block completely dissolve. During the bath, reverse the
mixture of 3 accelerated lotions;

3. A
short centrifuge collects droplets on the tube wall. Pack the HiPure DNA Mini Column in a 2ml centrifugal
tube. Transfer≤700μl of the solution to the column. 12,000×g centrifugal intervals 30 to 60
seconds;

4. Drop
the filter and put the columns back into the 2ml centrifugal tube. Transfer the remaining solution to the
column.12,000 × g centrifugal intervals 30 to 60 seconds;

5. Drop
the filter and put the columns back into the 2ml centrifugal tube. Add 300μl Buffer GDP to the column.
Stand still for 1 minute. 12,000×g centrifugal intervals 30 to 60 seconds;

6. Drop
the filter and put the columns back into the 2ml centrifugal tube. Add 600μl Buffer DW2;

7. Drop
the filter and put the columns back into the 2ml centrifugal tube. 12,000×g centrifugal intervals 30
to 60 seconds;

8. Drop
the filter and put the columns back into the 2ml centrifugal tube. Add 600μl Buffer DW2 to the column.
12,000×g centrifugal intervals 30 to 60 seconds;

9. Drop
the filter and put the columns back into the 2ml centrifugal tube.12,000xg centrifuge for 2
minutes;

10. Add
15 to 30 μl Elution Buffer to the center of the column membrane in the 1.5ml centrifugal tube. Leave
for 2 minutes. 12,000×g centrifuge for 1 minute. Drop the column and store the DNA at
-20°C;

11. Short
centrifuge, transferred to the sterilized 1.5 or 2ml centrifugal tube.

12. Add
a Buffer GDP of equal volume, invert the tube;

13. Pack
the HiPure DNA column in the collection tube. Transfer the mixture to the DNA column. 12,000×g off
30 to 60 seconds.

Chemical Transformation

A. **Apparatus**

1. Ice
Box

2. Hot
Tub

3. Shaker

B. **Materials**

1. Sensitive
cells

2. LB
culture medium

C. **Procedure**

1. Take
100 pL of the sensitive cells melted on the ice to add DNA gently mixed, on ice static 5min;

2. 42°C
hot tub 45~60s, rapidly transferred to the ice bath static 2min. Add 700 microliters of LB liquid culture
medium to the centrifugal tube;

3. Take
the appropriate volume of recovery liquid uniformly applied to the antibiotic culture base containing amp,
37 ° C culture box reversed to cultivate overnight.

Incite the monoclonal culture

A. **Apparatus**

1. Pipet

2. Tube

B. **Materials**

1. Culture
plate

2. LB
Broth

3. Amp

C. **Procedure**

1. Take
out the plate bacterial culture liquid;

2. Use
a small pipette to place the monoclonal into the bacterial tube;

3. Add
2ml LB medium and Amp.

Plasmid Extraction

A. **Apparatus**

1. Centrifuge

2. Tube

3. Absorbent
Paper

4. HiPure
EF Maxi Column

B. **Materials**

1. Buffer
E1

2. Buffer
E2

3. Buffer
E3

4. Buffer
E4

C. **Procedure**

1. Pour
the liquid to the 4 centrifugal tubes, 50 ml each;

2. Centrifuge,
4200rmp, 45min;

3. Pour
out the supernatant, invert the tube and place it on absorbent paper to drain;

4. Add
2.5 ml Buffer E1 to each tube, mix the liquid;

5. Whirl
the liquids for 10 seconds;

6.  Transfer
the mixed bacterial liquid to one tube;

7. Add
10 ml Buffer E2, mix the liquids gently;

8. Add
10 ml Buffer E3 to neutralize, mix the liquid violently;

9. Centrifuge
;

10. Filtrate,
remove the flocculent matter;

11. Add
Buffer E4, mix;

12. Transfer
half the liquid to the HiPure EF Maxi Column;

13. Centrifuge,
4200rmp, 3min;

14. Remove
the filtrate;

15. Measure
its concentration.

Transfection

A. **Apparatus**

1. Pipet

2. Tube

3. Super
Clean Bench

B. **Materials**

1. Reduced
serum culture medium CD05

2. Plasmid

3. PEI

C. **Procedure**

1. Prepare
liquid A: 1ml CD05+20ug plasmid, rest for 5 min;

2. Prepare
liquid B: 1ml CD05+60μl PEI, rest for 5 min;

3. Pour
liquid B into A, rest for 20 min;

4. Pour
liquid A and B into the cell HEK293F;

5. Shaker
incubation at 37 degrees Celsius for 5-6 days.

Protein Purification

A. **Apparatus**

1. Pipet

2. Tube

B. **Materials**

1. Ultrasounded
ddH2O

2. PBS
phosphate Buffer

3. Tris
HCl

4. Protein
after transfection

C. **Procedure**

1. Prepare
the ultrasounded pure water and PBS phosphate buffer;

2. Transmute
the collected protein after 6 days;

3. Mix
the three into the centrifuge 8,000 rpm centrifugal 30min;

4. Remove
the cells to cleanse;

5. Use
the ultrasounded pure water to wash Protein A for 3 times;

6. Wash
the nickle column with PBS for 3 times;

7. Wash
the protein with Tris HCl.

SDS-PAGE Analysis

A. **Apparatus**

1. Mini-PROTEAN
Tetra cell

2. Comb

B. **Materials**

1. Resolver
A

2. Resolver
B

3. APS

4. TEMED

5. Stacker
A

6. Stacker
B

C. **Procedure**

1. Prepare
Resolver solution: 2.5 ml Resolver A, 2.5 ml Resolver B, 25 μl 10% APS, 2.5 μl TEMED;

2. Fill
the solution to 0.5 cm below the bottom of comb teeth;

3. Prepare
Stacker solution: 1 ml Stacker A, 1 ml Stacker B, 10 μl 10% APS, 2 μlTEMED;

4. Fill
the solution the the top of the model;

5. Insert
the comb and allow polymerization for 30 min;

6. Run
Gel.

ELISA

A. **Apparatus**

1. Enzyme
calibration

2. Board-Washing
machine

B. **Materials**

1. PBST

2. Anti-huma
IgG FC-HRP

3. Casein

4. H2SO4

5. TMB

6. ADC

C. **Procedure**

1. Il13RA2-11His:
50 μl/well, 2 μg/ml;

2. 4°C,
coated overnight;

3. Prepare
0.1% PBST cleaning solution;

4. Prepare
1% Casein closure solution;

5. Close
for 1 hr, 37° C, 200μg/well;

6. Use
0.1%PBST to wash for three times;

7. Add
ADC, 37° C, incubating for 1 hr, 50μl/well;

8. Use
0.1%PBST to wash 3 times;

9. Anti-human
IgG FC-HRP 50μl/well. 37° C, incubating for 1 hr;

10. Use
0.1%PBST to wash 3 times;

11. Add
TMB to show the color: 50μl/well, coloration for 10 min away from dark;

12. Add
50 μl/well 2M H2SO4 to
end the coloration;

13. Detect
the OD value at 450nm using enzyme calibration machine.

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Overview

Antibody-drug
conjugate (ADC) is an emerging class of highly effective drugs that is a perfect combination of
chemotherapy and immunotherapy. As of September 2023, 16 ADC drugs have been marketed worldwide. With the
development of ADCs, it is crucial to test the stability of ADCs in plasma. Our product is an
anti-paclitaxel (ptx) antibody (33D9) that binds Antibody-drug conjugate coupled to ptx with high
specificity and affinity (eg:52B8hG1ptx). Thus, the stability of 52B8hG1ptx in plasma can be detected
using the anti-paclitaxel antibody (33D9), and by analogy, any ADC with ptx as a small-molecule drug can
be detected. in addition to this, the ability of 33D9 to bind not only ptx on an ADC, but also ptx alone,
expands the scope of its application. The most classical method of antibody production is the hybridoma
technique, and through the preliminary research in the laboratory, we have obtained the heavy chain
variable region and light chain variable region sequences of 33D9 respectively. The main task of this
experiment is to combine the heavy chain variable region and light chain variable region sequences of 33D9
with the constant region sequences (mIgG2a) on pEE12.4 and pEE6.4, respectively, by molecular cloning
technology, to form an expression vector that can fully express the antibody. Then, using the transfection
technique, the heavy chain expression vector (pEE12.4-33D9H) and light chain expression vector
(pEE6.4-33D9L) were packed with polyethyleneimine (PEI) transfection reagent, and the above complexes were
transfected into human embryonic kidney epithelial cells (293F) for antibody expression. After 5 days of
expression, we could purify the antibody 33D9 from the supernatant of the culture medium of 293F. finally,
an enzyme-linked immunosorbent assay (ELISA) was applied to detect the ability of 33D9 to bind ADC
(eg:52B8hG1ptx).

Target 1: Plasmid Construction

For
this experiment we designed two plasmids: antibody heavy chain (33D9H) protein expression plasmid, and
antibody light chain (33D9L) protein expression plasmid. Among them, the heavy chain variable region
sequence and light chain variable region sequence of the antibody were obtained from the genomic DNA of
hybridoma cells.

![](https://static.igem.wiki/teams/4961/wiki/results/results-1.png)

Figure
1 Principle of ClonExpress® Rapid Cloning Technology (Vazyme C115)

In
order to construct our complete antibody expression plasmid, we asked Gene Synthesis company synthesise
the DNA fragments, then, using the technique of molecular cloning, we inserted the heavy chain DNA
fragment of the antibody into the pEE12.4 vector and the light chain DNA fragment of the antibody into the
pEE6.4 vector. We transformed the expression plasmid into E. coli competent cells, and then coated them on
LB solid medium plates containing ampicillin and cultured them overnight at 37℃.

As
shown in Figure 2, we verified through DNA gel electrophoresis that we successfully obtained the pEE12.4
vector, pEE6.4 vector, the heavy chain DNA fragment of the antibody, and the light chain DNA fragment of
the antibody. We also successfully obtained a monoclonal clone of the recombinant plasmid.
![](https://static.igem.wiki/teams/4961/wiki/results/results-2.png)

Figure
2 Purity verification and colony inoculation

A:
Purity of the digested vector

B:
Purity of amplified fragments

C:
Overnight growth of Escherichia coli

Target 2 Pick monoclonal colonies, sequence, amplify correct colonies

We
selected two monoclonal clones separatelyinto 15ml LB (Amp+) medium, incubated for 6h, took 500 µl
of the bacterial solution and sent it to the company for sequencing, and then transferred the bacterial
solution with the correct sequencing result into 1L of fresh LB (Amp+) medium and incubated it at 37℃
overnight. The sequencing results were as follows.The
sequencing results in Figure 3 indicate that we successfully obtained two types of recombinant
plasmids.

![](https://static.igem.wiki/teams/4961/wiki/results/results-3a.png)

Figure
3A Heavy chain
sequencing results

![](https://static.igem.wiki/teams/4961/wiki/results/results-3b.png)

Figure
3B Light chain sequencing results

Target 3 Extraction of plasmids

After
constructing the antibody expression plasmid, it needs to be allowed to amplify inside E. coli, and after
the amplified E. coli is lysed, a large amount of antibody expression plasmid will be released, which
provides material for the subsequent transfection experiments. We can get the high purity and low
endotoxin antibody expression plasmid by using the company's HiPure Plasmid EF Maxi Kit (Megan).

Target 4 Transfection of 293F cells, expression and purification of antibody 33D9

At
present, we have obtained a large number of antibody light and heavy chain expression plasmids. The
pEE12.4-33D9H and pEE6.4-33D9L were encapsulated with PEI transfection reagent, and then the complex was
transfected inside a 293F cell line using a transfection method, and the cell supernatant was collected
after 5-7 days. Subsequently, we used protein A affinity purification to obtain the 33D9 antibody from
other proteins in the supernatant of the 293F cells.

![](https://static.igem.wiki/teams/4961/wiki/results/results-4.png)

Figure
4 Plasmid-vector complex formulation and protein expression

The
size of 33D9 antibody is 150kD, of which the size of light chain fragment is 25kD, and the size of heavy
chain fragment is 50kD.The results of polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purity
of the protein that we purified from the supernatant of 293F was high(Figure 5). Therefore, the 33D9
antibody was expressed and purified with high quality, which meets the requirements of our subsequent
experiments.

![](https://static.igem.wiki/teams/4961/wiki/results/results-5.png)

Figure 5 SDS-PAGE gel

![]( https://static.igem.wiki/teams/4961/wiki/results/results-6.png)

Figure
6 Work flow of Purity
of the 33D9 antibody(DetaiBio)

Target 5 Validation of 33D9 binding to 52B8hG1ptx by enzyme-linked immunosorbent assay double antibody
sandwich method

Now
we have the product we want to develop, the 33D9 antibody, which binds specifically to paclitaxel. With
the double antibody sandwich method (Figure 7)we
can detect the concentration of ADC coupled to ptx in different media. Before applying it to a real
scenario, we have to make a standard curve for our product to determine its upper and lower limits of
detection.

![](https://static.igem.wiki/teams/4961/wiki/results/results-7.png)

Figure7 Double
antibody sandwich method

The
results of the double-antibody sandwich method showed that we successfully made a standard curve that
could detect 52B8hG1ptx (Figure 8).
By analogy, any drug coupled to paclitaxel, which itself can be directly coupled to fluorescence or
indirectly coupled to fluorescence, can be detected in different solutions using our antibody. This
provides strong experimental data to support the development of our ELISA kit.

![](https://static.igem.wiki/teams/4961/wiki/results/results-8.png)

Figure
8 ELISA
results：Standard Curve
of 52B8G1PTX

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