TITLE: Cell DIVE™ Platform | Ab Conjugation: Initial Conjugation & Scale up Conjugation
AUTHORS: Anup Sood, Eric Williams, Liz McDonough

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Scope</span></div><div class = "text-block">The purpose of this protocol is to conjugate purified antibodies to Cy dyes. Antibody conjugation involves combining dyes with antibodies to achieve a fluorescently labeled antibody. Each antibody will be labeled with a molar equivalent of dye; the quantitative output of this is deemed D/P. In the initial conjugation step, each antibody will be labeled with differing D/P levels and validated to determine which level is optimal.</div></div>

[STEPS]
?. [ ]
Please select between the following cases:
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Note: In case if your institution does not have NanoDrop, please use a UV spectrophotometer of your choice.For Cy2 - measure absorption at 489 nm. The Cy2 extinction coefficient is 150,000. Divide absorption at 550 nm by 0.15 to get concentration in μM.Molar Concentration (mM) = (Average μM concentration X 250 dilution factor)/1000.For Cy3 - measure absorption at 550 nm. The Cy3 extinction coefficient is 150,000. Divide absorption at 550nm by 0.15 to get concentration in μM.Molar Concentration (mM) = (Average μM concentration X 250 dilution factor)/1000.For Cy5 - measure absorption at 650 nm. The Cy5 extinction coefficient is 250,000. Divide absorption at 650 nm by 0.25 to get concentration in μM.Molar Concentration (mM) = (Average μM concentration X 250 dilution factor)/1000.For Cy7 - measure absorption at 750 nm. The Cy7 extinction coefficient is 200,000. Divide absorption at 650 nm by 0.2 to get concentration in μM.Molar Concentration (mM) = (Average μM concentration X 250 dilution factor)/1000.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
From the home screen select “Proteins and Labels”.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Select IgG under “Type”, as per Figure 1.1.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Select the dye to be measured from the dropdown menu for “Dye1,” as per (1) on Figure 1.2.
NOTE 1: Cy2 is not a standard option in the NanoDrop software. You will need to add Cy2 to the dye list. Consult the instrument software manual for specific instructions.
NOTE2: Cy7 is incompatible with the “Proteins and Labels” Program. Use the “UV-Vis” program to read Cy7 dye and conjugates.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Measure  and place on NanoDrop pedestal to blank the instrument.
[fresh 1X PBS]
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Select “Blank.”
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
After the measurement completes wipe the pedestal with a Kimwipe.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Measure  and place on NanoDrop pedestal to measure the baseline.
[fresh 1X PBS]
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Select “Measure.”
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
After the measurement completes wipe the pedestal with a Kimwipe.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Refer to Figure 1.3: Ensuring a Stable Baseline on the NanoDrop.
A Stable Baseline should read an absorbance between 0.02 and –0.02, with no tilt in the horizontal line.An Unstable Baseline will look tilted or veer outside of the acceptable -0.02 and +0.02 acceptable range.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Measure  (Section 'Materials' for preparation, in μM) 5 times, taking 5 independent readings.
[of the dye concentration measurement solution]
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
The instrument will calculate the dye concentration.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Refer to (2) of Figure 1.4: Setting Dye Parameters on NanoDrop.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Cy7 measurements: The measurements for Cy7 dye and conjugates are slightly different.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
From the home screen select “UV-Vis”.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Change the “Baseline correction” wavelength to a wavelength where the signal is expected to be 0. Refer to Figure 1.4, Step 1.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
To calculate the molar concentration of the Cy dye 1X stock solution:Molar Concentration (mM) = (Average μM concentration X 250 dilution factor)/1000
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Write the calculated concentration on the aluminum storage sleeve along with the date. This Molar Concentration (mM) will be used as the Dye Conc. in Form-1 and Form-7.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Add 280 and 750 nm to the list of wavelengths to be measured. Refer to Figure 1.4, Step 2.
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
In the UV-Vis program, the absorbance is displayed for a 1 mm path length and the dye concentration is not automatically calculated. The dye concentration for Cy7 is calculated as follows: Dye concentration (μM) = Absorbance750nm*50
?. [Preparing calculations for pilot conjugations]
Refer to Table 2.1, for specific parameters for the antibody to be conjugated.  ABCDE1Dye# of D/P Ratios to testTarget Initial D/PTarget Initial EfficiencyAcceptable D/P Range2Cy223.5/5.075%3.0-6.03Cy323.5/5.075%3.0-6.54Cy521.5/3.075%1.5-4.55Cy721.5/3.075%1.5-4.5
ABCDE1Dye# of D/P Ratios to testTarget Initial D/PTarget Initial EfficiencyAcceptable D/P Range2Cy223.5/5.075%3.0-6.03Cy323.5/5.075%3.0-6.54Cy521.5/3.075%1.5-4.55Cy721.5/3.075%1.5-4.5
NOTE: In the event antibody purification yield is low or the vendor supplies insufficient quantity to perform 2 D/P per dye, inform the study coordinator and, if approved, continue conjugating 1 D/P per dye.
?. [Preparing calculations for pilot conjugations]
The pilot conjugations will be used to determine the optimal D/P for the specific antibody tested. Every antibody will have an optimal D/P.
?. [Preparing calculations for pilot conjugations]
Determine the minimum quantity to conjugate. Be sure to save at least  to be validated alongside the direct conjugate.Optimally,  reactions should be performed; however, a minimum of  is required for every conjugation, regardless of study.
[unconjugated primary antibody]
45 µg
30 µg
?. [Preparing calculations for pilot conjugations]
Use Form-7 to calculate the volume of antibody, 20X borate, 1X PBS and dye needed for the pilot reactionInput any cell that is colored blue“Dye purity” is the percent (%) reactive dye information that will be on the Certificate of Analysis and is lot dependent. Enter the information on Form 7 in decimal form (i.e. 90.2% = 0.902). If the information is not available, use the following website to look up the CoA using the lot#: https://www.cytivalifesciences.com/en/us/support/quality/certificates“Loading” is the initial ratio of dye to antibody that is used in the conjugation. The loading is calculated as: Initial D/P divided by expected efficiency (expressed as a decimal). Refer to Table 2.1 for the appropriate values. For example, if targeting a Cy3 labeling at a D/P of 3.5, the “Loading” would be 4.67 (3.5/0.75).Choose a dye dilution that will allow you to add a minimum of 2 μL of solution while not overly diluting the entire sample. As you adjust the “dye dilution” cell on Form-7, the “μL dye” will change accordingly. This volume is the amount of the diluted dye that you need to add. See Section 'Materials' for the preparation of the working dye solutions. If the dilution needed is not described in Section 'Materials', adjust the calculations accordingly. You should prepare at least twice the volume of working solution that will need to be added to the conjugation.If there is enough antibody, repeat the calculations for the second D/P target.Refer to Figure 2.1, for an example of calculating these values.On Form-7, there are two choices of calculations depending on your starting antibody concentration.“Dye purity” is the percent (%) reactive dye information that will be on the Certificate of Analysis and is lot dependent. Enter the information on Form 7 in decimal form (i.e. 90.2% = 0.902). If the information is not available, use the following website to look up the CoA using the lot#: https://www.cytivalifesciences.com/en/us/support/quality/certificates“Loading” is the initial ratio of dye to antibody that is used in the conjugation. The loading is calculated as: Initial D/P divided by expected efficiency (expressed as a decimal). Refer to Table 2.1 for the appropriate values. For example, if targeting a Cy3 labeling at a D/P of 3.5, the “Loading” would be 4.67 (3.5/0.75).Choose a dye dilution that will allow you to add a minimum of 2 μL of solution while not overly diluting the entire sample. As you adjust the “dye dilution” cell on Form-7, the “μL dye” will change accordingly. This volume is the amount of the diluted dye that you need to add. See Section 'Materials' for the preparation of the working dye solutions. If the dilution needed is not described in Section 'Materials', adjust the calculations accordingly. You should prepare at least twice the volume of working solution that will need to be added to the conjugation.If there is enough antibody, repeat the calculations for the second D/P target.Refer to Figure 2.1, for an example of calculating these values.On Form-7, there are two choices of calculations depending on your starting antibody concentration.Choose a dye dilution that will allow you to add a minimum of 2 μL of solution while not overly diluting the entire sample. As you adjust the “dye dilution” cell on Form-7, the “μL dye” will change accordingly. This volume is the amount of the diluted dye that you need to add. See Section 'Materials' for the preparation of the working dye solutions. If the dilution needed is not described in Section 'Materials', adjust the calculations accordingly. You should prepare at least twice the volume of working solution that will need to be added to the conjugation.If there is enough antibody, repeat the calculations for the second D/P target.Refer to Figure 2.1, for an example of calculating these values.On Form-7, there are two choices of calculations depending on your starting antibody concentration.
?. [Antibody Pilot Conjugation]
Remove the aluminum jacket containing the Cy Dye 1X Stock Solution from its storage location.
If the dye is being removed from the refrigerator allow it to thaw at   for approximately   , while remaining in the aluminum jacket.
?. [Antibody Pilot Conjugation]
Combine volumes specified in  for the antibody, fresh 1X PBS and borate into a 1.5 mL eppendorf tube using a pipette.
?. [Antibody Pilot Conjugation]
Vortex the mixture for  at a moderate speed to thoroughly mix.
?. [Antibody Pilot Conjugation]
Spin the mixture in the Fixed Speed Centrifuge for .
Do not allow the mixture to incubate for more than 5 minutes before adding the dye working dilution, as the antibody structure could be compromised.
?. [Antibody Pilot Conjugation]
Make the dye dilution calculated in  carefully by adding the calculated amount of Cy Dye 1X Stock Solution to 1X PBS.
To ensure that unwanted dye has not stuck to the outside of the pipette tip, gently wipe the outside of the pipette tip with a Kimwipe before diluting in 1X PBS. See image below:
?. [Antibody Pilot Conjugation]
Vortex the dye working solution for  at a speed setting of 9.
?. [Antibody Pilot Conjugation]
Spin the solution in the fixed speed centrifuge for .
?. [Antibody Pilot Conjugation]
Add the appropriate amount of working Dye Solution, calculated in , within 1 minute to the antibody/borate mixture to prevent dye hydrolysis.
?. [Antibody Pilot Conjugation]
Place solution in an antibody cardboard box in a drawer containing no light.
?. [Antibody Pilot Conjugation]
Incubate at  for .
0 Room temperature
Use a timer to ensure reaction does not go over 30 minutes.
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Begin this process immediately after conjugate incubation has commenced (step 33).
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Remove two Zeba Spin 0.5 mL columns from their packaging.
If doing multiple conjugations simultaneously, prepare 2 columns per conjugation.
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Remove and dispose of bottom closures and loosen caps.
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Place columns into 1.5 mL eppendorf tubes.
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Remove storage solution by spinning them into a microcentrifuge at .
Centrifuge: 1500 34, 1 min, 23 10
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
The storage solutions for the columns will have passed through and the chromatography resin will now be compacted.
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Discard the caps, storage buffers, and place a vertical mark where resin is slanted upwards.
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Add  to columns.
[fresh 1X PBS solution]
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Place the columns back in the microcentrifuge. Ensure the marked side is facing towards the outside of the centrifuge.
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Centrifuge at .
Centrifuge: 1500 34, 1 min
?. [Preparing Zeba Spin Columns for Direct Conjugate Purification]
Transfer prepared columns to new, clean eppendorf tubes labeled with the antibody name.
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
If needed, add fresh 1X PBS to the conjugation reaction after the 30 minute incubation period is over so that the total volume of the conjugation reaction is >.
70 µl
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
Apply the conjugation reaction solution to the center of one of the Zeba spin columns. Be sure not to disturb the surface of the compacted resin. Refer to Figure 5.1.
The minimum volume that can be loaded into a zeba column is  .
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
After the sample has been put on the column measure  and apply to the top of the column. This is referred to as a stacker and will help in overall antibody yield.
[fresh 1X PBS]
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
Spin in the microcentrifuge at . Be sure the black line on the column is facing outward.
Centrifuge: 1500 34, 2 min, 23 10
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
The purified direct conjugate will flow through the column and collect in the bottom of the 1.5 mL eppendorf tube.
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
Remove the purified DC from the first column tube and place the sample onto the center of the second prepared column, be sure not to apply sample to the side of the compacted resin.
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
Spin in the microcentrifuge at .
Centrifuge: 1500 34, 2 min, 23 10
This is to ensure complete removal of any unbound dye.
?. [Purifying the Direct Conjugate to Remove Unbound Dye via Zeba Spin Columns]
Discard the used Zeba columns and proceed to quantitative assessment.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Open the NanoDrop 2000c software version 7.2.1.1 program located on the desktop of the NanonDrop computer
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
From the home screen select “Proteins and Labels”. If Cy7 was used, follow alternative procedure described earlier ().
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Select IgG under “Type”. (Refer to (1) on Figure 6.1)
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Select dye under Dye 1: Refer to (1) of Figure 1.2 ().
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Measure  and place on NanoDrop pedestal to blank the instrument.
[fresh 1X PBS]
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Select “Blank.”
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
After the measurement completes wipe the pedestal with a Kimwipe.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Measure  and place on NanoDrop pedestal to measure the baseline.
[fresh 1X PBS]
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Select “Measure.”
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
After the measurement completes wipe the pedestal with a Kimwipe.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Refer to Figure 1.3 (): Ensuring a Stable Baseline on the NanoDrop 2000c.If the baseline is poor, repeat
A Stable Baseline should read and Absorbance between 0.02 and -0.02, with no tilt in the horizontal line.An Unstable Baseline will look tilted or veer outside of the acceptable -0.02 and +0.02 acceptable range.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Measure the purified direct conjugate 3 times, verify the A280 and ADye.Refer to Figure 6.1.
(2) is the protein absorbance at 280nm.(3) is the dye absorbance at the dye specified wavelength.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Calculate the direct conjugate D/P and efficiency. Use the appropriate form based on the dye conjugated#Form-2:Cy3 Direct ConjugateForm-3:Cy5 Direct ConjugateForm-5: Cy2 Direct ConjugateForm-6: Cy7 Direct ConjugateRefer to Figure 8.6.2.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Take a volumetric reading of the DC with a p200 pipette.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Input the volume of the DC into “Volume of DC (uL)” on the D/P calculation sheet to determine the yield of the direct conjugate.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
Ideally, the two conjugates should be separated by at least 1.5 D/P. If they are not, a new conjugate should be made utilizing the pilot efficiency to obtain the acceptable D/P levels.
?. [Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c]
The acceptable range of D/P is listed on Table 2.1 () for each dye. If the conjugates fall outside of this range consult the study coordinator to determine if the conjugate should be tested, or a new lot of conjugate should be made.
?. [Stabilizing the Conjugations ]
Refer to the appropriate D/P calculation form depending on what dye was used.
?. [Stabilizing the Conjugations ]
The template will calculate the direct conjugate concentration and amount of BSA/azide stabilizing solution to add.
?. [Stabilizing the Conjugations ]
The Desired Concentration will be calculated on the D/P Calculation Sheet.
Please note that the conjugates should be at a concentration  , adjust with calculated amount of fresh 1X PBS and stabilizing solution to reach appropriate concentration.
?. [Stabilizing the Conjugations ]
Transfer the stabilized DC to an amber tube and store at -.
2 °C
8 °C
?. [Stabilizing the Conjugations ]
Label the direct conjugate tubes with: biomarker name, dye, D/P, concentration, date of conjugation and vial #.
?. [Stabilizing the Conjugations ]
Input the conjugate into your reagent management system, if any.
?. [Stabilizing the Conjugations ]
Complete the Direct Conjugate Certificate of Analysis (see following section).
?. [Complete Direct Conjugate Certificate of Analysis ]
Antibody InformationConjugate InformationControlsBatch Record Verification: Conjugates are between Cy3 3.0 and 6.5 D/P:    ⬜ Yes    ⬜No    (check one)                                                                                            Cy3 1.5 and 4.5 D/P:    ⬜ Yes    ⬜No    (check one)Performance Verification: Conforms?     ⬜ Yes    ⬜No    (check one)We confirm that this product has been verified and conforms to previously outlined QC measures
?. [Determining molar concentration of 1X Cy Dye via NanoDrop 2000c]
Open the NanoDrop 2000c software version 7.2.1.1 program located on the desktop of the NanonDrop computer.
?. [Overview]
Initially perform a pilot conjugation (~-) to determine the optimal D/P of the antibody. Then assume a 100% efficiency for the scale up conjugation, targeting the optimal D/P selected after the initial conjugation. This will avoid the possibility of over labeling the antibody. Please save  for any trouble shooting.
35 µg
60 µg
[purified Ab]
?. [Pre-Experiment Check]
Antibody should be purified and in 1X PBS (w/azide).
?. [Pre-Experiment Check]
If the antibody was ordered as purified, confirm there aren't any incompatible additives by checking the data sheet and the Purification Guide section of the Chemistry SOP.
?. [Pre-Experiment Check]
Take a reading of the antibody using the NanoDrop (refer to section 'Quantitative Assessment of Pilot Direct Conjugate via NanoDrop2000c' of Pilot Conjugation) to check the concentration and ensure it is pure.
If the concentration is much higher than what the vial says, then this indicates the presence of BSA, which will interfere with the conjugation. The antibody will need to be purified if this is the case.If antibody concentration is below  , concentrate to   using Amicon filters. Refer to Section 7.1 ('Evaluate Need for Purification as per Formulation') of the Cell DIVE Antibody Purification manual.
?. [Pre-Experiment Check]
Use purified antibody (35 μg minimum) at a concentration of ≤ .
?. [Pre-Experiment Check]
Be sure to save at least  to be validated alongside the direct conjugate.
[unconjugated primary antibody]
?. [Scale-Up Conjugation Procedure]
Prepare the 1X dye solution and measure the concentration as described previously (Section 'Determining molar concentration of 1X Cy Dye via NanoDrop 2000c' of Pilot Conjugation).
?. [Scale-Up Conjugation Procedure]
Use the optimal D/P obtained after the pilot conjugation as a target. Assume a 100% efficiency upon scaleup. The “Loading” value used in Form-7 will be the optimal D/P. For example, if you determined the optimal D/P was 4.0, than the “Loading” parameter in Form-7 will be 4.0. This will prevent the possibility of overlabeling the antibody.
?. [Scale-Up Conjugation Procedure]
Measure the concentration of the antibody as described in Section 7.8 ('Confirming the complete removal of purified antibody via NanoDrop') in the Purification Manual.
?. [Scale-Up Conjugation Procedure]
Prepare the antibody conjugation solution as described previously (Section 'Preparing calculations for pilot conjugations' of Pilot Conjugation). Use Form-7 to determine the appropriate amounts of all the reagents.
?. [Scale-Up Conjugation Procedure]
Add the appropriate amount of dye while vortexing at low speed.
?. [Scale-Up Conjugation Procedure]
Leave the mixture in complete darkness at  for .
8 Room temperature
?. [Scale-Up Conjugation Procedure]
Prepare Zeba desalting columns (0.5 mL) immediately following the start of incubation. If you are conjugating more than 70 μg of antibody, then one Zeba column will not be enough. You can split the conjugation between two columns (4 columns total per conjugation). If you are conjugating more than 150 μg of antibody, switch to PD-10 columns.
?. [Purification using PD-10 columns]
PD-10 columns are gravity fed and will need a longer preparation time. The standard sample size is  - . If a lower volume is used, the conjugate will be further diluted.
?. [Purification using PD-10 columns]
Remove the top cap and discard the column storage solution.
?. [Purification using PD-10 columns]
Cut the sealed end of the column at the notch.
?. [Purification using PD-10 columns]
Fill the column with 1X PBS and let the buffer enter the packed resin.
?. [Purification using PD-10 columns]
Repeat this filling and elution 4 times, then discard the flow-through.
?. [Purification using PD-10 columns]
Add - to the top of the column. Dilute your conjugate with 1X PBS if volume is less than .
1 mL
[conjugation solution]
1 mL
?. [Purification using PD-10 columns]
Once the conjugate solution has completely entered the column, elute with 1X PBS until the colored conjugate has completely eluted.
NOTE: If the color is hard to see, the eluent fractions can be measured with the NanoDrop to determine if all the conjugate has been eluted.
?. [Scale-up Conjugate Evaluation]
Measure D/P and determine yield and efficiency using procedure described earlier.
?. [Scale-up Conjugate Evaluation]
If the D/P calculated is too low, more dye can be conjugated using the established procedure.
?. [Scale-up Conjugate Evaluation]
Once an acceptable D/P ratio has been achieved, add stabilizing solution, 1X PBS and determine final conjugate antibody concentration.
?. [Scale-up Conjugate Evaluation]
Label and store vials at .
4 °C
?. [Scale-up Conjugate Evaluation]
Complete paperwork.
?. [Scale-up Conjugate Evaluation]
Submit material for revalidation vs original direct conjugate.
?. [Antibody Pilot Conjugation]
Remove the antibody from it's storage location and allow it to equilibrate to  for
0 Room temperature