Protocol for culture, purification, and target validation of a hybridoma-generated monoclonal antibody targeting Aβ truncated species

Summary

Alzheimer’s disease (AD) is characterized by the deposition of full-length and truncated amyloid beta (Aβ) species within brain parenchyma and cerebral vessels. However, Aβ truncated species remain understudied. Here, we present a protocol for culture and characterization of a mouse monoclonal antibody targeting N-terminally truncated proteoforms starting at position 4. We describe a detailed procedure for hybridoma culture, antibody collection, and isolation via affinity chromatography. We then describe steps for target validation via dot blot, as well as potential applications.

For complete details on the use and execution of this protocol, please refer to Cabrera et al. and Rostagno et al.^1,2

Graphical abstract

Highlights

• •Protocol for culture of a hybridoma-generated antibody targeting Aβ 4-x species
• •Detailed procedure for antibody purification via affinity chromatography
• •Instructions for target validation via dot blot
• •Antibody validation using IP/MS and IHC on mouse brain tissue

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Alzheimer’s disease (AD) is characterized by the deposition of full-length and truncated amyloid beta (Aβ) species within brain parenchyma and cerebral vessels. However, Aβ truncated species remain understudied. Here, we present a protocol for culture and characterization of a mouse monoclonal antibody targeting N-terminally truncated proteoforms starting at position 4. We describe a detailed procedure for hybridoma culture, antibody collection, and isolation via affinity chromatography. We then describe steps for target validation via dot blot, as well as potential applications.

Before you begin

This protocol describes the production and purification of a monoclonal antibody targeting N-terminally truncated species starting at position 4 (Aβ 4-x). Validated applications include Immunohistochemistry (IHC), and Immunoprecipitation coupled with Mass Spectrometry (IP/MS). In the case of Immunohistochemistry, cross-reactivity may occur when using the mouse monoclonal antibody on mouse tissue, leading to background staining as the antibody binds to endogenous mouse immunoglobulins in the tissue. The protocol described can be applied to other hybridoma-generated monoclonal antibodies. Before beginning any experiment involving the use of the antibody, at least 6–8 weeks are needed for hybridoma cells to proliferate and to allow significant production of antibody.

Institutional permissions

Animal studies were conducted in accordance with protocols that were reviewed and approved by the NYU Institutional Animal Care and Use Committee (IACUC, IA18-01958). Female APPswe/PS1dE9 (APP/PS1) mice were housed in accordance with the National Institute of Health (NIH) guidelines in the NYU School of Medicine Animal Facilities. Mice were housed under a 12-h light/dark cycle and received regular diet and water ad libitum.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
4G8 antibody (1:3,000)	BioLegend	800703
6E10 antibody (1:3,000)	BioLegend	803017
HRP-conjugate goat anti-mouse antibody (1:30,000)	Invitrogen	G21040
Chemicals, peptides, and recombinant proteins
Hybri-care medium	ATCC	46-X
PFHM-II Protein-Free Hybridoma Medium	Gibco	12040077
Fetal bovine serum	Corning	35011 CV
Dimethyl sulfoxide (cell culture)	MP	194819
Penicillin/Streptomycin solution, 100×	Corning	30002CI
Sodium bicarbonate	Fisher Scientific	S233-500
Sodium hydroxide solution	Fisher Scientific	SS266-1
Sodium chloride	Fisher Scientific	S640-3
Hydrochloric acid	Fisher Scientific	SA48-1
Trypan blue solution	Sigma	T8154-100 mL
Protein G Sepharose 4 Fast flow	GE Healthcare	17061802
Tris base	Fisher Scientific	BP1521
Glycine	Fisher Scientific	BP3811
Tween 20	Sigma-Aldrich	P1379
Synthetic peptides (Aβ 1-34, Aβ 1-40, Aβ 1-42, Aβ 4-34, Aβ 4-40, Aβ 4-42)	ERI Amyloid Laboratory	Custom generated
HistoPrep Xylene	Fisher	HC-700
HistoPrep Alcohol	Fisher	HC-800
Cytoseal 60 Mounting medium	Epredia	8310–4
Dako Dual endogenous enzyme blocking reagent	Dako	S2003
Vecstatin anti-mouse ABC peroxidase kit	Vector Laboratories	PK-4002
3,3-diaminobenzidine (DAB) tablets	VWR	E733-100
Hematoxylin (Mayer’s solution)	Sigma	MHS1
Super Signal West Pico Chemiluminescent substrate	Thermo Scientific	34080
Dynabeads M-280 Sheep anti-mouse	Invitrogen	11202D
Ammonium bicarbonate	Fisher	A643-500
Formic acid 96% (HPLC grade)	Sigma	F0507-500
Water, LC/MS grade	Fisher	W6-1
2-propanol, LC/MS grade	Fisher	A461-500
Peptide Calibration Standard (low mass range 1,000 and 3,500 Da)	Bruker Daltonics	Part No: 8206195
Protein Calibration Standard 1 (low mass range between 4,000 and 20,000 Da)	Bruker Daltonics	Part No: 8206355
α-cyano-4-hydroxycinnamic acid matrix	Agilent Technologies	G2037A
Critical commercial assays
Mouse on Mouse (M.O.M.) Immunodetection kit	Vector Laboratories	BMK-2202
Experimental models: Cell lines
Hybridoma cell line, subclone 18H6C4	GenScript	Custom generated
Experimental models: Organisms/strains
APP/PS1 mice strain (1 year old, female)	Jackson Laboratories	MMRRC_034832-JAX
Other
Peristatic pump	Pharmacia	LKB Pump P-1
Nano Drop 2000 Spectrophotometer	Thermo Fisher Scientific	ND-2000
Filter paper, standard grade	Whatman	1001-150
Vivaspin 20 (10 KDa) ultrafiltration concentrator	GE Healthcare	28932360
Dot blot apparatus	Bio-Rad	1706545
Nitrocellulose membrane (0.45 μm)	Bio-Rad	1620168
ChemiDoc MP Imaging System	Bio-Rad	12003154
ImmEdge PAP pen	Vector Laboratories	H-4000
Epredia Coplin staining jar	Fisher Scientific	19–4
DynaMag-2 Magnet	Invitrogen Dynal	12321D
Multi-Purpose rotator/orbital shaker	Barnstead	2314
Savant SpeedVac concentrator	Thermo Fisher Scientific	SVC-100H
Mini Lab Roller	Labnet	H5500
MTP 384 massive target T plate	Bruker Daltonics	26755
AutoFlex MALDI-TOF mass spectrometer	Bruker Daltonics	-

Materials and equipment

Reagent preparation

• •Hybri-care medium for hybridoma culture (pH 7–7.5).

Reagent	Final concentration	Amount
HCM powder	1 X	12 g
NaHCO3	40 mM	3.41 g
Penicillin/Streptomycin Solution (100×)	1 X	10 mL
Fetal Bovine Serum (FBS)	1 X	100 mL
ddH2O	N/A	890 mL
Total	1 X	1 L

Store at 4°C for up to 3 months

CRITICAL: After mixing HCM powder with NaHCO₃ solution and ddH₂O, adjust pH to 7–7.5 with 1 M Hydrochloric Acid (HCl), sterilize using a rapid filter disposable unit (Thermo Fisher; 0.22-micron) inside a sterile cell culture biosafety cabinet. At this point, add antibiotics and FBS.

HCl is toxic and corrosive: handle using proper Personal Protective Equipment (PPE).

CRITICAL: Prepare HCM in advance. To verify lack of contamination, incubate a few ml of medium in a humidified incubator containing 5% CO₂ and warmed at 37°C for at least 48 h. Discard or re-filter if contamination is present.

• •Protein-Free medium for hybridoma culture (pH 7–7.5).

Reagent	Final concentration	Amount
Protein-Free Hybridoma Medium	1 X	1 L
Penicillin/Streptomycin Solution (100×)	1 X	10 mL
Total	1 X	1.01 L

Store at 4°C for up to 3 months

Note: The PFM used in this protocol is a complete medium that is ready-to-use and only requires the addition of antibiotics.

CRITICAL: Prepare PFM in advance. To verify lack of contamination, incubate a few ml of medium in a humidified incubator containing 5% CO₂ and warmed at 37°C for at least 48 h. Discard if contamination is present.

• •Freezing Medium for hybridoma culture.

Reagent	Final concentration	Amount
Fetal bovine serum (FBS)	90 % v/v	45 mL
Dimethyl sulfoxide (DMSO)	10% v/v	5 mL
Total	1 X	50 mL

Store at 4°C for up to 3 months

• •Elution Buffer (glycine solution) for affinity chromatography (0.1 M, pH 2–2.5).

Reagent	Final concentration	Amount
Glycine	0.1 M	1.2 g
ddH2O	N/A	200 mL
Total	0.1 M	200 mL

Store at 20°C–25°C for up to 3 months

CRITICAL: Adjust pH to 2–2.5 with 1 M Hydrochloric Acid (HCl).

HCl is toxic and corrosive: handle using proper PPE.

• •Tris(hydroxymethyl)aminomethane Base (1 M, pH 11).

Reagent	Final concentration	Amount
TRIS Base	1 M	6.06 g
ddH2O	N/A	50 mL
Total	1 M	50 mL

Store at 20°C–25°C for up to 3 months

CRITICAL: Adjust pH to 11 with 1 M Sodium Hydroxide (NaOH). Store at 20°C–25°C.

NaOH is toxic and corrosive: handle using proper PPE.

• •TRIS-buffered Saline (10×, pH 7–7.5).

Reagent	Final concentration	Amount
TRIS Base	200 mM	24.2 g
Sodium Chloride (NaCl)	1.5 M	88 g
ddH2O	N/A	Up to 1 L
Total	10 X	1 L

Store at 20°C–25°C for up to 3 months

CRITICAL: Adjust pH to 7–7.5 with 1 M Hydrochloric Acid (HCl). Store at 20°C–25°C.

HCl is toxic and corrosive: handle using proper PPE.

• •TBS- 0.1 % Tween 20 (1×).

Reagent	Final concentration	Amount
TBS 10×	1 X	100 mL
Tween-20	0.1 %	1 mL
ddH2O	N/A	899 mL
Total	1 X	1 L

Store at 20°C–25°C for up to 1 month

• •Ammonium bicarbonate (NH₄HCO₃) for IP/MS (50 mM).

Reagent	Final concentration	Amount
NH4HCO3	50 mM	0.197 g
ddH2O, LC/MS Grade	N/A	50 mL
Total	1 X	50 mL

Do not store. Make fresh every time

• •IP/MS buffer.

Reagent	Final concentration	Amount
2-propanol, LC/MS Grade	N/A	40 mL
ddH2O, LC/MS Grade	N/A	40 mL
Formic Acid, HPLC Grade	N/A	10 mL
Total	4:4:1 v/v/v	90 mL

Store at 20°C–25°C for up to 1 month

Step-by-step method details

Hybridoma culture and collection of antibody-containing supernatant

Timing: 6–8 weeks

Here, we describe in detail the procedure for growing hybridoma cells and harvesting the antibody-containing conditioned media.

Note: Hybridomas are hybrid cells derived from immortal myeloma cell lines fused with antibody-producing B lymphocytes isolated from spleen after immunization with a specific antigen. Once the desired antibody-producing hybridoma has been identified, single cells are selected and expanded (cloned) so that each of them produces an identical antibody which will be then referred to as monoclonal. After cloning, selected hybridomas are further characterized to confirm antibody production and target-specificity. Once specificity is verified, hybridomas can be cultured to produce the desired monoclonal antibody in large quantities.³ For this study, the hybridoma cells were generated at GenScript (Piscataway, NJ) based on immunization of mice with a peptide consisting of Aβ residues 4–9 plus a C-terminal cysteine residue (FRHDSGC) used for linking to keyhole limpet haemocyanin (KLH).¹ Among the different hybridoma cell lines generated and subcloned, clone 18H6C4 was selected for the study. The monoclonal antibody produced, labeled 18H6, is an IgG1k molecule. Hybridoma cells are cultured in suspension in 5% CO₂ 37°C humidified incubators. All cell culture-related steps must be performed in a sterile cell culture biosafety cabinet using sterile reagents.

• 1.
  Thawing of the desired hybridoma clone.

  • a.
    Warm 10 mL of HCM in a 25 cm² flask for at least 30 min.
  • b.
    Remove the cryovial containing the frozen hybridoma cells (about 10⁶ cells in 1 mL) from liquid nitrogen storage.
  • c.
    Thaw the cryovial at 37°C in a water bath gently agitating for a few minutes.
    This should take about 2–3 min.
  • d.
    Sterilize the exterior of the cryovial with 70% Ethanol (EtOH) and move it to a sterile cell culture biosafety cabinet.
  • e.
    Place vial content in the 25 cm² flask containing warm medium.
  • f.
    Incubate the flask in upright position in a humidified incubator containing 5% CO₂ and warmed at 37°C. Label this flask as “Maintenance flask”.

    CRITICAL: The use of HCM at this stage is critical to ensure cell growth. The medium is specifically formulated to support hybridoma growth during production, cloning and culture. It derives from DMEM and NCTC 135 with the addition of insulin and HEPES as buffering system.⁴

    It is also essential to culture cells in upright flasks as hybridomas grow in suspension. Cells will deposit at the bottom of the flask as shown in Figure 1A.

    Pause point: Let cells grow in a humidified incubator containing 5% CO₂ and warmed at 37°C. Monitor cell growth in the flask using an inverted bright-field microscope. Cells should be split once they reach a density of approximately 1 x 10⁶ viable cells/mL and viability assessed using Trypan blue staining.

    CRITICAL: Hybridomas are sensitive to overcrowding. Split and feed cultures as soon as supernatant color shifts to orange/yellow to avoid cell death. Hybridomas typically have a doubling time of 24–50 h that varies depending on the specific cell lines and culture conditions. In our case, doubling time was 48 h.

• 2.
  Passaging of maintenance flask.

  • a.
    Transfer half of the content of maintenance flask (5 mL) to a new 25 cm² flask.
  • b.
    Add 5 mL of HCM to maintenance flask as well as to the new flask.
    Label this flask as “New flask” (100% HCM).
  • c.
    Incubate maintenance and new flask in a humidified incubator containing 5% CO₂ and warmed at 37°C.

    Pause point: Let cells grow in the maintenance flask in a humidified incubator containing 5% CO₂ and warmed at 37°C until they reach a density of approximately 1 x 10⁶ viable cells/mL. Cells in the maintenance flask can be maintained for another 5 days with the addition of 10 mL of HCM. Excess cells can be frozen for storage in liquid nitrogen using the freezing solution.

• 3.
  Adapting the new flask of hybridomas to protein-free medium (PFM).

  Note: PFM is a serum free, protein-free medium specifically designed to support growth of hybridoma cells and antibody production, as well as to facilitate purification of the monoclonal antibodies compared to serum-supplemented media.

  CRITICAL: Gradually adapt cells to the new medium by slowly increasing the PFM/HCM ratio at each passage as shown in Figure 1B. It is recommended to keep a backup culture (maintenance flask) in HCM until cells are fully adapted to the new medium. As the purpose of the maintenance flask is to maintain the culture in a stable proliferating state, only HCM must be used for this purpose. Never add PFM to the maintenance flask.

  CRITICAL: Monitor cell growth in the flask using an inverted bright-field microscope. Cells will deposit at the bottom of the flask. Assess the degree of cell density and absence of bacterial and fungal contamination.

  • a.
    Warm HCM and PFM at 37°C in water bath for at least 30 min.
  • b.
    Add 1.25 mL of PFM to the 5 mL of cells cultured in HCM (flask labeled 100% HCM).
    This is now the 75% HCM/25% PFM mixture (see Figure 1B).

    Pause point: Let cells grow in a humidified incubator containing 5% CO₂ and warmed at 37°C until they reach a density of approximately 1 x 10⁶ viable cells/mL.

  • c.
    Add 1.56 mL of PFM to the 6.25 mL to the flask labeled 75% HCM/25% PFM.
    This is now the 50% HCM/50% PFM mixture (see Figure 1B).

    Pause point: Let cells grow in a humidified incubator containing 5% CO₂ and warmed at 37°C until they reach a density of approximately 1 x 10⁶ viable cells/mL.

  • d.
    Add 1.95 mL of PFM to the 7.8 mL to the flask labeled 50% HCM/50% PFM.
    This is now the 25% HCM/75% PFM mixture (see Figure 1B).

    Pause point: Let cells grow in a humidified incubator containing 5% CO₂ and warmed at 37°C until they reach a density of approximately 1 x 10⁶ viable cells/mL.

  • e.
    Replace the 25% HCM/75% PFM conditioned media with 10 mL 100% PFM (see Figure 1B).

    Pause point: Let cells grow in a humidified incubator containing 5% CO₂ and warmed at 37°C until they reach a density of approximately 1 x 10⁶ viable cells/mL.

  • f.
    Transfer cells to a 75 cm² flask and continue expanding the hybridoma culture in PFM until reaching a volume of 100–120 mL of PFM per flask.
    Excess hybridoma cells can be frozen using a freezing solution.
• 4.
  Collection of antibody-containing culture supernatant

  • a.
    Using a 25 mL disposable pipette, collect harvested media (70–90 mL) from each flask expanded in 100% PFM in a new separate container.

    Note: Be sure to leave around 30 mL of supernatant inside the cell culture flask to avoid disturbing cell deposit at the bottom of the flask

  • b.
    Add 15 mL of fresh PFM to each flask for a total of 45 mL and return cells to the incubator.

    Pause point: Let cells grow for 2–3 days in a humidified incubator containing 5% CO₂ and warmed at 37°C until they reach a density of approximately 1 x 10⁶ viable cells/mL.

  • c.
    Add PFM when needed upon cell expansion and continue hybridoma culture as described in 3f. Once culture flasks have reached a volume of 100–120 mL PFM , repeat supernatant collection. Supernatants can be stored at −20°C until the desired volume is reached for antibody purification.

    Note: The number of cultured flasks can be expanded accordingly to avoid storing media prior to antibody purification (we recommend approximately 500 mL of harvested media).

Figure 1.

Hybridoma cell culture

(A) 18H6C4 hybridoma cells were incubated in flasks with vented caps in upright position in a 5% CO₂ 37°C humidified incubator. Arrow indicates cell deposits at the bottom of the flask (right panel).

(B) Passaging protocol. (HCM= Hybri-Care Medium, PFM= protein free medium). Figure created in BioRender (Valle, M. 2024 https://BioRender.com/d68o411).

Antibody isolation via affinity chromatography

Timing: 2 days

In this section, we describe in detail how protein G affinity chromatography is used to isolate the monoclonal antibody from the hybridoma culture supernatants.

Note: Affinity chromatography is commonly used for antibody purification due to its high specificity and selectivity.^5,6 In the case of antibody isolation, affinity chromatography techniques are typically based on the use of matrices targeting either the antigen binding site or the constant region of the immunoglobulin molecule. An advantage of using protein G is its high affinity binding to the constant Fc region of immunoglobulins allowing capture of these molecules without disrupting the Fab region which could result in loss of affinity. Due to the property of Protein G binding to ligands at neutral pH, an acidic glycine hydrochloride solution (pH 2–2.5) was used as elution buffer.

Note: Start antibody collection with at least 500 mL of cell culture supernatant.

• 5.
  Preparation of samples and Protein G Sepharose column

  • a.
    Take a 5 mL aliquot of the supernatant to be analyzed via dot blot before starting the purification to ensure it contains the immunoreactive antibody.
    If previously stored at −20°C, keep hybridoma harvested medium at 4°C for 16–18 h or at 20°C–25°C for a few hours. Start isolation when the medium is completely thawed.
  • b.
    Prepare 1× PBS.
  • c.
    Add 1 mL of Protein G Sepharose to an empty chromatography column.
    A column of 1 cm diameter was used in this protocol.
  • d.
    As Protein G Sepharose is stored in 20% EtOH, wash the column with PBS 1× at 1 mL/min rate. Use 20–30 mL of PBS 1× to ensure complete EtOH removal.
  • e.
    Stop the flow and discard the flow-through.

    CRITICAL: Be sure to leave about 1 mL of PBS 1× above the column surface to avoid drying off the affinity matrix.

  • f.
    Fill the column with harvested media.

    CRITICAL: Before starting the antibody isolation, check that the column is not clogged and supernatant flows easily as shown in Figure 2B. Monitor the process to ensure that the Sepharose matrix does not dry throughout the incubation with the media.

  • g.
    Connect the pump to link the column with the reservoir containing the harvested media to ensure re-circulation as shown in Figure 2A. Start the flow at 1 mL/min rate.

    Pause point: Despite the high binding capacity of the Protein G matrix (∼20 mg of human IgG per milliliter of resin), passing the entire volume of cell supernatant through the column at the recommended 1 mL/min rate for sample application will take considerable time. If 500 mL of harvested media are used for antibody purification, as in this example, more than 8 h will be required. As a practical tip, it is suggested to recirculate harvested media for 16 h at 4°C , as shown in Figure 2A.

  • h.
    The next morning, move column and the connected pump at 20°C–25°C . Increase the flow rate to 2 mL/min and let the supernatant pass through the column until approximately 1 mL is left above the resin surface.

    CRITICAL: Before starting the antibody isolation, check that the column is not clogged and supernatant flows easily as shown in Figure 2B. Monitor the process to ensure that the Sepharose matrix does not dry throughout the incubation with the media.

• 6.
  Isolation of antibody from cell culture harvested media

  • a.
    Wash the column with 100 mL 1× PBS at 1–2 mL/min rate.

    CRITICAL: Do not continue until the column has been cleared of the culture media as media residuals may result in antibody impurities.

    CRITICAL: Do not discard the unbound portion of harvested media at this stage. Store it at 4°C for further testing of residual antibody.

  • b.
    In the meantime, prepare 1.5 ml Eppendorf tubes and label them in numerical order.
    Add 20 μL TRIS Base 1 M to each tube to be used for collection of eluted fractions to assure immediate neutralization after low pH elution.
  • c.
    Let the last 1 mL PBS flow through the column and then stop the flow.
  • d.
    Gently add 6 mL of Glycine hydrochloride 0.1 M to the affinity matrix and set the flow rate to 1 mL/min.
  • e.
    Start the pump and collect 0.5 mL fractions.
  • f.
    Stop the pump after all the desired fractions are collected.

    Note: In our case, typically 12 fractions of 0.5 mL are enough to collect the antibody from the harvested media. Absorbance values should peak around fractions 4–8 and slowly decrease.

    CRITICAL: It is essential to add the TRIS Base solution to neutralize eluted bound fractions from the acidic pH of glycine and avoid antibody denaturation.

• 7.
  Concentration of antibody fractions

  • a.
    Set spectrophotometer absorbance at 280 nm using glycine solution as blank. Measure absorbance of 1.5 μL aliquots from each fraction.

    CRITICAL: Wash column with 1× PBS as soon as all antibody has been eluted. Do not leave Protein G Sepharose in glycine hydrochloride buffer for more than 10 min since this will cause denaturation of the affinity matrix. Store Protein G Sepharose in 20% EtOH at 4°C.

    CRITICAL: Before continuing, block an ultrafiltration concentrator device (10 kDa Molecular Weight Cut Off, MWCO) by incubation with 0.1% BSA for 30 min. Then, spin at 500 g for 30 min. Add PBS 1× to the BSA-blocked filter and store at 4°C.

  • b.
    Combine the antibody-containing fractions with absorbance higher than 0.5 and add them to a BSA-blocked ultrafiltration concentrator.
  • c.
    Centrifuge at 500 g until the volume containing the purified antibody is about 1 mL.
  • d.
    Add 1 mL of PBS 1× and repeat the centrifugation procedure for buffer exchange at least 3 times.

    Note: Confirm the buffer exchange to PBS by assuring the pH of the purified antibody is neutral (6.5–7).

  • e.
    Measure absorbance at 280 nm using PBS 1× as blank to assess antibody concentration using human IgG extinction coefficient of 1.36 (molar extinction coefficient of 210,000 M⁻¹ cm⁻¹). This fraction contains the purified antibody. Store at −20°C.

Figure 2.

Monoclonal antibody isolation using affinity chromatography

Hybridoma cell culture harvested media was recirculated through a Protein G Sepharose column for 16 h at 4°C to ensure complete capture of the antibody by the affinity matrix (A). Check column flow before starting antibody isolation procedures ensuring column filter is not clogged and column is properly packed (arrow, B).

Target validation via dot blot

Timing: 2 days

Before any biological application, it is critical to validate the antibody specificity for its target protein. Here, we describe a detailed protocol for dot blot, a rapid method for qualitative analysis to assess whether a protein of interest is present in a sample. See Figures 3A and 3B for details on dot blot apparatus.

Note: Alternatively, specificity may be determined by western blot.

• 8.Preparation of target protein

Figure 3.

Target validation via dot blot

Dot blot apparatus components (A) and order of assembly (B). Dot blot assay illustrates a positive signal for 18H6 hybridoma cell culture harvested media (C, left image) and no signal for media from hybridoma cells no longer producing and secreting the monoclonal antibody (C, right image). Dot blot highlighting immunoreactivity of Aβ peptides with a mixture of 4G8 and 6E10 antibodies recognizing Αβ species starting at position 1 as well as those beginning at residue 4 (Aβ1-40 and Aβ4-40, respectively) (D, left panel). Peptide immunoreactivity with 18H6 antibody that only recognizes Αβ species starting at position 4 (Aβ4-40) and is blind for full length Aβ species (D, right panel).

For this protocol, synthetic homologues of Aβ 1–40 and Aβ 4–40 were prepared and used as target proteins as previously described.¹ Each peptide was solubilized in 5 μL of DMSO and diluted in ddH₂O to 500 μg/mL. The protein load for each peptide was 500 ng per well in a dot blot apparatus.

• 9.Dot blot Analysis (Day 1).
  • a.
    Wet nitrocellulose membrane by incubating for 10 min with 15 mL TBS 1× on an orbital shaker at low speed (20 rpm).
  • b.
    Assemble apparatus including the membrane as shown in Figure 3A.
  • c.
    Add 100 μl per well of the respective target proteins, containing 500 ng each, according to the desired layout.
  • d.
    Fill the remaining wells with comparable volume of 1 X TBS and incubate for 30 min.
  • e.
    Connect apparatus to a vacuum source and aspirate wells content.
  • f.
    Block all wells with 200 μl of filtered 1 % milk in TBS-T and then incubate for 1 h.
  • g.
    Repeat step 2e.
  • h.
    Block all wells with 200 μl of filtered 5 % milk in TBS-T and then incubate for 1 h.
  • i.
    Repeat step 2e.
  • j.
    Disassemble the dot-blot apparatus and take out the membrane.
    Wash it with 15 mL TBS-T (3 times for 3 min) until all the milk has been removed.
  • k.
    Cut membrane according to the desired layout.
  • l.
    Incubate membrane for 16–18 h with pertinent antibodies at 4°C on an orbital shaker at low speed (20 rpm). The antibodies used in this protocol are the hybridoma-derived antibody 18H6 (10 mL, 1:1,000 in TBS-T) and a mixture of Aβ-targeting antibodies 4G8 and 6E10 (10 mL, 1:3,000 in TBS-T).

    CRITICAL: It is crucial to block membrane in situ prior to removal from the dot bot apparatus to avoid background signal caused by peptide diffusion and/or run off. Always add loading controls for each experiment. For this protocol, a mixture of Aβ-targeting antibodies 4G8 (recognizing residues 17–24 of Αβ) and 6E10 (recognizing residues 1–16 of Αβ) was used (1:3,000 each in TBS-T). As shown in Figure 3D, 4G8+6E10 mix recognizes Aβ 4–40 as well as full-length peptide (1–40) while the hybridoma-derived antibody 18H6 only recognizes Aβ 4–40.

    Note: If testing culture supernatants instead of antibodies, incubate the membrane directly with the undiluted harvested media (Figure 3C). If testing the purified 18H6 antibody, optimize final dilution to assure optimal signal with Aβ 4-x peptides and no background (1:1000 dilution was used for this protocol). If testing multiple samples with different antibodies, cut the membrane into smaller parts and incubate separately.

• 10.Dot blot Analysis (Day 2).
  • a.
    Wash the membranes 3 times with 15 mL TBS-T for 5 min each with constant shaking on an orbital shaker (40 rpm).
  • b.
    Incubate with 10 mL HRP-conjugated secondary antibody anti-Mouse diluted with TBS-T (1:30,000) for 1 h at RT with on an orbital shaker at low speed (20 rpm).
  • c.
    Discard secondary antibody and wash membranes 3 times with 15 mL TBS-T for 5 min each.
  • d.
    Analyze membrane using an HRP chemiluminescent detector system and a ChemiDoc MP Imaging System. Optimal exposure time in this protocol is 30–60 s.

    Note: Final primary and secondary antibody concentrations as well as membrane exposure time need to be optimized for each system. Antibody aliquots that tested positive for target validation can be pooled together and further concentrated with an ultrafiltration concentrator, if needed.

Tested applications, immunohistochemistry

Timing: 2–3 days

Purified antibodies that have been successfully target-validated can be used in a variety of applications. Here, we describe steps for immunohistochemistry (IHC).

Note: For this application, purified 18H6 antibody was evaluated on brain tissue samples obtained from APP/PS1 mice (1 year old, females). Modifications from the original protocols^1,2 include the use of Mouse on Mouse (M.O.M.) kit (Vector Laboratories, Burlingame, CA) in accordance with the manufacturer’s instructions (Mouse On Mouse (M.O.M.) Detection Kit, Basic see User guide) to decrease background staining resulting from the use of mouse monoclonal antibody on mouse tissue specimens.

• 11.
  Sample preparation.

  • a.
    Prepare 7 μm sequential paraffin embedded sections.
  • b.
    Perform deparaffinization in 100% xylene and rehydration in decreasing grades of ethanol (100%, 95%, and 70%) in separate jars (50 mL each). Then, rinse abundantly in water.
• 12.
  Antigen retrieval and blocking.

  • a.
    Pre-treat slides by incubation with 50 mL formic acid 96% in a new jar for 10 min.
  • b.
    Circle tissue section using a PAP pen.

    Note: A PAP pen provides a hydrophobic barrier that keeps reagents localized on tissue specimens, preventing reagent mixing.

  • c.
    Incubate tissue section with 250 μL of endogenous enzyme blocking reagent for 10 min.
  • d.
    Incubate 1 h in 250 μL M.O.M. blocking reagent followed by 5 min in 250 μL M.O.M. diluent.
• 13.
  Primary antibody incubation.

  • a.
    Incubate with desired antibody in M.O.M. diluent.

Note: For this experiment, sequential slides were incubated with purified 18H6 antibody recognizing Αβ 4-x species (1:100 in 250 μL M.O.M. diluent, 16–18 h) or a combination of 4G8+6E10 monoclonal antibodies recognizing all Αβ species (1:1,000 each in 250 μL M.O.M. diluent, 30 min). Dilution and incubation time of antibodies must be optimized for each system.

• 14.
  Secondary antibody incubation.

  • a.
    Wash with 50 mL PBS-T 1X in a new jar (3 times for 5 min each).
  • b.
    Incubate sections for 10 min with 250 μL biotinylated secondary antibody diluted in M.O.M.

Note: For this experiment, sequential slides were incubated in M.O.M. biotinylated anti-Mouse IgG reagent (1:250 in 250 μL M.O.M. diluent, 10 min). Dilution and incubation time of antibodies must be optimized for each system.

• 15.
  Signal amplification.

  • a.
    Repeat washes as above (14 a) and incubate with 250 μL Vecstatin ABC reagent for 30 min for signal amplification.

    Pause point: At this point, prepare 3,3-diaminobenzidine (DAB) chromogen by dissolving 1 tablet in 10 mL PBS 1X. Vortex until the tablet has completely dissolved and keep protected from light. Aliquot and store unused solution in dark at −20°C.

  • b.
    Repeat washes and incubate with 250 μL of the solution of DAB chromogen.
  • c.
    Rinse thoroughly with water.
  • d.
    Counterstain tissue section in 250 μL hematoxylin for 12 min.

    Note: For this experiment slides were incubated with DAB for 1 min for 4G8+6E10 staining and 5 min in the case of 18H6.

• 16.
  Dehydration and mounting.

  • a.
    Dehydrate sections in increasing grades of ethanol (70%, 95%, and 100%, 5 min each) followed by incubation in 100% xylene in separate jars (50 mL each).
  • b.
    Mount slides using low viscosity mounting medium (10–20 μL per section) avoiding formation of bubbles.

Note: For this experiment, sequential slides were scanned using a Leica AT2 bright-field whole-slide scanner at the NYU Langone Experimental Pathology Research core. Results for this application are shown in Figure 4.

Figure 4.

Tested application: Immunohistochemistry

Sequential sections from hippocampus (A and B) and cortex (C and D) of an APP/PS1 Tg mouse (female, 1 year old) stained with either a combination of 4G8+6E10 antibodies (A and C) or purified 18H6 monoclonal (B and D). Bars represent 200 μm (top panels), 100 μm (middle panels) and 50 μm (bottom panels). Note that 18H6 immunoreacts with only a subset of plaques with a tendency to highlight the central amyloid cores, as reported.¹

Tested applications, immunoprecipitation coupled with MALDI-TOF mass spectrometry

Timing: 2–3 days

Purified antibodies that have been successfully target-validated can be used in a variety of applications. Here we describe the steps for Immunoprecipitation coupled with Mass spectrometry (IP/MS) analysis.

Note: Modifications from the original protocol¹ include the use of a mixture of synthetic Αβ peptides for specificity validation as well as of a mixture of isopropanol, water, formic acid (4:4:1) solution (IP/MS Elution Buffer) for elution of the immunoprecipitated material. The Aβ peptides are soluble in most salt-free acidic solutions containing water, either methanol or isopropanol, and either formic acid or trifluoroacetic acid. All reagents should be of the highest purity available to minimize MALDI-TOF background. See Figure 5A for complete workflow.

• 17.
  Washing beads.

  • a.
    Resuspend the anti-mouse Dynabeads in the original vial by gently end-over-end mixing.
  • b.
    Transfer 50 μL of Dynabeads suspension to a 1.5 mL Eppendorf tube.
  • c.
    Add 0.5 mL of PBS 1X and mix by manual end-over-end rotation.
  • d.
    Place the tube in the magnet for 1 min.

    CRITICAL: Do not vortex the beads as they will be damaged by the procedure.

    CRITICAL: Use specially designed racks equipped with potent magnets that allow the complete retrieval of the beads in 0.5 μL–2 mL Eppendorf tubes and avoid losses in the subsequent steps of the IP procedure.

  • e.
    Remove supernatant.
  • f.
    Remove the tube from the magnet and repeat steps 2c–2e three times.
• 18.
  Coating beads with anti-Aβ antibodies.

  • a.
    Add 1 mL of the desired antibody dilution to the 50 μL of washed beads. Total volume of the diluted antibody should be at least 500 μL to facilitate the mixing in step 3b.
  • b.
    Incubate for 2–3 h at 20°C–25°C with bi-directional mixing.
  • c.
    Collect the beads by placing the tube on the magnet for 1 min.
  • d.
    Remove the supernatant.

Note: For the experiments illustrated herein, purified 18H6 antibody recognizing Αβ 4-x species (12 μL, 1.0 mg/mL), or a combination of 4G8+6E10 monoclonal antibodies recognizing multiple Αβ species (3 μL each, 1.0 mg/mL) were attached to the 50 μL of washed sheep anti-mouse coated Dynabeads and used for IP-MS. These proportions are enough to complete for one immunoprecipitation but, if more samples are to be processed, the protocol should be scaled up accordingly.

• 19.
  Blocking and washing antibody-coated paramagnetic beads.

  • a.
    Remove the tube from the magnet and resuspend the beads in 500 μL 0.1% BSA in PBS 1X to block any remaining binding sites on the Dynabeads.
  • b.
    Incubate for 30 min at 20°C–25°C.
  • c.
    Wash the beads 3 times with 500 μL 0.1% BSA in PBS 1X.
  • d.
    Collect the beads by placing the tube on a magnet for 1 min.
  • e.
    Remove the supernatant.
• 20.
  Immunoprecipitation.

  • a.
    Add the desired sample to be analyzed (peptide mixture in this example) to the antibody-coated beads. Total volume should be at least 500 μl to facilitate the mixing in step 5c.

    Note: For the preparation of the peptide mixture, weigh the different Αβ homologues (Aβ 1-34, Aβ 1-40, Aβ 1-42, Aβ 4-34, Aβ 4-40 and Aβ 4-42 in this example), and dissolve them individually using the IP/MS Buffer to a concentration of 40 μM. Combine equal volumes (4 μL) of each of the stock peptide solutions, creating a working mixture containing all species at a final concentration of 6.6 μM each. Neutralize the peptide mixture by diluting it 1000-times with 50 mM ammonium bicarbonate pH 8.3 (take 1 μL of working mixture and dilute it with 999 μL ammonium bicarbonate) prior to IP.

  • b.
    Resuspend the beads pellet by gentle end-over-end mixing.
  • c.
    Incubate the beads with 1 mL of neutralized peptide mixture using bi-directional mixing for 16–18 h at 4°C.

    CRITICAL: It is essential to check that the pH of all peptide samples prior to incubation with antibody-coated beads ranges from 6.5 to 8.5 to ensure proper antigen-antibody interaction.

  • d.
    Remove the tube from the bi-directional mixer and place it in a magnet for 1 min. Then, collect the supernatant in a separate tube without disturbing the beads pellet.
  • e.
    Wash the beads 2 times with 500 μL PBS 1X each, followed by one wash with the same volume of 50 mM ammonium bicarbonate.

    Note: For this experiment, 1 mL of neutralized peptide mixture containing 6.6 pmol of each peptide was used in step 5a.

• 21.
  Elution.

  • a.
    Place the tube on a magnet for 1 min to pellet beads and remove the supernatant.
  • b.
    Add 50 μL of the IP/MS buffer.
  • c.
    Place on bi-directional mixer for 10–15 min.
  • d.
    Place tube on a magnet to retrieve the eluant and transfer it to a separate 0.5 mL tube.
  • e.
    Place tube containing the eluent in a Speedvac.
  • f.
    Follow Speedvac operational instructions to dry the eluent.

Pause point: Eluent consisting of 50 μL volume should dry in about 3 h. Avoid over drying as it will enhance the peptides’ aggregation properties compromising their solubility.

• 22.
  Preparation for MALDI-TOF mass spectrometry analysis.

  • a.
    Resuspend dried sample in the IP/MS elution buffer so that the final concentration is twice the expected concentration to be observed by MALDI-TOF MS.

    Note: For this specific example, samples were redissolved in 3 μL of IP/MS elution buffer for a concentration of 2.2 μM per peptide.

  • b.
    Mix 1 μL of the redissolved sample with 1 μL of Agilent ACCA Matrix.
  • c.
    Apply 1 μL of the mixture sample-matrix to be analyzed in single spot of a MALDI target stainless steel plate.
  • d.
    Wait a few minutes until the solution has crystalized/dried.

    Note: For this experiment, each spot acquired in the MALDI represents 1.1 pmol/peptide.

  • e.
    Perform external calibration using Peptide Calibration Standard (low mass range 1,000 and 3,500 Da ) and Protein Calibration Standard 1 (Low mass range between 4,000 and 20,000 Da).

    Note: Before each data acquisition, the instrument is further fine calibrated and tuned, using the synthetic products of Amyloid beta full length and truncated homologs of (Aβ 1-42) and (Aβ 1-40), as described.⁷

  • f.
    Load spotted plate into the MALDI-MS instrument and proceed with data acquisition.

    Note: While the mixture of 4G8+6E10 monoclonal antibodies will recognize multiple Αβ species (all the full length and truncated species used in this experiment), IP with the purified 18H6 antibody, due to its specificity for Αβ4-x species, will capture only those peptides truncated at position 4. Thus, the tandem IP depicted in Figures 5C and 5D to illustrate antibody specificity uses both beads coated with 18H6 monoclonal antibody (12 μL, 1.0 mg/mL) as well as beads coated with a combination of 4G8+6E10 monoclonal antibodies (3 μL each, 1.0 mg/mL). Figure 5B shows the theoretical and observed mass of the various peptides used in this experiment

    Note: The stainless-steel target plate is reusable. To re-condition the plate, remove any residual material by washing sequentially with 80% glacial acetic acid followed by MS grade H₂O, MS grade methanol, and a final wash with MS grade acetonitrile. This procedure will prevent diffusion of sample droplets across the MALDI target plate prior to crystallization.

Figure 5.

Tested application: Tandem immunoprecipitation coupled with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis

(A) IP/MS workflow. The Aβ peptide mixture was incubated with 18H6-coated paramagnetic beads and the unbound material re-immunoprecipitated with 4G8+6E10-coated beads. As a control, the peptide mixture was immunoprecipitated with 4G8+6E10-coupled beads and the unbound material re-immunoprecipitated with the same 4G8+6E10-coated beads to re-capture any potentially unbound Aβ peptides.

(B) Theoretical and experimental masses for Αβ species employed in the IP/MS experiments.

(C) MALDI-TOF MS profile of peptides immunoprecipitated with purified 18H6 antibody that recognizes only N-terminally truncated species starting at position 4 (Aβ 4-34, Aβ 4-40, Aβ 4-42; left panel), and uncaptured material re-immunoprecipitation with 4G8+6E10-coated beads, illustrating the recovery of Aβ species with intact N-terminus (Aβ 1-34, Aβ 1-40 and Aβ 1-42; right panel).

(D) MALDI-TOF MS spectra of peptides immunoprecipitated with 4G8+6E10-coated beads illustrating recovery of all peptides in the mixture (left panel) and absence of signal upon re-immunoprecipitation of uncaptured material with the same 4G8+6E10-coated beads (right panel). Created in BioRender. Valle, M. (2024) https://BioRender.com/v23z063.

Expected outcomes

Upon effective execution of this protocol, the user will be able to successfully culture hybridoma cell lines and isolate a monoclonal antibody specific for its target protein. After 6–8 weeks from the start of the hybridomas culture – or 6–10 weeks depending on cells proliferation rate – sufficient cell culture harvested media should have been collected to start antibody isolation.

The total yield of purified antibody from a 0.5–1 L batch of harvested media is expected to range between 2–4 mg although these values may substantially vary. Among the factors affecting antibody production and secretion, the type of hybridoma and the cell subculture number are critical contributors. Genetic instability of the different hybridomas together with specific growth and metabolic characteristics of the cultures are known to affect the robustness of antibody production.⁸

Figure 3C illustrates the outcome of the antibody purification from 0.5 L of supernatant. Antibody specificity for Aβ4-x species is verified via dot blot (Figure 3D) and further validated by IHC, and IP/MS (Figures 4 and 5). While the combination of 4G8+6E10 antibodies immunoreacted with multiple Aβ species and can be considered representative of the total Aβ content, 18H6 immunoreactivity representing Aβ 4-x species concentrated primarily to the cores of the amyloid plaques (Figure 4), as previously reported.¹

Note: Specificity of the purified antibody can be tested by tandem IP/MS. As illustrated in Figure 5, if beads coated with 18H6 antibody are used in the first step of the procedure, only Aβ fragments truncated at position 4 are retrieved, whereas the rest of the peptides present in the mixture are recovered when 4G8 + 6E10 coated-beads beads are used (Figure 5C). On the contrary, if beads coated with 4G8+6E10 antibodies are used in the first step, no peptides should remain in the supernatant under the conditions described, as illustrated by the blank MS profile after a second IP using with beads coated with the same pan-Aβ antibody combination (Figure 5D).

Limitations

The methodology described for hybridoma-derived antibody culture, collection, and purification has certain limitations. The procedure is time-consuming and requires close follow-up of the cell culture. Time required to produce a significant amount of antibody may vary depending on the hybridoma type and growth characteristics thus, we recommend that before proceeding with antibody purification the conditioned media is tested via dot blot analysis to verify the antibody presence. This is particular important because after several subcultures, the cells may reduce the antibody production in the media and their presence cannot be assessed by simple visual observation as there is no color or pH change that could be used as an indicator. Thus, bi-weekly validation of antibody content in the harvested media via dot blot is highly recommended.

Additional characterization of the antibody can contribute to the in-depth characterization of the purified antibody. This may include assessment of impurities or antibody fragmentation via SDS-PAGE, Western blot analysis, and size exclusion chromatography. If required by the application, evaluation of the antibody binding affinity via assessment of dissociation constant (Kd) using the Biacore SPR system could also be of interest.

The use of the antibody in in vivo experiments including passive immunization protocols requires sterilization which can be achieved by filtration through 0.22 μm disposable units (Thermo Fisher; 0.22-micron). If needed, before any in vivo applications, endotoxins could be removed with Endotoxin Removal Resin Spin Columns (#88276, Thermo Scientific) followed by evaluation of residual levels using Endotoxin Quantification assays (#A39552S, Thermo Scientific).

Troubleshooting

Problem 1

Hybridoma cultures do not expand sufficiently or exhibit frequent contamination (related to steps 2 and 3).

Potential solution

• •Increase cell density in the starting culture by combining additional vials from hybridoma frozen stocks and/or decreasing the initial volume of HCM.
• •Test different batches of HCM or PFM media.
• •Double antibiotic concentration within each medium.
• •Improve sterility conditions and procedures.

Problem 2

Hybridoma cultures stopped producing and secreting antibody (related to steps 2 and 3).

Potential solution

• •Re-start hybridoma culture from new frozen stocks.

Problem 3

Low antibody yield after purification (related to step 4).

Potential solution

• •Repeat purification using the unbound fraction of the Protein-G Sepharose chromatography procedure to collect residual antibody in the harvested media. One batch of harvested media can be re-purified until no more than 0.1 mg antibody is retrieved from the column.
• •Prepare a new Protein G-Sepharose column.
• •Verify that the pH of the glycine hydrochloride elution buffer is within the 2–2.5 range.

A more basic pH may compromise antibody elution from the affinity matrix.

• •Prepare a new ultrafiltration concentrator to ensure the device is not clogged and that the device is properly blocked to prevent non-specific binding.

It is advisable to change the concentrator at least every 10 uses.

• •Consider using an ultrafiltration concentrator with higher MWCO (e.g. 20–30 kDa).
• •Thaw a new hybridoma cryovial and re-start cultures if the hybridomas have been sub-cultured for more than 3 months.

Problem 4

High background on IHC sections (related to steps 13–15).

Potential solution

• •Optimize incubation times with primary and/or secondary antibody dilution in M.O.M.
• •Decrease incubation time with DAB chromogen.
• •Increase length and frequency of inter-step washes.

Problem 5

Low or no peptide signal present in MALDI-TOF MS spectra (related to step 22).

Potential solution

• •Check the pH of all samples prior to IP to ensure proper antigen-antibody interaction (optimal pH range: between 6.5–8.5).
• •The use of certain solutions can introduce non-volatile salts into samples during the elution/wash steps which may interfere with subsequent MALDI-TOF analysis. Prepare new fresh solutions using MS quality reagents to minimize background or wash the dried sample spots several times by adding and removing 1 μL 0.1% trifluoroacetic acid by pipette.
• •Other matrices besides the α-cyano-4-hydroxycinnamic acid are commercially available and may be more suitable for your specific experiment.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Jorge Ghiso (Jorge.Ghiso@nyulangone.org).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Maria Luisa Valle (maria.valle@nyulangone.org).

Materials availability

Reagents generated within this study are available upon reasonable request.

Data and code availability

All data associated with this protocol can be obtained from the lead contact, Jorge Ghiso, upon request. This protocol does not involve the creation or use of any original code.

Acknowledgments

The work was supported in part by the NIH grants AG059695 and 5U24CA126485.

Author contributions

M.L.V. carried out hybridoma culture, antibody isolation, target validation, IHC applications, and manuscript writing. B.G. performed hybridoma culture, antibody isolation, and target validation. J.L. performed IP/MS experiments. C.W. participated in scientific discussions, evaluation of IHC data, and manuscript editing/review. T.A.N. and H.E.-B. oversaw MS experiments, participated in scientific discussions, and edited the article. J.G. and A.R. conceptualized the project (devised the project and the main conceptual ideas), supervised scientific work and data interpretation, and participated in the writing, editing, and review of the manuscript.

Declaration of interests

The authors declare no competing interests.