Detection of HuCAL derived antibodies

Since HuCAL derived antibodies are human Fab molecules, they can be detected by antisera developed against human IgG F(ab’)2 preparations. In addition, HuCAL antibodies are usually equipped with one or two tags (genetically fused to the C-terminus of the antibody heavy chain), and can be detected by using commercially available anti-tag antibodies. We recommend the use of the following detection reagents:

Detection of Fab scaffold
Detection of C-terminal His₆-tag (HHHHHH)
Detection of C-terminal Myc-tag (EQKLISEEDL)
Detection of C-terminal FLAG M2-tag (DYKDDDDK)
Detection of C-terminal StrepII-tag (NWSHPQFEK)

Assay	1. FAB	2. His₆	3. Myc	4. FLAG	5. Strep II
Indirect ELISA
Western Blot
IHC
FACS
Immuno- precipitation

Please click on the symbol to display the protocol!

Indirect ELISA

The detection of antigen immobilized on microtiter plates is the standard procedure used by AbyD for quality control of purified antibodies. The antigen is incubated in the well at a concentration of 5 μg/ml, and the purified HuCAL antibody (final concentration of 1 μg/ml) is used as primary antibody. Typically, Maxisorp ELISA plates from Nunc are used.

Indirect ELISA – Detection of Fab

Coating: Antigen 5μg/ml, o/n @ 4°C
Wash: 2 x PBST
(PBST buffer with 0.05 % Tween 20)
Blocking: 5 % MP-PBST
(PBST containing 5 % milk powder)
Wash: 2 x PBST
Primary AB: HuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec Buffer
Wash: 5 x PBST
Secondary AB: α-Fab-AP conjugate¹ (1:5 000 in HiSpec Buffer), 1h @ RT
Wash: 5 x PBST
Detection: AttoPhos² (1:10 in H₂O)

Indirect ELISA - Detection of c-terminal His₆-tag

Coating: Antigen 5 μg/ml, o/n @ 4°C
Wash: 2 x PBST
(PBST buffer with 0.05 % Tween 20)
Blocking: 5 % MP-PBST
(PBST containing 5 % milk powder)
Wash: 2 x PBST
Primary AB: HuCAL antibody (2 μg/ml) 1h @ RT in PBST or HiSpec Buffer
Wash: 5 x PBST
Secondary AB: α-His₆-HRP ³
(1:400 in HiSpec Buffer), 1h @ RT
Wash: 5 x PBST
Detection: QuantaBlu Peroxidase Substrate, soluble⁴

Indirect ELISA - Detection of c-terminal myc-tag

Coating: Antigen 5 μg/ml, o/n @ 4° C
Wash: 2 x PBST (PBS buffer with 0.05 % Tween 20)
Blocking: 5 % MP-PBST (PBST containing 5 % milk powder)
Wash: 2 x PBST
Primary AB: HuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec Buffer
Wash: 5 x PBST
Secondary AB: α-c-myc-HRP⁵
(1:100 - 1:500 in HiSpec Buffer), 1h @ RT
Wash: 5 x PBST

Detection

: QuantaBlu Peroxidase Substrate²

Indirect ELISA – Detection of c-terminal FLAG M2-tag

Coating: Antigen 5 μg/ml, o/n @ 4°C
Wash: 2 x PBST
(PBS buffer with 0.05% Tween 20)
Blocking: 5% MP-PBST
(PBST containing 5% milk powder)
Wash: 2 x PBST
Primary AB: HuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec Buffer
Wash: 5 x PBST
Secondary AB: α-FLAG M2-HRP⁷
(1:100 - 1:1000 in HiSpec Buffer), 1h @ RT
Wash: 5 x PBST
Detection: QuantaBlu Peroxidase Substrate⁴ (1:10 in H₂O)

Indirect ELISA – Detection of c-terminal Strep-tagII

Coating: Antigen 5 μg/ml, o/n @ 4°C
Wash: 2 x PBST
(PBS buffer with 0.05% Tween 20)
Blocking: 5% BSA-PBST
Wash: 2 x PBST
Primary AB: HuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec Buffer
Wash: 5 x PBST
Secondary AB: Strep-Tactin HRP conjugate⁸
(1:4000 in 0.5% BSA-PBST), 1h @ RT
Wash: 5 x PBST
Detection: QuantaBlu Peroxidase Substrate⁴

Western Blot

The amount of antigen material loaded on the gel depends on the sensitivity of the detection system. For initial testing, use 200 μg of cell lysate proteins or 300ng of pure antigen. The purified HuCAL antibody should be used at a final concentration of 1 to 10 μg/ml. We recommend a starting concentration of 5 μg/ml.

For chemiluminescent detection, the system of choice is the use of an HRP conjugate in combination with a chemiluminescent substrate (e.g. ECL Plus, GE Healthcare). Alternatively, the BCIP/NBT substrate (AbD Serotec #BUF045A) can be used for alkaline phosphatase conjugates.

Western Blot - Detection of Fab

SDS-PAGE: load 10ng-1μg antigen/lane
Blot: transfer to a PVDF membrane according to standard protocols
Blocking: 5 % MP-TBST (TBS containing 0.05 % Tween 20 and 5 % milk powder)
Wash: Rinse blot with TBST
Primary AB: HuCAL antibody (1 to 10μg/ml), 1 h @ RT in 1 % MP-TBST
Wash: 3 x 5 min with generous amount of TBST
Secondary AB: α-Fab-HRP conjugate¹
(1:5000 in 1 % MP-TBST), 1h @ RT
Wash: 3 x 5 min with generous amount of TBST
Detection: ECL Plus chemilumenescence substrate¹⁰

Western Blot - Detection of c-terminal His₆-tag

SDS-PAGE: load 10ng-1μg antigen/lane
Blot: transfer to a PVDF membrane according to standard protocols
Blocking: 5 % MP-TBST (TBS containing 0.05 % Tween 20 plus 5 % milk powder)
Wash: Rinse blot with TBST
Primary AB: HuCAL antibody (1 to 10μg/ml) 1h @ RT in 1 % MP-TBST
Wash: 3 x 5 min with generous amount of TBST
Secondary AB: α-His-HRP₆-POD³
(1:500 - 1:1000 in 1 % MP-TBST), 1h @ RT
Wash: 3 x 5 min with TBST
Detection: ECL Plus chemilumenescence substrate¹⁰

Western Blot - Detection of c-terminal myc-tag

SDS-PAGE: load 10ng-1μg antigen/lane
Blot: transfer to a PVDF membrane according to standard protocols
Blocking: 5 % MP-TBST (TBS containing 0.05 % Tween 20 and 5 % milk powder)
Wash: Rinse blot with TBST
Primary AB: HuCAL antibody (1 to 10μg/ml), 1h @ RT in 1 % MP-TBST
Wash: 3 x 5 min with generous amount of TBST
Secondary AB: α-c-myc-HRP⁵
(1:100 - 1:500 in 1 % MP-TBST), 1h @ RT
Wash: 3 x 5 min with generous amount of TBST
Detection: ECL Plus chemilumenescence substrate¹⁰

Western Blot – Detection of c-terminal FLAG M2-tag

SDS-Page: load 10ng-1μg antigen/lane
Blot: transfer to a PVDF membrane according to standard protocols
Blocking: 5% MP-TBST (TBS containing 0.05% Tween 20 and 5% milk powder)
Wash: Rinse blot with TBST
Primary AB: HuCAL antibody (1 to 10μg/ml), 1h @ RT 1 % MP-TBST
Wash: 3 x 5 min with generous amount of TBST
Secondary AB: α-FLAG M2-HRP⁷
(1:100 - 1:1000 in 1 % MP-TBST), 1h @ RT
Wash: 3 x 5 min with generous amount of TBST
Detection: ECL Plus chemilumenescence substrate¹⁰

Western Blot – Detection of c-terminal Strep-tagII

SDS-Page: load 10ng-1μg antigen/lane
Blot: transfer to a PVDF membrane according to standard protocols
Blocking: 10% BSA-TBST, 1h @ RT (TBS buffer with 0.05% Tween 20 and 10% BSA)
Wash: Rinse blot in TBST
Primary AB: HuCAL antibody (1to 10μg/ml), 1h @ RT in 1% BSA-TBST
Wash: 3 x 5 min with generous amount of TBST
Secondary AB: Strep-Tactin HRP conjugate⁸
(1:4000 in 1% BSA-TBST), 1h @RT
Wash: 3 x 5 min with generous amount of TBST
Detection: ECL Plus chemilumenescence substrate¹⁰

Immunohistochemistry (IHC)

The protocols have been developed for paraffin-embedded tissue sections. The methods have been successfully tested both with manual staining and with staining using the DAKO auto-stainer. The recommended format for IHC is Fab-dHLX-MH due to its bivalency.

Before staining, the tissue section needs to be de-paraffinized. In addition, an antigen retrieval procedure should be performed:

1. De-Paraffinization (by Xylene or Rotihistol)

Pour a sufficient volume of Xylene or Rotihistol into 4 cuvettes. The tissue should be covered completely. Incubate for 5 min each by shaking gently.
Place the slides in 100 % ethanol for two times 2 min
Place the slides in 90 % ethanol for two times 2 min
Place the slides in 80 % ethanol for two times 2 min
Place the slides in 70 % ethanol for two times 2 min
Place the slides in 50 % ethanol for two times 2 min
Wash 2 x 2 min with TBS

2. Heat-Induced Epitope Retrieval (HIER)

Pre-heat the buffer (10 mM citrate-buffer pH 6.0) and the slides in the microwave oven at 900 W for 5 min.
Boil slides for another 5-15 or 30 min at 500 W. Add buffer, if necessary.
Allow the sections to cool for 15 min in their retrieval buffer.
Wash sections in TBS for 2 x 2 min.

IHC²⁵ - Detection of c-terminal His₆-tag

Tissue section: preparation (e.g. de-paraffinization) and antigen retrieval
Wash: 3 x TBST (TBS buffer with 0.05 % Tween 20)
Biotin blocking: Ultra V Block¹², 5 min @ RT (do not exceed 10 min)
Wash: 3 x TBST
(TBS buffer with 0.05 % Tween 20)
Protein blocking: Power Block¹³, 15min @ RT
Primary AB: HuCAL antibody (5-25μg/ml in antibody-diluent), 1h @ RT
Wash: 3 x TBST
Secondary AB: mouse α-His6-antibody¹⁴
(1:200 in antibody-diluent), 30 min
Wash: 3 x TBST
Tertiary AB: biotinylated goat-α-mouse¹⁵
(ready to use), 15 min @ RT
Wash: 3 x TBST
Conjugate AB: Streptavidin-HRP conjugate¹⁶, 15 min @ RT
Wash: 3 x TBST
Stain: DAB substrate¹⁷, 10 min @ RT
Wash: 3 x TBST
Counterstain: Haematoxylin, 1 min @ RT

IHC - Detection of c-terminal₆myc-tag

Tissue section: preparation (e.g. de-paraffinization) and antigen retrieval
Wash: 3 x TBST
(TBS buffer with 0.05 % Tween 20)
Biotin blocking: Ultra V Block¹², 5 min @ RT (do not exceed 10 min)
Wash: 3 x TBST
(TBS buffer with 0.05 % Tween 20)
Protein blocking: Power Block¹³, 15min @ RT
Primary AB: HuCAL antibody
(5-25μg/ml in antibody-diluent), 1h @ RT
Wash: 3 x TBST
Secondary AB: α-c-myc⁵ (1:80 in antibody-diluent), 30 min @ RT
Wash: 3 x TBST
Tertiary AB: biotinylated goat-α-mouse¹⁵
(ready to use), 15 min @ RT
Wash: 3 x TBST
Conjugate AB: Streptavidin-HRP conjugate¹⁶, 15 min @ RT
Wash: 3 x TBST
Stain: DAB substrate¹⁷, 10 min @ RT
Wash: 3 x TBST
Counterstain: Haematoxylin, 1 min @ RT

Fluorescence Activated Cell Sorting (FACS)

The purified HuCAL antibody should be used at a final concentration of 100μg/ml for monovalent Fab, and 50μg/ml for bivalent Fab mini-antibody.

FACS - Detection of Fab

Antigen expr. cells: suspension of single cells (receptor density: as high as possible; make sure the cells are viable), wash once with cold FACS buffer (PBS with 3 % FCBS and 0.05 % Sodium Azide), adjust the cells to 10⁷ cells/ml (= 0.5 x 10⁶ cells/50μl) in cold buffer
Primary AB: HuCAL antibody (100 μg/ml Fab; 50 μg/ml Fab-dHLX), 1h @ 4° C
Wash: 2 x cold FACS buffer
Secondary AB: α-human F(ab`)2R-PE conjugate ¹⁸ (1:100 in FACS buffer), 1h @ 4° C in the dark
Wash: 2 x cold FACS buffer
Detection: Flow cytometer

FACS - Detection of c-terminal myc-tag

Antigen expr. cells: suspension of single cells (receptor density: as high as possible; make sure the cells are viable), wash once with cold FACS buffer (PBS with 3 % FCBS and 0.05 % Sodium Azide), adjust the cells to 107 cells/ml (= 0.5 x 106 cells/50μl) in cold buffer
Primary AB: HuCAL antibody
(100 μg/ml Fab; 50 μg/ml Fab-dHLX), 1h @ 4° C
Wash: 2 x cold FACS buffer
Secondary AB: α-c-myc⁵
(1:2000 in FACS buffer), 1h @ 4° C
Wash: 2 x cold FACS buffer
Tertiary AB: α-mouse lg FITC conjugate¹⁹
(1:200 in FACS buffer), 1h @ 4° C in the dark
Wash: 2 x cold FACS buffer
Detection: Flow cytometer

FACS-Detection of c-terminal FLAG M2-tag

Antigen expr. cells: suspension of single cells (receptor density: as high as possible; make sure the cells are viable), wash once with cold FACS buffer (PBS with 3% FCBS and 0.05% Sodium Azide), adjust the cells to 10⁷ cells/ml (= 0.5 x 10⁶ cells /50μl) in cold buffer
Primary AB: HuCAL antibody
(100μG/ML FAB; 50μg/ml FAB-dHLX), 1h @ 4°C
Wash: 2 x cold FACS buffer
Secondary AB: α-FLAG M2⁷ (1:2000 in FACS buffer), 1h @ 4°C
Wash: 2 x cold FACS buffer
Tertiary AB: α-mouse lg FITC conjugate¹⁹
(1:200 in FACS buffer), 1h @ 4°C in the dark
Wash: 2 x cold FACS buffer
Detection: Flow cytometer

Immunoprecipitation (IP)

HuCAL antibodies can be used for immunoprecipitation by first applying secondary antibodies and capturing the complexes with sepharose beads coupled with reagents specific to the secondary antibody. The purified HuCAL antibody should be used at a final concentration of 2 to 10 μg/ml.

Immunoprecipitation - Detection of c-terminal myc-tag

Sepharose conj.: couple α-c-myc⁵ (2.5 μg) to Protein L Plus²⁰ (15 μl) in TNEC buffer²¹, 1h @ 4° C on an overhead rotator
Wash: 3 x TNEC buffer, centrifuge (8000g for 2 min @ 4° C)
Primary AB: add HuCAL antibody (2-10 μg/ml) to pre-formed complex, 16 hrs @ 4° C on an overhead rotator
Wash: 3 x TNEC buffer, centrifuge (8000g for 2 min @ 4° C)
Immunoprecipitation: add lysate (antigen concentration: as high as possible), over-night @ 4° C on an overhead rotator
Centrifugation: collect sepharose pellet (14 000g for 2 min @ 4° C), discard the supernatant
Wash: 3 x TNEC buffer and 1 x PBS, centrifuge (8000g for 2 min @ 4° C)
Detection: add 2 x SDS reducing gel loading buffer, incubate 5 min @ 100° C, re-centrifuge and load supernatant onto a 12 % SDS gel

Immunoprecipitation – Detection of c-terminal FLAG M2-tag

Sepharose conj.: couple α-FLAG M2⁷ (10μg) to Protein G²² (3-5mg) in TNEC buffer²¹, 2hrs @ 4°C on an overhead
Wash: 3 x TNEC buffer, centrifuge (8000g for 2min @ 4°C)
Primary AB: add HuCAL antibody (2-10μg/ml) to pre-formed complex, 2hrs @ 4°C on an overhead rotator
Wash: 3 x TNEC buffer, centrifuge (8000g for 2min @ 4°C)
Immunoprecipitation: add lysate (antigen concentration: as high as possible), 2hrs @ 4°C on an overhead rotator
Centrifugation: collect sepharose pellet (8000g for 2min @ 4°C), discard the supernatant
Wash: 3 x TNEC buffer and 1 x PBS, centrifuge (8000g for 2min @ 4°C)
Detection: add 2x SDS reducing gel loading buffer, incubate 5min @ 100°C, re-centrifuge ans load supernatanr onto a 12% SDS gel

Footnotes

1

Goat anti-human lgG F(ab’)2 specific antibody-Alkaline Phosphatase conjugate; AbD Serotec #0500-0100

2

AttoPhos substrate for Alkaline Phosphatase, Roche #1681982

3

Anti-His₆-HRP; AbD Serotec #MCA1396P

4

QuantaBlu Peroxidase Substrate; Pierce #15169

5

Anti-c-myc-HRP, Mouse monocional antibody (clone 9E10), AbD Serotec #MCA2200P

6

HiSpec Buffer, AbD Serotec [BUF049]

7

ANTI-FLAGM2-HRP Monoclonal Antibody mouse; Sigma #A8592

8

Strep -Tactin HRP conjugate, IBA #2-1502-001

9

BCIP/NBT Alkaline Phosphatase Substrate; AbD Serotec #BUF045A

10

ECL PLus, GE Healthcare

11

This protocol is valid for paraffin-embedded tissue slides

12

Ultra V Block; LabVision #TA-125-UB

13

Power Block; BioGenex #HK085-5K

14

Mouse α-His6antibody; AbD Serotec #MCA1396

15

Goat anti-mouse, biotinylated; LabVision #TM-125-BN

16

Streptavidin-HRP conjugate; LabVision #TS-125-HR

17

DAB substrate; DakoCytomation #K3468

18

Goat Anti-Human lgG (H+L) R-Phycoerythrin conjugate; Dianova/Jackson Immuno Research #109-116-088

19

FITC-conjugated rabbit anti-mouse lg, (Fab´)2 fragment, DakoCytomation #F0313

20

Protein L Plus, Pierce #20520

21

TNEC buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA; Roche #1332473

22

Protein G; Pierce #20398