Blocking and labeling with Fab fragments

Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and Double Labeling Primary Antibodies from the Same Host Species

Monovalent Fab fragments of affinity-purified secondary antibodies are offered to cover (block) the surface of immunoglobulins for double labeling primary antibodies from the same host species, or to block endogenous immunoglobulins in tissue sections or on cell surfaces. They can be used for these purposes because Fab fragments have only a single antigen binding site (i.e. they are monovalent).

In contrast, divalent antibodies (whole IgG and F(ab’)₂ fragments) have two antigen binding sites. After labeling the first primary antibody, some antigen binding sites on the first secondary antibody may remain open which could capture the second primary antibody introduced in a subsequent step. Consequently, it will appear as overlapping labeling, even though there may not be overlapping antigens. Therefore, divalent antibodies should not be used for blocking or for double labeling two primary antibodies from the same species.

Monovalent Fab secondary antibodies are not necessary when primary antibodies from the same host species are different classes of immunoglobulins, such as IgG and IgM, or different subclasses of IgG, such as Mouse IgG1 and Mouse IgG2a. In these cases, it is much easier and more effective to use class specific or subclass specific antibodies, respectively, to distinguish between the two primary antibodies.

Caution: Whole IgG and F(ab’)₂ fragments are divalent antibodies, with two antigen binding sites, therefore they cannot be used in the following protocols which specifically require Fab fragments.

Detection of two unlabeled primary antibodies from the same host species

Example A

Labeling with two unconjugated primary antibodies of the same host species.

A Detection method using conjugated Fab fragments and whole IgG secondary antibodies.

Example B

Labeling with two unconjugated primary antibodies of the same species.

A blocking method using unconjugated fab fragments to “change” the species of one antibody.

Example C

Labeling using two unconjugated primary antibodies of the same species after blocking for endogenous Ig.

A blocking method to allow use of two same species primary antibodies following blocking for endogenous Ig with unconjugated fab fragments.

Important Note:

The monovalent Fab fragments have not been adsorbed to remove cross-reactivities to other species. If the experimental sample contains endogenous immunoglobulins Example C should be used. Example A or B could introduce background.

Detection of one unlabeled and one or more labeled primary antibodies from the same host species

Example D

Labeling using unconjugated and conjugated primary antibodies of the same host species.

A blocking method using unconjugated fab fragments to prevent detection of the conjugated primary by subsequent antibodies.

Example E

Labeling using conjugated and unconjugated primary antibodies of the same species.

A blocking method using whole Ig antibodies to prevent non-specific labeling.

Example A

Example A: Use of conjugated Fab fragments for labeling and blocking

Examples A, B and C show some of the possible protocols used for double labeling two unconjugated primary antibodies from the same host species.

The success of these experimental designs will require some empirical manipulations. Optimizing reagent concentrations in each step or switching the labeling sequence of the two antigens may influence the outcome.

Labeling the less abundant primary antibody first increases blocking efficiency.
Blocking with an appropriate normal serum helps to reduce background.
To avoid displacement of the Fab antibody by the labeled secondary antibody, a light post-fixation with glutaraldehyde may be used, provided that it does not affect antigenicity of the target proteins.

Step 1. After blocking with normal serum, incubate with the first primary antibody, in this example Rabbit Anti-Antigen X. Wash.

Step 2. Incubate with excess conjugated secondary antibody, in this example Alexa Fluor® 488-Fab fragment Goat Anti-Rabbit IgG (H+L). Wash.

Step 3. Incubate with the second primary antibody, Rabbit Anti-Antigen Y.

Step 4. Incubate with a second conjugated secondary antibody, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L). Wash.

Application Notes:

Monovalent Fab fragments have not been adsorbed against other species, so they may cross-react with endogenous Ig. Use Example C to avoid detection of endogenous Ig.
Example A may require a high concentration of conjugated Fab to saturate the first primary antibody. If this results in unacceptable background, try a lower concentration of the conjugated Fab, followed by further blocking with unconjugated Fab.

Example B

Example B: Use of unconjugated Fab fragments to cover the first primary antibody, presenting it as a different species

Examples A, B and C show some of the possible protocols used for double labeling two unconjugated primary antibodies from the same host species.

Labeling the less abundant primary antibody first increases blocking efficiency.
Blocking with an appropriate normal serum helps to reduce background.
To avoid displacement of the Fab antibody by the labeled secondary antibody, a light post-fixation with glutaraldehyde may be used, provided that it does not affect antigenicity of the target proteins.

Step 1. After blocking with normal serum, incubate with the first primary antibody, in this example Rabbit Anti-Antigen X. Wash.

Step 2. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibody, in this example unconjugated Fab fragment Goat Anti-Rabbit IgG (H+L). This presents the rabbit IgG as goat Fab. Wash.

Step 3. Incubate with conjugated tertiary antibody directed against the host species of the Fab antibody. The tertiary antibody must not recognize the host species of either the primary antibodies or the second secondary antibody. This example used Alexa Fluor® 488-Mouse Anti-Goat IgG (H+L) (min X Ms, Hu, Rb Sr Prot). Wash.

Step 4. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.

Step 5. Incubate with second conjugated secondary antibody, that does not recognize the host species of either the Fab antibody used in step 2 or the tertiary antibody used in step 3. In this example, Rhodamine Red™‑X-Mouse Anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) was used. Wash.

Application Note:

Monovalent Fab fragments have not been adsorbed against other species, so they may cross-react with endogenous Ig. Use Example C to avoid detection of endogenous Ig.

Example C

Example C: Use of unconjugated Fab fragments for blocking after the first secondary antibody step.

Examples A, B and C show some of the possible protocols used for double labeling two unconjugated primary antibodies from the same host species.

Labeling the less abundant primary antibody first increases blocking efficiency.
Blocking with an appropriate normal serum helps to reduce background.
To avoid displacement of the Fab antibody by the labeled secondary antibody, a light post-fixation with glutaraldehyde may be used, provided that it does not affect antigenicity of the target proteins.

Step 5. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.

Step 6. Incubate with the same secondary antibody as used in step 2, conjugated to a different probe, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.

Step 3. Incubate with normal serum from the same host species as the primary antibodies, in this example normal rabbit serum. The purpose of this step is to saturate open binding sites on the first secondary antibody with IgG so that they cannot capture the second primary antibody. Wash.

Step 4. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibodies, in this example Fab Goat Anti-Rabbit IgG (H+L). The host species of the Fab antibody should be the same as the host species of the conjugated secondary antibody. This step covers the rabbit IgG so that the second secondary antibody will not bind to it. Wash.

Step 1. After blocking with normal serum, incubate with the first primary antibody, in this example Rabbit Anti-Antigen X. Wash.

Step 2. Incubate with conjugated secondary antibody, in this example Alexa Fluor® 488-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.

Example D

Example D: Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies

Examples D and E illustrate multiple labeling protocols that include a directly labeled and an unlabeled primary antibody from the same host species. It is advisable to incubate the less abundant primary first. In Example D, the directly labeled primary antibody is incubated first, then blocked with Fab fragments prior to applying the unlabeled primary antibody.

If the unlabeled primary antibody is incubated first (Example E), double labeling can be achieved without using Fab fragments. Following incubation with the labeled secondary antibody, normal serum is used to block open binding arms of the secondary, preventing capture of the labeled primary.

Step 1. After blocking with normal serum, incubate with conjugated primary antibody, in this example Alexa Fluor® 488-Rabbit Anti-Antigen X. Wash.

Step 2. Incubate with an excess of unconjugated Fab Goat Anti-Rabbit IgG (H+L). Wash.

Step 3. Incubate with the unconjugated primary antibody, in this example Rabbit Anti-Antigen Y. Wash.

Step 4. Incubate with conjugated secondary antibody, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L). Wash.

Example E

Example E: Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments

Step 1. After blocking with normal serum, incubate with the unlabeled primary antibody, in this example Rabbit Anti-Antigen X. Wash.

Step 2. Incubate with conjugated secondary antibody, in this example Alexa Fluor® 488-Goat Anti-Rabbit IgG (H+L). Wash.

Step 3. Incubate with normal serum from the host species of the primary antibody, in this example normal rabbit serum. Wash.

Step 4. Incubate with conjugated primary antibody, in this example Rhodamine Red™‑X-Rabbit Anti-Antigen Y. Wash.

Blocking and labeling with Fab fragments

Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and Double Labeling Primary Antibodies from the Same Host Species

Caution: Whole IgG and F(ab’)₂ fragments are divalent antibodies, with two antigen binding sites, therefore they cannot be used in the following protocols which specifically require Fab fragments.

Detection of two unlabeled primary antibodies from the same host species

Example A

Labeling with two unconjugated primary antibodies of the same host species.

Example B

Labeling with two unconjugated primary antibodies of the same species.

Example C

Labeling using two unconjugated primary antibodies of the same species after blocking for endogenous Ig.

Important Note:

Detection of one unlabeled and one or more labeled primary antibodies from the same host species

Example D

Labeling using unconjugated and conjugated primary antibodies of the same host species.

Example E

Labeling using conjugated and unconjugated primary antibodies of the same species.

Example A

Example A: Use of conjugated Fab fragments for labeling and blocking

Application Notes:

Example B

Example B: Use of unconjugated Fab fragments to cover the first primary antibody, presenting it as a different species

Application Note:

Example C

Example C: Use of unconjugated Fab fragments for blocking after the first secondary antibody step.

Example D

Example D: Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies

Example E

Example E: Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments

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Blocking and labeling with Fab fragments

Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and Double Labeling Primary Antibodies from the Same Host Species

Caution: Whole IgG and F(ab’)2 fragments are divalent antibodies, with two antigen binding sites, therefore they cannot be used in the following protocols which specifically require Fab fragments.

Detection of two unlabeled primary antibodies from the same host species

Labeling with two unconjugated primary antibodies of the same host species.

Labeling with two unconjugated primary antibodies of the same species.

Labeling using two unconjugated primary antibodies of the same species after blocking for endogenous Ig.

Important Note:

Detection of one unlabeled and one or more labeled primary antibodies from the same host species

Labeling using unconjugated and conjugated primary antibodies of the same host species.

Labeling using conjugated and unconjugated primary antibodies of the same species.

Example A: Use of conjugated Fab fragments for labeling and blocking

Application Notes:

Example B: Use of unconjugated Fab fragments to cover the first primary antibody, presenting it as a different species

Application Note:

Example C: Use of unconjugated Fab fragments for blocking after the first secondary antibody step.

Example D: Use of unconjugated Fab fragments for detection of one unlabeled and one or more labeled primary antibodies

Example E: Detection of one unlabeled and one or more labeled primary antibodies without the use of Fab fragments

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Caution: Whole IgG and F(ab’)₂ fragments are divalent antibodies, with two antigen binding sites, therefore they cannot be used in the following protocols which specifically require Fab fragments.