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Primary and Secondary Antibody

posted Aug 18, 2019, 12:51 PM by Danny Xu   [ updated Aug 26, 2019, 7:56 PM ]


https://resources.rndsystems.com/images/site/rnd-systems-ihc-protocol-br.pdf
  1. How do I decipher the secondary antibody nomenclature?
    Anti-Goat IgG (H+L), highly cross-adsorbed, CF™ 568 antibody produced in donkey

Immunoglobulin Ig Classes and Isotypes

There are five mammalian immunoglobulin (Ig) classes where the heavy chain differs in amino acid composition and number: IgA (alpha), IgD (delta), IgE (epsilon), IgG (gamma) and IgM (mu). As there are five different Ig antibody classes, it can be advantageous to select a secondary raised against a specific class. Immunoglobulin classes, subclasses and their binding valency are described fully here.

The table below shows the different Ig classes and subclasses/isotypes in human and mouse.

Ig Class 

Human Ig Subclasses

Mouse Ig Subclasses

IgA

IgA1

IgG1

IgD

IgA2

IgG2a

IgE

IgG1

IgG2b

IgG

IgG2

IgG3

IgM

IgG3

 

 

IgG4

 

IgG Antibody

IgG Antibody IgG Structure

  • Two heavy chains: each chain composed of VH, CH1, hinge region, CH2 and CH3
  • Two light chains: each chain composed of VL and CL. There are two types of light chain, called kappa and lambda, always identical for each antibody
  • Two antigen binding sites: found at the end of the VH and VL chain, known as the paratope
Key  

Fv = Fragment, variable region

Fc = Fragment, crystallisable region

VH = Variable heavy chain

VL = Variable light chain

CH1 = Constant heavy chain 1

CL = Constant light chain

CH2 = Constant heavy chain 2

CH3 = Constant heavy chain 3

Fab = Fragment, antigen binding region

Fd = Heavy chain of the Fab

Lc = Light chain of the Fab

 

Click here for further information on IgG and the antibodies available to this Ig class.

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IgA Antibody

IgA Antibody IgA Structure

  • Four alpha heavy chains
  • Four light chains
  • Tetravalent
  • One secretory protein chain
  • One joining (J) chain

 

 

Click here for further information on IgA and the antibodies available to this Ig class.

IgD Antibody

IgD Antibody IgD Structure

  • Two delta heavy chains
  • Two light chains
  • Bivalent
  • Does not bind to Fc receptors

 

 

 

Click here for further information on IgD and the antibodies available to this Ig class.

IgE Antibody

IgE Antibody IgE Structure

  • Two epsilon heavy chains
  • Two light chains
  • Bivalent
  • Heavy chains contain a unique CH4 domain not found in other classes

 


 

Click here for further information on IgE and the antibodies available to this Ig class.

IgM Antibody

IgM Antibody IgM Structure

  • Pentamer of bivalent subunits
  • Ten Mu heavy chains
  • Ten light chains
  • One Joining (J) chain
  • Decavalent
  • Considerably higher molecular mass than other classes
     

 

Click here for further information on IgM and the antibodies available to this Ig class.


In what species was the primary antibody developed?
Secondary antibodies are directed against the species of the primary antibody. Therefore, you will need a secondary antibody that is raised in a species different than the host species of the primary antibody. For example if your primary antibody is raised in a mouse, you will need an anti-mouse secondary antibody raised in goat, rabbit, etc.

What is the class (isotype) and/or subclass of the primary antibody?
This question is primarily important when working with monoclonal antibodies. Polyclonal antibodies, however, are typically IgG class immunoglobulins. For this reason, the secondary antibodies will mainly be an anti-IgG antibody.

Monoclonal antibodies are most commonly developed in mice and occasionally in rats, hamsters or rabbits. For example, if the primary monoclonal antibody is mouse IgM, one would want a secondary antibody that reacts with mouse IgM (anti-Mouse IgM).

If the primary monoclonal is one of the mouse IgG subclasses, almost any anti-mouse IgG secondary antibody should bind to it. If the subclass of the primary antibody is not known, then anti-Mouse IgG F(ab) secondary antibodies may be used since they recognize most mouse immunoglobulin subclasses.

Primary ABs:
Mark W. Recommended HCS-1 is mouse Monoclonal IgG2a, http://dshb.biology.uiowa.edu/hcs-1 

Storage and Handling Recommendations: Although many cell products are maintained at 4°C for years without loss of activity, shelf-life at 4°C is highly variable. To ensure retention of antibody activity, we recommend aliquotting the product into two parts: 1) a volume of antibody stored at 4°C to be used within two weeks. 2) the remaining product diluted with an equal volume of molecular grade glycerol and stored at -20°C.

P.W.: Anti-Parvalbumin Millipore Sigma MAB1572 is Mouse Monoclonal  IgG1   Storage and Stability  Stable for 1 year at -20ºC from date of receipt.
mainly use the mouse monoclonal from Millipore Sigma (MAB1572). The one in rabbit would work as well. Apparently the trick is to block with goat serum when staining zebrafish; other serums do not work as well.  We usually order our secondaries from Molecular Probes through Thermo Fisher, although we're trying out a new source that's slightly cheaper, Jackson Immunoresearch.


Secondary Abs: Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG (H+L) https://www.jacksonimmuno.com/catalog/products/115-545-003

Physical State: Freeze-dried solid 
Storage and Rehydration: Store freeze-dried solid at 2-8°C. Rehydrate with the indicated volume of dH2O and centrifuge if not clear. Prepare working dilution on day of use. Product is stable for about 6 weeks at 2-8°C as an undiluted liquid.
Extended Storage after Rehydration: Aliquot and freeze at -70°C or below. Avoid repeated freezing and thawing.

Alternatively, add an equal volume of glycerol (ACS grade or better) for a final concentration of 50%, and store at -20°C as a liquid.

Expiration date: one year from date of rehydration. The expiration date may be extended if test results are acceptable for the intended us


There are many classes and subclasses of human and mouse IgG(s). Choosing a secondary may be difficult. However, one common factor among these IgG(s) are the light chains (kappa and lambda). In other words, IgG, IgM, IgA, IgD and IgE all have either kappa or lambda light chains. The heavy chain, however, is class specific.

Summary of immunoglobulin classes and subclasses:
         Immunglobulin Classes: IgG, IgM, IgA, IgE, IgD
         Human Immunoglobulin Subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
         Mouse Immunoglobulin Subclasses: IgG1, IgG2a, IgG2b, IgG3
         Light Chains: Kappa, Lambda
         Heavy Chains: IgG (gamma), IgM (mu), IgA (alpha), IgD (delta), IgE (epsilon)


Additional information on antibody reactivities:
  • Polyvalent: react with all classes
  • Anti-Fc and heavy-chain specific: react with heavy chains only, therefore, they are class specific.
  • Anti-Fab and whole molecule specific: react with heavy and light chains. Due to the light chain reactivity, they can react with all classes.
  • Light chain (kappa, lambda) specific: react with all classes, since all classes use the same kappa or lambda light chains.

Secondary Antibody Specific Binding Locations

The nature of the epitope recognized by the secondary antibody can have an impact on the success of an experiment, and it is worth taking the time to consider if this may affect your data. Here we have divided our secondary antibody range into four groups based on the epitope recognized.

1. Universal

H/L chain – these polyclonal secondary antibodies have a broad target binding area as they recognize both the heavy and light chains, which are found in all formats (full Ig, F(ab)2’ and Fab’) and classes of primary antibodies.
 

Example of use - due to their broad reactivity, universal polyclonal secondary antibodies can be used in most applications when specificity is not an issue. See the example of western blotting in the following section:  “Detection of light and heavy chains of a mouse monoclonal IgG1 by western blot analysis”. 

2. Light chain

Ig lambda light chain or kappa light chain – light chain specific secondary antibodies will target 25 kDa polypeptides without detecting the heavy chain. Light chain ratios vary greatly between species, and secondary antibodies are not generally cross-reactive, so it is important to establish the specific light chain in your primary antibody when using this type of secondaries.

Example of use - western blotting of immunoprecipitates may cause detection problems if the antigen is of a similar size to the 50 kDa heavy chain of IgG, this is because the secondary may detect this component of the precipitating primary antibody. The use of an anti-light chain secondary antibody will prevent this, as only the 25 kDa light chain will be co-detected in the blot. See an example blot in the following section: “Detection of light and heavy chains of a mouse monoclonal IgG1 by western blot analysis”. 

3. Heavy chain

  1. Gamma chain – these secondary antibodies target the heavy chain found in full IgG, F(ab)2’ and Fab’ formats. They do not cross-react with IgG light chains.
  2. Mu chain – these secondary antibodies only recognize the heavy chain on IgM, and as a result, no cross-reactivity is seen with other Ig class heavy chains or any Ig light chains.
  3. Alpha chain and alpha 1 chain – these secondary antibodies recognize the alpha chain on IgA subclass, and have minimal cross-reactivity with other human immunoglobulin subclasses.
  4. Fc – these secondary antibodies recognize the Fc region of a specific and specified Ig subclass. They can be useful to capture primary antibodies with their antigen binding domains clearly presented.
  5. CH2 domain (CH3 in IgM and IgE) – these secondary antibodies recognize the CH2 domain on the heavy chain in the Fc region of a specific and specified Ig subclass. The CH2 domain in an antibody has a role in preventing degradation in vivo, and may be added to therapeutic proteins to increase their half-life. Read secondary antibodies to CH2 and CH3 domains for further information about how secondary antibodies specifically targeted to the CH2 and CH3 domains of immunoglobulins enable the study of the Fc fragments in the development of new therapeutic antibody fragments.
  6. CH3 domain – these secondary antibodies recognize the CH3 domain on the heavy chain in the Fc region of a specific and specified Ig subclass. As above, this region plays a role in antibody stability.
  7. Hinge region – these secondary antibodies recognize the hinge region of a specific and specified Ig subclass.
  8. Fd – these secondary antibodies recognize the Fd region of human IgG. The Fd region is the heavy chain of the Fab, i.e. approximately the first 220 amino acids from the N-terminus of the heavy chain comprised of the VH and CH1 regions.  Typically these antibodies show minimal cross-reactivity to the Fc region of human IgG and other human immunoglobulins.
     

Example of use - in an analogous situation to the one described above, the use of an anti-heavy chain secondary can prevent interference with the detection of antigens of a similar size to the 25k Da IgG light chain of the precipitating primary antibody by only detecting the larger heavy chain component. See example western blot data in the following section: “Detection of light and heavy chains of a mouse monoclonal IgG1 by western blot analysis”. 



When should I use a pre-adsorbed secondary antibody?
Using pre-adsorbed antibodies will reduce non-specific background when working with tissues and cells. The process involves passing the secondary antibody over immobilized serum proteins from potentially cross reactive species. Therefore, if you are working with human tissues, choose a secondary antibody that has been adsorbed with human serum or human IgG.

Cross-Adsorbed Antibodies
  1. Cross reactions are often seen with secondary antibodies. For example, anti-mouse IgG may cross react to some degree with rat IgG, or an anti-rat IgG may show some cross reaction with IgM, and an anti-mouse IgG2a may show some cross reaction with mouse IgG2b. In some cases cross reactivity is not significant, but in others a high degree of specificity is required for accurate data.

    The initial way to achieve specificity is to use a cross-adsorbed polyclonal secondary. Here unwanted cross-reactivity is removed by pre-adsorption of the secondary antibody with the cross reacting antigen, to yield a more specific secondary and therefore reduce non-specific background staining. As an alternative to this, a monoclonal secondary may be selected that binds to an epitope unique to the chosen isotype.

    Read our overview on cross-adsorbed secondary antibodies to discover why and how you should use them, how they are generated, and a list of the cross-adsorbed secondary antibodies available now.



  2. When is a F(ab) or F(ab')2 fragment antibody necessary?
    If working with tissues or cells that have Fc receptors, choose a F(ab) or F(ab')2 fragment when possible to eliminate non-specific binding.  In more detail, F(ab')2 antibody fragments are used in assay systems where the presence of the Fc region may cause problems. Samples such as lymph nodes, spleen, and peripheral blood preparations contain cells with Fc receptors (macrophages, B lymphocytes, and natural killer cells) which could bind the Fc region of intact antibodies, causing high background staining. Use of F(ab')2 fragments ensures that any antibody binding observed is not due to Fc receptors.
Andy Groves Lecture@BIE 2019




Blocking Explained


Preventing Non-specific Staining

Once tissue or cell samples have been appropriately prepared and fixed, the samples are ready to be stained. All IHC/ICC studies are dependent on specific antibody-epitope binding, which is governed by hydrophobic interactions, ionic interactions, hydrogen bonding, and other intermolecular forces. However, the same attractive forces can also result in non-specific staining, i.e. binding of the primary antibody to amino acids other than those within the desired epitope of the antigen. This is a common problem that occurs in IHC/ICC experiments. The challenge is to reduce non-specific interactions without impairing antibody-epitope binding. Causes of non-specific staining include interactions of the primary and secondary antibodies with serum proteins, ionic interactions between antibodies and tissues, and interactions with endogenous molecules capable of affecting the IHC detection system used. These issues can result in high background and an inability to visualize the antigen of interest in its appropriate cellular location. Staining problems of this type can be addressed by blocking non-specific interactions using a blocking reagent. These steps are performed prior to incubation of the sample with the primary antibody.

Preventing Non-specific Hydrophobic Interactions

Although hydrophobic interactions play an essential role in epitope-antibody binding, these forces can also promote non-specific binding. Most proteins have some degree of hydrophobicity due to the neutral side chains of several amino acids. Tissue incubation with heat-inactivated normal serum or bovine serum albumin (BSA) is a common procedure used to reduce non-specific hydrophobic binding. Selection of the type of normal serum is important to prevent interactions with the primary or secondary antibodies, or with the tissues/cells being stained. For example, goat serum would not be advised as a blocking reagent for use with a goat-derived primary antibody. Instead, a serum identical to the host animal of the secondary antibody or from an unrelated species is recommended. BSA and non-fat dry milk are also frequently used as blocking reagents. One of these reagents is typically included in the diluents for the primary and secondary antibodies. The addition of non-ionic detergents including 0.3% Triton X-100™ or Tween 20™ can also reduce non-specific hydrophobic interactions.

Blocking Non-specific Binding with Serum. A. CD14 was detected in paraffin-embedded human tonsil tissue using anti-human CD14 biotinylated affinity-purified polyclonal antibody (Catalog # BAF383). Tissue was subjected to antigen retrieval using the Basic Antigen Retrieval Kit (Catalog # CTS013). Tissue was stained using high sensitivity streptavidin conjugated to HRP (HSS-HRP) and DAB, and counterstained with hematoxylin (blue). B. Non-specific background staining is markedly reduced in a parallel experiment which included a blocking step using animal serum for 15 minutes at room temperature prior to incubation with the primary antibody. HSS-HRP and animal blocking serums are included in all R&D Systems Cell and Tissue Staining Kits.

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