1. Secondary antibody란?
Target antigen을 detection, sorting, purification 하기 위한 primary antibody에 binding 하는 항체입니다. Primary antibody의 종과 isotype에 특이성을 갖기 때문에, 원하는 단백질의 검출에 활용이 됩니다. 각각 다른 enzyme, dye가 conjugation 되어 있고, 여러분의 실험 목적에 따라서 secondary antibody를 선택하게 됩니다.
2. Secondary antibody performance는 어떻게 결정될까요?
- Specificity (특이성) - target을 얼마나 정확하게 인지하는지
- Sensitivity(민감성) - target을 인지할 수 있는 양으로, binding affinity와 labeling 정도
- Consistency(일관성) - 다른 batch를 사용할 때에 결과에 흔들림이 없는 일정한 결과
3. Antibody 구조
Antibodies exist as one or more copies of a Y-shaped unit, composed of four polypeptide chains. Each Y contains two identical copies of a heavy chain, and two identical copies of a light chain, which are different in their sequence and length. The top of the Y shape contains the variable domain(V) also known as the fragment, antigen-binding(F(ab))region. This region binds tightly and specifically to an epitope on a given antigen. The base of the antibody consists of constant domains(C)and forms what is known as the fragment, crystallizable region(Fc). This region is important for the function of the antibody during an immune response.
4. Conjugation 선택
5. Enzyme detection methods
|Horseradish Peroxidase(HRP)||Chromogenic, soluble(TMB, ABTS, OPD..)||ELISA||Easy to use||Light sensitive coloration|
|Chromogenic, precipitating(CN, AEC, DAB..)||WB, SB, IHC||Easy to use||Backgrounin blood samples and some other tissues. Staining stability lower than AP|
|Fluorogenic(ADHP/resotufin)||ELISA||High sensitivity||Need fluorescence equipment|
|Luminol||WB, SB, IHC||High sensitivity||Need radiographic equipment or light scanner|
|Alkaline Phosphatase(AP)||Chromogenic, soluble(pNNP)||ELISA||Linear kinetic. Often more sensitive than HRP||Unstable|
|Chromogenic, precipitating(BCIP/NBT..)||WB, SB, IHC||Staining stability higher than HRP||Interference with nuclear counteratin|
|Fluorogenic(4-MUP)||WB, IHC||Sensitivity||Need fluorescence equipment|
6. Fluorescent detection methods
Alexa 형광이 conjugation 된 secondary antibody는 주로 Immunostatining, Flow cytometry 등의 application에서 double 혹은 triple staining 에 활용이 됩니다. 이러한 실험의 경우, 실험 계획 단계에서 부터 secondary의 형광 조합을 신중하게 고려해야 합니다.
아래 compability 표를 참고하여최적의 결과를 위한 조합을 선택할 수 있습니다.
7. Class/subcalss of antibody
In mammals, antibodies are divided into five isotypes: IgG, IgM, IgA, IgD and IgE, based on the number of Y units and the type of heavy chain. The isotypes differ in their biological properties, functional locations and ability to deal with different antigens:
|Isotype||Heavy chain||Light chain||MW (kDa)||Structure||Function|
|λ or κ||150–600||Monomer - tetramer||Most produced Ig. Found in mucosal areas, such as the gut, respiratory and urogenital tract, and prevents their colonization by pathogens. Resistant to digestion and is secreted in milk.|
|IgD||δ||λ or κ||150||Monomer||Function unclear. Works with IgM in B cell development; mostly B cell bound|
|IgE||ε||λ or κ||190||Monomer||Binds to allergens and triggers histamine release from mast cells and is involved in allergy. Also protects against parasitic worms.|
|γ1, γ2, γ3, γ4||λ or κ||150||Monomer||Major Ig in serum. Provides the majority antibody based in immunity against invading pathogens. Moderate complement fixer. IgG3 can cross placenta.|
|IgM||μ||λ or κ||900||Pentamer||First response antibody. Expressed on the surface of B cells and in a secreted form with very high avidity. Eliminates pathogens in the early stages of B cell mediated immunity before there is sufficient IgG.|
The type of heavy chain present defines the class of an antibody. There are five types of mammalian Ig heavy chain denoted by Greek letters: α, δ, ε, γ and μ. These chains are found in IgA, IgD, IgE, IgG and IgM antibodies, respectively. Heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids.
Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region composed of three tandem Ig domains and a hinge region for added flexibility, heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The variable region of the heavy chain differs depending on the B cell that produced it, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.
In mammals there are only two types of light chain, λ and κ. A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211–217 amino acids. Each antibody contains two light chains that are always identical. Other types of light chains, such as the iota (ι) chain, are found in lower vertebrates like Chondrichthyes and Teleostei.
The Y-shape of an antibody can be divided into three sections: two F(ab) regions and an Fc region. The F(ab) regions contain the variable domain that binds to cognate antigens. The Fc fragment provides a binding site for endogenous Fc receptors on the surface of lymphocytes, and is also the site of binding for secondary antibodies. In addition, dye and enzymes can be covalently linked to antibodies on the Fc portion of the antibody for experimental visualization.
These three regions can be cleaved into two F(ab) and one Fc fragments by the proteolytic enzyme pepsin. Antibody fragments have distinct advantages in certain immunochemical techniques. Fragmenting IgG antibodies is sometimes useful because F(ab) fragments (1) will not precipitate the antigen and (2) will not be bound by immune cells in live studies because of the lack of an Fc region. Often, because of their smaller size and lack of crosslinking (due to loss of the Fc region), F(ab) fragments are radiolabeled for use in functional studies. Fc fragments are often used as Fc receptor blocking agents in immunohistochemical staining.
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