Polymerase Secondary Antibody Introduction

Introduction

The peroxidase-labeled antibody (Ab) method,introduced in 1968, was the first practical application of antibodies to paraffin-embedded tissues for most clinical and research studies (Nakane, 1968). However, conventional secondary Ab (IgG)-HRP conjugates are basically limited to not more than 3 molecules of HRP per single IgG molecule, This molar ratio of conjugation is sufficient in routine Western blot or elisa, but may lack the sensitivity required for detecting targets present in low picogram to femtogram amounts without the use of additional steps to amplify the signal.,in particular, immunohistochemistry.

During the past few decades, improvements in the reagents and protocols used for immunohistochemistry have led to increased sensitivity of detection systems. A several-fold higher antigen detectability than those achieved in enzyme–anti-enzyme immune complex techniques (PAP and APAAP) (Sternberger et al., 1970) or in standard avidin/streptavidin–biotin-complex (ABC or SABC) and Labeled StreptAvidin-Biotin (LSAB) protocols can be gained with the chain polymer-conjugate technology developed in the last decade of the former century.

However, PAP and APAAP method which will form a huge skeleton structure in aqueous environment appears to create spatial hindrance, thus reducing the penetrative ability of the detection reagent, especially for hidden nuclear antigens.  (for review see: Shi et al., 1999 and citations therein).

Since biotin (vitamin H) which is contained in many tissues has been introduced in ABC (SABC) method, it can combine with ABC (SABC), thereby generate nonspecific staining.

Meanwhile, to reduce experimental step, enhence the signal, reduce nonspecific reaction, Fine Biotech adopts a new polymerase-mark system (including poly- HRP Goat Anti-Rabbit IgG and poly- HRP Goat Anti-Mouse IgG). More enzymes will connect to the single antibody through the arm structure. Meanwhile, the optimal structure of new system enhences the signal during immunochemistry and reduces the experimental steps.

Data of various detection systems

 

Amplifier systemsizecompositionThe experimental stepsSignal
PAP, APAAPVery large, >5000kd
  • Anti-HRP-igG
  • HRP
  • HRP-igG
  1. first antibody
  2. PAP or APAAP
medium
ABC(SABC)Large, >2000kd
  • Avidin(StreptAvidin)
  • Biotin-HRP
  1. first antibody
  2. biotin- second antibody
  3. ABC(SABC)
Strong
Poly-HRP-igGMedium, 700~1200kd
  • HRP(multiple)-igG
  • (second antibody)
  1. first antibody
  2. second antibody
Very Strong
HRP-igG(normal)Small, 200~282kd
  • HRP(1-3)-igG
  • (second antibody)
  1. first antibody
  2. second antibody
Weak or none

 

Schematic diagram:

 

IHC Test results

Two kinds of antibodies form Fn-test (Fine Biotech) are tested, high concentration targets (Loading Control Antibodies) and lower level targets.

Second antibody (Poly-HRP Goat anti rabbit IgG(Catalogue No.ES-0004)and Poly-HRP Goat anti mouse IgG(Catalogue No.ES-0003) form Fn-test (Fine Biotech).

DAB kit form Fn-test(Catalogue No.IHC0005)

(high level targets)

AntibodiesDilutionlocationCatalogue No.Figure mark
Histone H3.31:150nucleusFNab03888A
PCNA1:200nucleusFNab06216B
a-Tubulin1:300Cytoplasm, CytoskeletonFNab00333C
GAPDH1:500Cytoplasm, nucleusFNab03342D
Lamin A/C1:400nucleusFNab04681E
VDAC11:400Cell membrane, MitochondrionFNab09826F
β-actin(monoclonal)1:250Cytoplasm, CytoskeletonFNab00873G

 

(lower level targets)

Antibodies Dilution location Catalogue No. Figure mark
TRX-11:300Cytoplasm, NucleusFNab09033H
PXMP21:150Peroxisome membraneFNab06965I
TTK1:200CytoskeletonFNab09094J
MEK31:300Cytoplasm, nucleusFNab05114K
MAP3K31:300CytoplasmFNab09830L
HDAC51:250Cytoplasm, nucleusFNab03802M
P2RX71:250Cell membraneFNab06071N

 

Tissue samples immunostained in this test: human kidney (A,D,F,N),

human liver cancer(B,E), human breast tumor (C,J,K) ,human colon cancer (H,I,L,M)

rat heart (G). Poly-HRP Goat anti rabbit igG with 1:100 dilution and Poly-HRP Goat anti mouse igG with 1:100 dilution. Staining with DAB at 10 mins, Nuclei were counterstained with Mayor `s hematoxylin.

Negative control test

Tissue samples immunostained in this negative control test: rat heart (1), human colon cancer (2),rat liver (3), human breast tumor (4). Negative serum as first antibody, Poly-HRP Goat anti rabbit igG and anti-mouse igG with 1:50 dilution mixture. Staining with DAB at 10 mins, Nuclei were counterstained with Mayor `s hematoxylin.

Conclusion

The poly-HRP conjugates showed excellent signal amplification, without non-specific results, and greatly simplified the experimental procedure.

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