Rabbit UltraAvidin HRPO Staining Kit

The avidin-biotin complex method, utilizing peroxidase-labeled avidin, has revolutionized immunostaining by significantly enhancing the detection of minute antigen quantities and streamlining the overall process._1,2 This approach involves a biotin-labeled secondary antibody targeting the primary antibody's species-specific immunoglobulin. Subsequently, peroxidase-conjugated UltraAvidin binds, creating a complex with numerous horseradish peroxidase molecules indirectly linked to each secondary antibody._3,4 The exceptional affinity between avidin and biotin, characterized by a dissociation constant of 10-15M, ensures remarkable stability. Detection is achieved through the enzyme's capacity to transform a soluble substrate/chromogen into an insoluble colored product.

Leinco's UltraAvidin, a modified avidin variant, minimizes non-specific binding to cell surfaces by preventing both lectin-like receptor adherence and electrostatic interactions. The F(ab/)2 fragment secondary Goat anti-rabbit IgG antibody further reduces non-specific binding by eliminating Fc receptor interactions, making it ideal for immunohistochemical studies. Additionally, affinity isolation using Rabbit IgG and adsorption against human serum proteins and mouse serum minimize cross-reactivity with human or mouse tissues or specimens. The biotin conjugation of the antibody is optimized for performance through careful molar ratio control. The resulting kit delivers exceptional sensitivity, enabling the detection of low antigen levels or the utilization of lower primary antibody concentrations.

1. UltraBlockTM Blocking Buffer - 4ml
2. Reagent A: Goat Anti-Rabbit IgG-Biotin - 1 ml
3. Reagent B: UltraAvidin Peroxidase - 1ml
4. Mixing Bottles with drop dispenser tips: These are used to prepare the working solutions for the staining kit and to dispense the required amount on each tissue section or well. (Note: If held vertically upside down the volume of each drop is 45-50 ul. Holding the bottle at an angle would increase drop volume.)
Note: Store all reagents at 2-8^C. Keep bottles closed to prevent microbial or dust contamination. Do not reuse working solutions.

Standard Procedure for ELISA
ELISA is a widely used method for detecting soluble antigen/ antibody by sandwich or bridge method where the antigen/ antibody is immobilized on to a 96-well plate.

Materials Required But Not Supplied
1. Antigen solution
2. 0.1M Sodium bicarbonate buffer, pH 9.2
3. Suitable diluting buffer such as 0.01M Phosphate buffered Saline, pH 7.2 (PBS) or 0.05M Tris Buffered Saline, pH 7.4 (TBS).
4. Tween 20
5. Bovine Serum Albumin
6. Primary Antibody
7. Peroxidase Substrate: TMB, OPD and ABTS are the most commonly used substrates of which TMB is considered the most sensitive (Leinco Part No.: T118).

Working Solutions
1. Coating Buffer: Dilute the antigen to an appropriate concentration with 0.1M Sodium bicarbonate buffer (10ug/ml is recommended for immunoglobulin).
2. Wash buffer: 0.05% Tween 20 and 0.1% BSA in PBS.
3. Blocking Buffer: Add 4 drops of Blocking Buffer from the Rabbit UltraAvidin Peroxidase Staining Kit to 10ml PBS in the first mixing bottle (Bottle 1). Snap on drop dispenser tip.
4. Primary Antibody: Adjust to required concentration with Wash buffer.
5. Biotinylated secondary antibody: Add 1 drop of Reagent A from the Rabbit UltraAvidin Peroxidase Staining Kit to 10ml PBS in Mixing Bottle 2.
6. UltraAvidin-Peroxidase: Add 1 drop of Reagent B from the Rabbit UltraAvidin-Peroxidase Staining Kit to 10ml PBS in Mixing Bottle 3.
7. Substrate: If using the Leinco Substrate Kit Prod. No. T118 (see page 4) dilute according to kit protocol, preferably just before use.
8. Stop solution: Depending on substrate choice.

Method
1. Pipet 100ul of the antigen solution in coating buffer into each well.
2. Incubate at 37C for 1 hour or overnight in the refrigerator.
3. Discard solution and wash the plate 3 times in 300ul Wash Buffer.
4. Discard buffer and flick on a paper towel to dry.
5. Add 200ul (4 drops, if using the dropper bottle) of Blocking Buffer to each well and incubate for 1 hour at room temperature.
6. Discard buffer and wash the plate 3 times in 300ul Wash Buffer, flick dry on a paper towel.
7. Add 100ul of primary antibody to each well and incubate for 1 hour at room temperature.
8. Discard solution and wash the plate 3 times in 300ul Wash Buffer, flick dry on a paper towel.
9. Add 100ul (2 drops, if using the dropper bottle) of the diluted biotinylated secondary antibody to each well and incubate the plate for 30 mins. at room temperature.
10. Discard solution and wash the plate 3 times in 300ul Wash Buffer, flick dry on a paper towel.
11. Add 100ul (2 drops, if using the dropper bottle) of the prepared UltraAvidin-Peroxidase to each well and incubate the plate for 30 mins. at room temperature.
12. Discard solution and wash the plate 3 times in 300ul Wash Buffer, flick dry on a paper towel.
13. Add 100ul of the prepared substrate solution and incubate in the dark for 15 minutes or until color develops.
14. Add 50ul of a stop solution, if necessary.
15. Read results in the ELISA reader at appropriate wavelength.

Note: It is recommended to keep wells A1 and A2 as substrate controls, where you add only the substrate buffer. This serves as your blank.


Standard Procedure for Immunohistochemistry
Tissue antigens can be localized in histologic sections using immunohistochemical methods. The antigen is indirectly labeled by first binding to a primary antibody which in turn is bound to a secondary antibody-biotin-avidin-peroxidase complex. It is possible to stain two antigens using this localization method with different substrate/ chromogen. This method may be used with both cryostat or paraffin sections.⁷

Materials Required But Not Supplied
1. Cryostat or paraffin tissue sections.
2. Xylene
3. Ethanol
4. Methanol
5. 30% Hydrogen Peroxide
6. Suitable diluting buffer such as 0.01M Phosphate buffered saline, pH 7.2 (PBS).
7. Primary Antibody
8. Peroxidase Substrate: DAB, 4-Chloronaphthol and AEC are the most commonly used substrates of which DAB is considered the most stable.
9. Hematoxylin/Methyl green/Nuclear fast red
10. Mounting Medium

Working Solutions
1. 3% Hydrogen peroxide in methanol.
2. Blocking Buffer: Add 3 drops of Blocking Buffer from the Rabbit UltraAvidin Peroxidase Staining Kit to 10ml PBS in the first mixing bottle (Bottle 1). Snap on drop dispenser tip.
3. Primary Antibody: Adjust to required concentration with PBS.
4. Biotinylated secondary antibody: Add 1 drop of Reagent A from the Rabbit UltraAvidin Peroxidase Staining Kit to 10ml PBS in Mixing Bottle 2.
5. UltraAvidin-Peroxidase: Add 1 drop of Reagent B from the Rabbit UltraAvidin Peroxidase Staining Kit to 10ml PBS in Mixing Bottle 3.
6. Substrate: If using the Leinco Substrate Kit Prod. No. K107 (see page 4) dilute according to kit protocol.

IHC Method
All the steps are carried out at room temperature in moist chambers.
1. For cryostat sections fix in acetone for 10 mins and air dry. Go directly to Step 5.
2. For paraffin sections fix at 35oC for 2-3 minutes.
3. Deparaffinize in xylene for 20 mins.
4. Keep in absolute ethanol for 10 mins.
5. Put in 3% Hydrogen peroxide in methanol for 10 mins. (This step may be deleted if there is no problem with endogenous peroxidase).
6. Wash under running tap water for 10 mins.
7. Put in PBS for 10 mins.
8. Wipe slide and add the blocking serum dropwise onto the slide, covering the section. Incubate for 20 mins.
9. Drain out blocking buffer and add primary antibody. Incubate for 1 hour.
10. Drain out antibody and wash in PBS for 10 mins.
11. Wipe slides and add the biotinylated secondary antibody, incubate for 30 mins.
12. Drain out antibody and wash in PBS for 10 mins.
13. Wipe the slides and add the UltraAvidin-Peroxidase, incubate for 30 mins.
14. Drain out solution and wash in PBS for 10 mins.
15. Add prepared substrate solution and incubate for 15 seconds or until color develops. Leinco's Metal Enhanced DAB substrate kit Prod. No. K107 develops within 10 seconds.
16. Remove excess substrate solution with two washes under running tap water for 5 mins. 17. Counterstain and mount.

(Note: Background staining due to endogenous avidin binding activity can be suppressed with Leinco's Avidin Binding Suppressing Kit Prod. No. K108).

These instructions are for the use of Leinco's Rabbit UltraAvidin Peroxidase Staining Kit for immunohistology, ELISA and immunoblotting. The rabbit primary antiserum/antibody is supplied by the end-user. Reagents supplied are sufficient for at least 1000 tissue sections in immunohistochemical staining or 2000 ELISA or immunoblotting tests. When not in use the reagents should be stored at 2-8^C.

1. Green, M. A.; et al. (1989) "Improved method for immunoperoxidase detection of membrane antigens in frozen sections" J. Clin. Pathol. 42:875-880
2. Giorno, R. A. (1984) "Comparison of two immuno-peroxidase staining methods based on the avidin-biotin interaction" Diag. Immunol. 2:161-166
3. Heyderman, E. (1979) "Immunoperoxidase techniques in histopathology: Applications, methods and controls" J. Clin. Pathol. 32:971-978
4. Guesdon, J. L. et al. (1979) "The use of avidin-biotin interaction in immunoenzymatic techniques" J. Histochem. Cytochem. Vol.27, No.8:1131-1139
5. 5.Hsu, S. M. et al. (1980) "A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies" Am. J. Clin. Pathol. Vol. 75, No.5:73-738
6. Young, P. R. (1989) "An improved method for the detection of peroxidase conjugated antibodies on immunoblots" J. Virol. Methods 24:227-236
7. Peter, J. H. and Baumgarten, H. (Eds.) (1992) Monoclonal Antibodies Springer-Verlag, New York,