References
48.Engvall, E. and Perlmann, P.O. (1971). Enzyme linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G.
Immunochemistry 8, 871-875.
ELISA
ELISA assays are methods for determination of substances such as peptides, proteins, antibodies and hormones, in which a crucial element of the detection is an antigen-antibody interaction. The acronym stands for Enzyme Linked ImmunoSor-bent Assay. Other names are also ud, such as EIA (Enzyme ImmunoAssay). Most commonly,ELISAs are performed in microtiter plates, which are also called microwell plates. The plates are usually made of polystyrene, which will bind anti-bodies spontaneously. It is this binding that makes ELISAs so easy to design and perform, as first de-scribed by Eva Engvall, et al.48Smaller molecules such as peptides and drugs require chemically ac-tivated ELISA plates such as Maleic Anhydride Ac-tivated Plates (Product #’s 15100, 15110). Some rearchers have found that the binding of anti-bodies to ELISA plates caus some denaturation.This can be solved in veral ways. Either an Fc binding protein, such as Protein A or Protein G,can be coat
ed in the plate, or the antibody may be biotinylated and coated in commercially available Streptavidin Coated Plates (Product #’s 15120,15124). The fact that one reactant of the ELISA is immobilized allows for easy paration of bound and unbound material during the assay. This makes possible the quential reaction of compo-nents, which are added, allowed to react and then washed away.
A key feature in all ELISAs is the fact that one of the reactants is labeled with an enzyme. A conjugation between the component and an appropriate en-zyme is made by any of a number of chemical or other methods. Pierce offers a wide lection of cross-linkers ud to generate conjugates that can
be ud in ELISA. Plea e the Cross-linking Section for additional information. The most com-monly ud enzymes are horradish peroxida (HRP) and alkaline phosphata. Some other en-zymes have been ud, but they have not gained widespread acceptance. The include ß-galactosi-da, acetylcholinestera and catala. When per-forming the ELISA with an HRP conjugate, a large lection of substrates is available that vary in n-sitivity and wavelength. When an alkaline phos-phata conjugate is ud in ELISA, the only com-monly ud substrate is PNPP (p -nitrophenolphosphate). Usually, an antibody is labeled with the enzyme, allowing detection of the antigen against which the antibody is raid. For tho wishing to make their own enzyme-antibody
conjugates, plea refer to the Enzymatic Labeling and Detection Section for further information on the pre-activated enzymes available from Pierce.For Pierce’s complete line of enzyme-labeled anti-bodies, plea e the Antibodies Ordering Sec-tion.
Types of Assays
Figure 7 illustrates a typical “sandwich” assay. This type of assay is called a “sandwich” assay becau the analyte to be measured is bound between the two antibodies–the “capture” antibody and the “detector” antibody. The vast majority of ELISAs performed are of the “sandwich” type, which is nsitive and robust.49
Competitive assays are often ud when the anti-gen is small and has only one epitope, or antibody binding site.50Often the ligand is labeled instead of the antibody (Figure 8). The unlabeled antigen and the labeled antigen compete for binding to the cap-ture antibody.
Both monoclonal and polyclonal antibodies have successfully been ud as the “capture” or the “de-tection” antibody in sandwich ELISA systems. Monoclonals have an inherent monospecificity to-ward a single epitope that allows fine detection and quantification of small differences in a target. Many rearchers u a polyclonal in one side of the “sandwich” assay and a monoclonal in the other.
The ELISA assay can be designed with the u of a condary antibody instead of a labeled primary antibody. Figure 9 is a schematic of how the as-says may be designed. In principle, the incubation step with the labeled primary antibody is replaced by two incubations, first with the primary (unla-beled) antibody, then with the labeled condary antibody.
For ELISA it is important that the enzyme antibody conjugate is of high specific activity. This is achieved when the antibody is affinity purified and the conjugation chemistry is an effective one. All Pierce antibody-enzyme conjugates fulfill the re-quirements. See the Antibodies Ordering Section
for the complete offering.
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“detector”
Figure 7: Typical “Sandwich” Assay.
References
49.Palomäki, P. (1991). Simultaneous u of poly- and monoclonal antibodies as enzyme tracers in a one-step enzyme immunoassay for the detection of
hepatitis B surface antigen. J. Immunol. Methods . 145, 55-63.
50.Rao, P.N. and Taraporewala, I.B. (1992). A nsitive enzyme-linked immunosorbent assay (ELISA) for testosterone. Steroids 57
hodoo
, 154-161.
Enzyme-labeled
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antigen
Antigen
Figure 8: Competitive Assay.
A Typical ELISA Protocol
Coating of antibody or antigen to the ELISA plate:•Dilute the protein to be coated to a
concentration of 10 µg/ml in a buffer
such as PBS (Product #28374) or Car-
bonate-Bicarbonate (Product #28382)•Incubate for up to 20 hours at room
temperature or 4°C
•Block unoccupied sites with a blocker
such as Blocker™ BSA (Product
#’s 37520, 37525) or SuperBlock™
Blocking Buffer (Product
#’s 37535, 37515)
•Store plate for future u (it can be
stored dry if SuperBlock™ Blockers are ud) Table 11 lists the BupH™ Dry Blend Buffers avail-able from Pierce.
kite怎么读Perform Actual Assay:
•Add sample to be tested, and incubate
for one or more hours
•Wash with PBS, 0.05% Tween or
TBS with 0.05% Tween
•Add enzyme-antibody conjugate and
incubate for 1 hour
•Wash again
•Add substrate
Determination of Individual Concentrations and Dilutions
For each assay, individual levels of reagents must be established by “trial and error.” Fortunately, wide ranges are usually acceptable. For example, most ELISA protocols bad on enzyme-antibody conjugates call for u of a 1:5,000 dilution of the conjugate, but they will work equally well at 1:2,000 or 1:20,000. To establish the correct lev-els, checkerboard titrations are performed. A checkerboard titration is simply a single experi-ment in which the concentration of two compo-nents is varied in a way that will result in a pattern (Figure 10). This method may be ud to optimize your r
eagents when performing an indirect ELISA, which us a labeled condary antibody. The one reagent is rially diluted across the plate, and the other reagent is rially diluted down the plate. This design permits you to analyze different con-centrations of the two reagents in each well and to obtain the optimal combination of both reagents. This type of experiment is also called a two-dimen-sional rial dilution.
There are veral important points to consider when coating an ELISA plate. It is important to as-sure that the coating solution is absolutely free of detergents, becau detergents will compete for binding and cau low and/or uneven binding. If you are doing a competitive assay, a lower concen-tration usually must be chon to ensure that the antibody is the limiting factor.
Also, excessive concentrations of coating protein occasionally may lead to less coating (the “hook”effect). After coating, the excess binding capacity (if any) is usually blocked using a blocking buffer. Blocking buffers were discusd earlier in this c-tion.
Many rearchers have found that the enzyme-an-tibody conjugates can yield the best results (low background and high signal) when diluted in the same material that was ud for blocking, with the addition of high quality 0.05% Tween®20 (Product #28320). Figure 11 compares Super-Block™ Bloc
king Buffer with Tween®20 to other proteins for u as conjugate diluents. It has been demonstrated that the Tween®should not be in-cluded during blocking, but should be included as a component of the diluent. Your particular system may be different, and not all coated proteins will maintain adherence when expod to buffers con-taining Tween®20, although most do.
The quality of Tween®20 ud for ELISA may be of importance to your system. Most commercial Tween®20 preparations have been found to con-tain up to 200 mM H2O2. This high concentration of
a powerful oxidant may be the cau of some ELISA artifacts. Pierce offers the highest quality of Tween®20 available. Pierce Surfact-Amps®20
(Product #28320) is free of peroxides and car-bonyls, and it is well-suited for u in ELISA and other immunotechnology applications. Pierce Tween®20 is packaged under nitrogen in aled ampules.
Most ELISA assays can be performed quite rapidly (in less than 1 hour). Some key components of a speedy assay are:
•The u of a plate mixer to create a
vortex in each well at all times
•The u of high quality conjugates.
See the Antibodies Ordering Section
for a listing of the affinity-purified
antibody-enzyme conjugates avail-
able from Pierce
•The u of blocking and other reagents
must be optimized with respect to sig-
nal-to-noi ratio
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c
t
#
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r
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p
t
i
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Table 11: BupH™ Dry Blend Buffers.
1:1
1:2
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1:2
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Figure 10: A Checkerboard Titration.
SuperBlock™
沪江考研Blocking Buffer
with .05%
Tween® 20绵羊猪
3% BSA10% skim milk
0.0
0.5
1.0
O
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4
9
n
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Figure 11: SuperBlock™ Blocking Buffer vs. other proteins for
u as conjugate diluents. SuperBlock™ Blocking Buffer with
.05% Tween®20 is the superior diluent.
ELISA Starter Kits
ELISA Starter Kits are designed for the rearcher who is first starting to work with ELISAs and needs all the reagents except the primary antibody.Pierce ELISA Starter Kits contain a balanced and controlled lection of some of our ELISA prod-ucts, as well as a detailed instruction booklet to get you going on your ELISA in the least possible amount of time. After using one of our ELISA Starter Kits, you will have gained the experience needed to start modifying and optimizing the assay for your particular application. Table 12 lists the ELISA Starter Kits available from Pierce. Kits are available for u with either HRP or Alkaline Phos-phata. The components of the ELISA Starter Kits are:
•Peroxida or Phosphata conjugated condary antibody
控股英文•Buffer Pack consisting of:
BupH™ Modified Dulbecco’s PBS BupH™ Carbonate-Bicarbonate Surfact-Amps ®Tween ®20Blocker™ BSA in PBS •1-Step™ ABT or
•Phosphata Substrate Kit •Complete instructions
Selecting an ELISA Plate
If you are binding an antibody or similar protein, an ELISA plate with high binding for the proteins s
hould be lected. We recommend 400 ng/cm 2as the value for which to look. Also, it is of impor-tance to most rearchers that the CV value of the binding is below 5%. Pierce ELISA plates undergo extensive QC testing and controlled manufacture,and are guaranteed to have CVs less than 5%. If you need to work in an 8-well, strip format, we rec-ommend our ImmunoWare™ 8-Well EIA Strip Plates (Product #15030), which hold the wells so tightly that they do not drop on inversion. The characteristics of the ImmunoWare™ 8-Well EIA Strip Plates are listed in Table 13. The Im-
NOTE: min x __________ indicates minimum cross-reactivity with the indicated rum proteins: Bv = Bovine Hn = Human Hs=Hor Ms=Mou Rt=Rat
t i b o d y i f i c i t y t
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e
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Table 12: ELISA Starter Kits.
