ELISA Kits and Antibody Pairs Support—Getting Started
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We offer full ELISA kits, Reagent Sets and Antibody Pairs. ELISA kits are complete, ready-to-use kits with pre-coated plates, all buffers, capture, and detection antibodies included. Most kits are single plate format, but some are available in 2- or 5-plate formats. Reagent Sets are for researchers who need the core kit components but prefer to make their own buffers and coat their own plates. Antibody Pairs are matched pairs of detection and capture antibodies for researchers who need to process large numbers of samples.
Our ELISA kits can be categorized into several different groups (see Table 2, Page 35 of the Protein Analysis Handbook), based on a number of factors: target protein class, sensitivity, readout method, or ability to detect specific phosphorylation states of the target protein.
- Standard colorimetric ELISA kits use a standard colorimetric readout and allow excellent sensitivity and detection range. Typical sensitivity is <10 pg/mL and standard curve range is around 10-250 pg/mL, although there are some kits with wider ranges.
- Ultrasensitive ELISA kits use a standard colorimetric readout but enable detection and analysis of proteins to levels as low as 0.5 pg/mL. With measurement range of 0.5-20 pg/mL, these kits are especially useful with highly diluted samples.
- Chemi ELISA kits use chemiluminescence detection for high sensitivity (<1 pg/mL) and are highly flexible with a wide measurement range from 0.5 to 2000 pg/mL.
- Phospho ELISA kits enable the specific detection of phosphorylation of key signaling proteins with high specificity, and are often used to supplement western blot results and provide quantitative data.
Running internal controls along with the blanks and the standard curve is an excellent way to monitor the performance of any ELISA kit. Internal controls are samples containing known amounts of the analyte being measured with the kit. A key attribute of internal controls, and what differentiates them from the samples of the standard curve, is that they duplicate the composition of your actual samples as closely as possible. This is important because your samples may contain components that affect detection of the analyte differently than does the Standard Diluent provided in the kit. In other words, internal controls allow you to determine if assaying the same amount of analyte in the Standard Diluent (the standard curve) and in the sample matrix (internal controls) gives the same results. It’s also important to remember that internal controls give you confidence in the accuracy of your results. Internal controls help ensure that the assay is performing consistently from assay to assay, every time you run it.
One source of internal controls is naturally occurring samples (such as serum or plasma) containing known amounts of the analyte you want to measure. Such positive control samples can be run as-is, or they can be diluted to various known concentrations, preferably with a naturally occurring negative sample of the same type. It’s a good idea to run at least 2 internal controls, if you can. One usually has an analyte concentration between the lowest point on the standard curve and the curve’s midpoint, while the other has a concentration between the midpoint and the highest concentration. Some researchers also run an internal control that falls close to the midpoint of the curve.
If you don’t have access to naturally occurring controls, you can prepare them yourself. Let’s say that you will be measuring interleukin-6 (IL-6) in human serum samples with an ELISA kit, Cat. No. KHC0061. First you will need to obtain some pooled normal human sera, which will be used as the sample matrix for your internal controls. Preferably, the IL-6 content of this serum should be negligible (but known) or too low to measure. Next you should add known amounts of human IL-6 to this serum to create the internal controls, using the human IL-6 standard provided in the kit. After you prepare the standard curve as described in the product manual, add some of the leftover concentrated IL-6 standard to the serum matrix. As described above, you can prepare controls with high and low IL-6 levels, or high, medium, and low, if you have enough wells in your ELISA plate.
Even if your sample type is not serum and you’re not measuring IL-6, follow the general procedure outlined above to prepare your internal controls with your protein and sample matrix of interest. A valuable resource describing Spike and Recovery and Linearity of Dilution assays can be found here. If you need more help, please contact Technical Support at email@example.com.
The chromogen blank and zero standard provide you with different indicators of assay performance. The chromogen blank—comprising only chromogen and stop solution—should have an A450 of <0.03. The A450 of the chromogen blank can be used to blank your plate reader and, in any case, it should be subtracted from all other A450 values you obtain from the plate.
If the chromogen blank is higher than ~0.03, you should check the color of the chromogen reagent straight from the bottle. It can range from clear and bluish to clear and slightly yellow. If it is a stronger blue color, then it is most likely contaminated and should not be used. The most frequent cause of contamination is transfer of unused chromogen solution back into the bottle. The chromogen solution should always be transferred to a clean reservoir in the first place.
The other blank wells that you run are the zero standard wells, which are sometimes referred to as the “zero wells”. The A450 of the zero standard wells in your assay should be <0.35, and preferably lower than this. Although the zero standard A450 value should be low, it is usually higher than the chromogen blank. It serves as the critical first point on the standard curve, so if the zero standard value is too high, it reduces the dynamic range covered by the standard curve. Expected values for the zero standard are provided as part of typical data in the kit manual. If the zero standard value is higher than expected, this usually indicates that there is some other problem with the ELISA.
We strongly recommend that you include both the chromogen blank and the zero standard when you run an ELISA. Since we recommend that all blanks, standards, controls, and samples be tested in duplicate, these blanks take up only 4 wells. If you are short of wells for samples, you can omit the highest standard in the standard curve as well as the next-highest one, if absolutely necessary. You should still make all of the standard dilutions as instructed in the manual, but don't add the one(s) that you're omitting to the plate. This recommendation is based on the assumption that the A450 values for your samples will still fall on a standard curve drawn with the remaining points. If they do not, then you will need to run the assay again with all of the standards or dilute the samples so that they fall on the abbreviated standard curve.
Yes. However, because the sample characteristics can influence your ELISA results, you need to consider effects of the sample “matrix”. The matrix is the liquid environment in which the target analyte is found, and its composition can influence the results of the ELISA. In particular, sample types containing low protein concentrations usually have the most effect. The quantitation of analytes in low-protein samples such as cerebrospinal fluid (CSF), tissue culture supernatant, bronchoalveolar lavage (BAL) fluid, and urine may produce results that are not truly comparable with those derived from our standard curves. This is because our standard curves are typically prepared with a serum-based diluent as the matrix (Standard Diluent).
Sample matrices with low protein concentrations should be modified to more closely resemble the Standard Diluent. Our researchers have found that addition of just a single protein component such as bovine serum albumin (BSA) to low-protein samples is not sufficient to adequately replicate the Standard Diluent. We’ve found that serum, in particular, is a key component.
For CSF and similar low-protein sample types, we adjust the sample matrix so that it more closely resembles the Standard Diluent. To do this, we dilute the samples 1:4 (one part sample plus three parts Standard Diluent, provided in the kit). This diluent is a suitable serum-containing, protein-based matrix. You can also use serum pooled from normal donors in place of Standard Diluent. If pooled serum is used, you need to know how much of the analyte is already present in it. This is so that the appropriate correction factor can be applied to your results.
Sometimes you need to replicate the composition of your sample type for use as a negative control or as a diluent for preparing samples for an ELISA. Pooled serum or plasma from normal donors, or unconditioned cell culture medium with or without added serum, is easy to obtain. When the sample is CSF, you may want to use artificial CSF, which has the following composition: 129 mM NaCl, 3 mM KCl, 1.25 mM NaHPO4, 1.8 mM MgSO4, 1.6 mM CaCl2, 21 mM NaHCO3, and 10 mM glucose, pH 7.4 (Paris D et al. (2002) Prostaglandins Other Lipid Mediat 70(1-2):1-12 [PMID: 12428674]).
We would recommend using the buffers and specific diluents provided in our ELISA kits. All of them are optimized for use in these kits. We cannot guarantee that our kits will perform as advertised if any other buffers or components are used. Also, please don’t use reagents from one type of kit when you are running a different one. Many of the reagents we provide are kit- and even lot-specific. If you need more help, please contact Technical Support at firstname.lastname@example.org.
All ELISA kits are provided in the sandwich ELISA format with capture antibody already coated onto a 96 well plate. Typical detection uses a biotinylated detection antibody followed by Streptavidin-HRP and HRP substrate. Most kits are available as single 96-well plate kits, some are available as 2- and 5-plate kits. Kits typically contain:
- 96 Well Strip Plate coated with capture antibody
- Standard protein
- Wash buffer, 10x
- ELISA buffer/Diluent, 10x
- Detection antibody (in most kits, biotinylated)
- HRP Reagent (either secondary antibody or streptavidin conjugated to HRP)(100x)
- Substrate (usually TMB)
- Stop solution
- Adhesive plate covers
Reagent Sets contain Capture Antibody, Detection Antibody, Recombinant Standard, HRP Conjugate, TMB Substrate and Stop Solution. Each contains enough reagents to process five 96-well plates. Reagent Sets are included in the main list of ELISA kits (search by “Reagent Set”).
Antibody pair kits contain capture antibody, detection antibody, recombinant standard and HRP conjugate. Each contains enough reagents to process forty 96-well plates. A list of Antibody Pair Kits is available by target.
Each Antibody Pair kit contains capture (coating) antibody, biotinylated detection antibody, recombinant standard, and streptavidin-HRP. Other reagents required are listed in the Antibody Pair manual included with the kit, and can also be purchased separately (Antibody Pair Buffer Kit, Cat. No. CNB0011; 5X Assay Buffer, Cat. No. DS98200; etc.). The manual also provides a specific procedure and illustrates an example of a standard curve that can be obtained when the specific procedure is followed.
A general procedure is summarized here:
1) Coat the microplate with diluted capture (coating) antibody overnight at 2–8°C; wash the plate.
2) Incubate the standards or samples in the coated microplate; wash the plate.
3) Incubate diluted biotinlyated detection antibody in the plate; wash the plate.
4) Incubate streptavidin-HRP in the plate for 15–45 min; wash the plate.
5) Incubate the plate with TMB substrate for 10–60 min, and then stop the reaction with Stop solution.
6) Read the microplate at 450 nm.
We recommend determining optimal buffer formulations, concentrations, and incubation times for individual applications.
In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), all of the wells of the 8-well strips provided are coated with a goat polyclonal anti-FITC antibody, which serves as a general capture reagent. When you add the individual FITC-labeled, subclass-specific monoclonals to the wells (step 2 in the assay procedure on page 2 of the manual), they bind to the goat capture antibody. This second layer of the ELISA sandwich now performs the subclass-specific capture function. When you add samples (i.e., serum, standards, and controls) to the wells, any human IgG present in the samples will bind to the subclass-specific antibodies that are captured on the plate via their FITC labels. The subclass-specific detection is enabled by each of these subclass-specific mouse monoclonal anti–human IgG antibodies provided in the kit. The actual subclass detection occurs after you add the HRP-labeled anti–human IgG antibody and the TMB chromogen solution to the wells. The amount of each IgG subclass is determined separately in its own set of wells.
An example of one of these subclass-specific sets is shown under step 1 in the assay procedure on page 2 of the manual. In this case, the setup shown is for IgG1, but if you wanted to measure only IgG4, for example, you would follow the same setup. However, instead of loading these wells with mouse anti–human IgG1, you would use the mouse anti–human IgG4 instead. If all you want to detect is IgG4, the rest of the wells in the plate can be used for other samples instead of additional standard curves for the other subclasses and their respective samples. However, we suggest running a standard curve for the IgG subclass of interest on each plate that you prepare, and each time you run the assay.
The control in the kit consists of lyophilized human serum, and the human IgG standard provided contains all four subclasses at the concentrations indicated on the lot-specific manual. Even though you may want to measure only 1 or 2 subclasses, you'll be using a standard that contains all of them. The other 3 subclasses don't interfere with detection of IgG3, for example, because the capture antibody is IgG3-specific. The detection antibody in the kit is an HRP conjugate of an anti–human IgG that detects all of the subclasses equally effectively and detects all of the human IgG captured in the wells.
Note that the antibody-coated plates in the kit come as 8-well strips that you snap into the frame provided. You do not have to run an entire plate or both plates at one time. Store any unused 8-well strips at 2–8°C and keep them dry. Unused wells in individual strips should be sealed securely to prevent the entry of moisture while running the assay.
In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), Part Numbers 50270HK, 50271HK, 50272HK, and 50273HK are monoclonal antibodies with the clone numbers HP6069, HP6002, HP6047, and HP6023, respectively. These are the clones referred to in the IUIS/WHO study that we cite (Jefferis R et al. (1985) Immunol Lett 10:223–252 [PMID: 3899923]). HP6069 is an IgG1κ with 100% specificity for the human IgG1 Fc; it has a pI of 6.4 (6.3–6.8). HP6002 is an IgG1 with 100% specificity for the human IgG2 Fc; it has a pI of 7.1 (6.8–7.4). HP6047 is an IgG1 with 100% specificity for the IgG3 hinge region; it has a pI of 6.6 (6.5–6.7). HP6023 is an IgG3κ with 100% specificity for human IgG4 Fc, but it cross-reacts by about 18% with IgG3; it has a pI of 7.7 (7.5–7.9). In Cat. No. 99-1000, each of these mouse anti–human IgG subclass antibodies is labeled with FITC, which is necessary for the capture step.
Three of these subclass-specific antibodies are sold as stand-alone, unlabeled products: Cat. No. A10630 (HP6069), Cat. No. 05-3500 (HP6002), and Cat. No. 05-3600 (HP6047). Clone HP6023 is only available biotinylated (Cat. No. MH1542) and conjugated to HRP (Cat. No. MH1742).
In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), the human IgG Subclass Standard is Part Number 50287HK. Each batch of 50287HK is calibrated against a WHO reference standard designated 67/97. More details about this standard are in the abstract from Klein F et al. (1985) Clin Chem Acta 150 (2):119–127.
No. A customer notified us early in 2014 that Cat. No. 99-1000 did not detect IgG subclasses from cynomolgus monkeys. As far as other nonhuman primates are concerned, we do not know if this kit will work with samples from these animals.
Phosphospecific ELISA Kits
Both types of ELISA kits capture total protein, regardless of its phosphorylation state, within the wells of a plastic 96-well plate. This is done by coating the wells with a “pan-antibody” that does not distinguish between the phosphorylated and non-phosphorylated forms of a protein and does not block the phosphorylation site to be studied. In addition, a phosphospecific ELISA kit quantifies the amount of that same protein that is phosphorylated on one or more specific amino acids. Instead of a second pan-antibody for detection, this assay uses an antibody that specifically recognizes an epitope that is only present on a protein when it is phosphorylated specifically (i.e., it is phosphospecific).
We recommend running the total and phosphospecific ELISAs simultaneously with the same samples. If this is not possible, make sure to test the same samples with both kits as soon as possible.
The results of our total ELISAs are given in pg/mL of sample, or sometimes ng/mL. This measurement is always given in mass units because standards of known mass are used to prepare the standard curve. The results of the phosphospecific ELISAs are given in “units”, which we do not relate to a particular mass of protein. We use units because it is difficult to precisely know the efficiency of a particular phosphorylation reaction, and therefore the ratio of phosphorylated to unphosphorylated protein, in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phosphospecific ELISA and are described within the product manual that accompanies each kit.
For example, a typical unit description would be “1 unit = the amount of FAK [pY397] derived from 300 pg of auto-phosphorylated FAK protein”. Since there is no guarantee that the FAK in our standard preparation is 100% phosphorylated, we refrain from making the statement that this corresponds to 300 pg of phosphorylated FAK. Instead, we validate a large batch of phosphorylated protein and use this to develop our unit definition and standard curve for our original assay. Subsequent preparations of our protein standards are normalized to the original batch of protein to ensure that our unit definitions remain constant from lot to lot.
In order to evaluate phosphorylation levels, we report comparative levels of protein phosphorylation in units of phosphoprotein per pg or ng of total protein. The total ELISA kit quantifies the mass of protein per sample, and the phosphospecific ELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) of various samples are similar or different.
Example: Two samples are tested for total CREB and CREB [pS133]
Sample 1 results: The total assay (KHO0231) shows 100 pg/mL of CREB in the sample. The phosphospecific ELISA (KHO0241) shows 50 units/mL of CREB [pS133]. In this sample, CREB is phosphorylated at serine 133 to the level of (50 units/mL)/(100 pg/mL) = 0.5 units/pg of total CREB.
Sample 2 results: The total assay shows 95 pg/mL of CREB in the sample. The phosphospecific ELISA results show 5 units/mL of CREB [pS133]. In this sample, CREB is phosphorylated at serine 133 to the level of (5 units/mL)/(95 pg/mL) = 0.053 units/pg of total CREB. When you compare sample 1 with sample 2, you see a 10-fold difference in the level of phosphorylation of CREB at serine 133, even though the amount of total CREB protein is nearly unchanged.
Our phosphospecific ELISA kits have several advantages, including ease of use and increased sensitivity. Phosphospecific ELISA kits are typically 2–10 times more sensitive than western blots, so they are particularly useful for the detection of “low-expressing” proteins or for small sample sizes. In addition, with the use of the recombinant standards provided in the kit, phosphospecific ELISAs provide quantitative results without having to perform densitometry.
For Research Use Only. Not for use in diagnostic procedures.