ELISA

Developing sandwich ELISAs requires careful selection of a matched antibody pair (a detector and capture antibody) that both bind specifically to the target protein. Screening performance of these antibodies as a pair is crucial for their validation. Only antibody combinations that show high specificity, selectivity, and consistent linearity of dilution for the target are used for further development.

We validate the performance of each antibody pair in plasma, serum, or tissue lysates, using spike-recovery experiments to validate antibody selectivity. This method involves ‘spiking’ purified recombinant target proteins into the biological matrix, which should be recovered and detected within -/+ 20% variation (80–120%) of the kit’s expected protein standard signal in the provided diluent. The recovery observed for the spike should be almost identical in both the biological matrix and the standard diluent for a sample matrix to be considered valid for our ELISA kits.
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Linearity studies of sample dilutions are also carried out using sandwich ELISA to ensure that our antibody pairs recognize not only purified recombinant protein, but also native target protein. For this, we measure a standard protein and the native signal of a protein in over a series of dilutions in parallel. If both the standard protein and native signal dilute proportionally, interpolated sample values will have an identical value at all doses tested once corrected for dilution. The concentration of the target protein is determined by multiplying the dilution factor by the calculated concentration (Figure 1).

^{Figure 1. Interpolated concentrations of native PSA in human serum and plasma (citrate) samples.}

^{The concentrations of PSA were measured in duplicates, interpolated from the PSA standard curves and corrected for sample dilution. Undiluted samples are as follows: serum 100% and plasma (citrate) 50%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean PSA concentration was determined to be 630 pg/mL in serum, 1,300 pg/mL in plasma (citrate).}

To further verify antibody specificity for use in ELISA, we test to what degree the antibodies bind to related proteins or family members. This allows us to gauge any cross-reactivity and interference. For example, to confirm the specificity of our CXCL2 antibodies for use in ELISA, we test using CXCL3 and CXCL1, which share 82% and 63% amino acid identity with CXCL2, respectively. In this case, the related family member proteins show similar-to-background absorbance values. To determine species reactivity, we will also check the same protein obtained from different species, ie mouse, rabbit, goat, rat, etc, to confirm that the antibody is specific for the human protein. Acceptable results for us will show less than 5% cross-reactivity.

Recombinant technology is used to produce the antibodies for our antibody pairs and ELISA kits. This means that once developed, our antibody pairs and ELISAs show high batch-to-batch consistency.

Western blot

Antibodies are validated in western blot using lysates from cells or tissues that we have identified to express the protein of interest. Once we have determined the right lysates to use, western blots are run and the band size is checked for the expected molecular weight. We will always run several controls in the same western blot experiment, including positive lysate and negative lysate (if possible, Figure 2).

When possible, we also include knock-out (KO) cell lines as a true negative control for our western blots. We are always increasing the number of KO-validated antibodies we provide. For any new batches of cell lines or antibodies, we run the old stock against the new to ensure the new batch matches our exacting standards.

If the western blot result gives a clear, clean band and we are happy with the result from the control lanes, we proceed to the next validation step.

^{Figure 2: Western blot: Rabbit Monoclonal[MJF-R30-104] to RAB29 ab256527 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 23 kDa in Wild-type U-87 MG ab278079 cell lysates with no signal observed at this size in RAB29 knockout U-87 MG ab306765 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.}

Immunohistochemistry

Immunohistochemistry (IHC) uses antibodies to detect the presence and location of proteins (antigens) within tissue sections. The interaction between the antibody and antigen can be visualized using either a colored enzyme substrate (chromogenic detection) or a fluorescent dye (fluorescent detection). For any given target, our scientists will review the literature, transcriptomics, and proteomics datasets to determine the suitable tissues for antibody validation.

Using our custom-built tissue microarrays (TMAs), we regularly validate our recombinant antibodies against up to 50 different tissue samples from three species (human multi-normal and multi-cancer, mouse, and rat TMAs), each tested in duplicate. This high-throughput testing enables us to examine multiple tissues of varying target expression while ensuring experimental uniformity.

Depending on the target’s complexity, we use cell pellets that either overexpress or are knock-outs for the target of interest, enabling detailed specificity testing. Disease-specific tissues (eg Glioblastoma for EGFRvIII and Alzheimer's disease for APOE4) are employed when necessary.

Our antibodies are routinely IHC validated using fully automated staining platforms like BOND™ RX Research Stainer (Leica®) and DISCOVERY ULTRA system (Roche Diagnostics). Slides are then scanned using NanoZoomer S360 (Hamamatsu Photonics K.K.) for the image analysis.  As part of the antibody validation process, our scientists will analyze the whole slide scans to ensure the antibody meets the sensitivity and specificity criteria, making it suitable for use in IHC.

^{Figures 3 and 4: IHC image of ab109186 staining Olig2 in formalin fixed paraffin embedded human cerebrum stained using DISCOVERY ULTRA system (Figure 3) and BOND™ RX system (Figure 4).}

Immunocytochemistry

Antibodies validated in immunocytochemistry are tested using a combination of immortalized cell lines and/or primary cells isolated from tissue (Figure 5).  Our scientists will review the available literature to identify positively and negatively expressing cells and the subcellular localisation/s of the target protein.

^{Figure 5. ab32127 staining Synaptophysin in primary mouse neurons/glia, DIV14 (prepared from E18 mouse hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. C57EHP) cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab32127 at 0.1µg/ml and ab192757, Mouse mono Anti-PSD95 antibody [K28/43] - Synaptic Marker. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).}

^{Also suitable in cells fixed with 100% methanol (5 min).}

^{Image was acquired with a high-content analyzer (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.}

We always test our antibodies in cells fixed with 100% methanol and 4% formaldehyde, as antigens are preserved and presented differently in each. We also identify optimal cell permeabilization, blocking, and antibody concentration for our testing. Counterstains are also included to help visualize the cells and their subcellular localisations for easier analysis. Where relevant, co-staining antibodies are also used (Figure 5).

Where conjugated secondary antibodies are required to visualize staining, a secondary-only control is always included. A positive control consisting of a different primary antibody of the same species, also expressed by the cells, and the same secondary is also run to confirm that any staining seen is due to the primary antibody.

Where possible, we include knock-out (KO) cell lines as a true negative expression control for our ICC (Figure 6.). We are always increasing the number of KO-validated antibodies in our portfolio.

^{Figure 6. ab195254 staining ALDH1A1 in A549 wildtype cells and ALDH1A1 KO A549 cells. The cells were fixed with 100% Methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4ºC with ab195254 at 1µg/ml (shown in green) and ab190573, Alexa Fluor® 647 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).}

^{Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.}

^{Figure 7. ab288063 staining ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) in untreated, PMA treated and PMA + LP treated HeLa cells. The cells were fixed with 100% Methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4ºC with ab288063 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).}

^{Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.}

^{The image shows increased nuclear staining after 24hr serum starvation followed by treatment with PMA (200nM, 15min) of HeLa cells. The LP treatment then removes all staining of antibody with no phospho ERK1/2 remaining.}

^{Ab184699 (ERK1 + ERK2) was used as a Pan control for ab288063. The results showed nuclear staining on untreated, with no increase after PMA treatment and no reduction after PMA + LP treated in HeLa cells.}

Peptide array

Peptide array is a very high-throughput method of antibody validation that allows us to test the specificity of our antibodies against over 500 peptides at one time (Figure 8). We predominantly use peptide array when we test our histone modification antibodies as it allows us to check cross-reactivity between different modifications.

We use a liquid handler to perform six serial dilutions of the peptides, which are then printed onto nitrocellulose slides in triplicate and used to assess the binding specificity of an antibody to all these peptides simultaneously.

Each nitrocellulose slide that we run contains several essential positive and negative controls to assess antibody specificity. We run old stock batches alongside new antibody test batches for side-by-side comparison. We also run peptide array validation of our own antibodies alongside external antibody batches from competitors to compare the performance of antibodies.

^{Figure 8. ab176916 was tested in peptide array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate). Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as the area under the curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.}

Dot blot

Dot blot was frequently used to validate our histone modification antibodies before we began to use peptide array. Many of the histone modification antibodies on our website are still suitable for use in dot blot, with full validation information provided.

The technique uses a similar principle to peptide array. Serial dilutions of several peptides are plotted onto a nitrocellulose membrane and we check the specificity of our antibody of interest against these control peptides (Figure 9).

Each membrane that we run contains several essential positive and negative controls to assess antibody specificity. When we produce new batches of antibody, we run them against the old batch to ensure specificity. We also run peptide array validation of our own antibodies alongside external antibody batches from competitors to compare the performance of antibodies.

^{Figure 9. Dot blot analysis of AAV8 + AAV3B using ab315827 at 1:2000 (0.5 ug/ml) followed by a Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1:5000 dilution.}

^{Dot blot was performed using 1 ng of each capsid loaded per well.}

^{Anti-AAV9 antibody [HL2368] (Anti-AAV9 antibody [HL2368]ab315818) (1:2000), Anti-AAV9 antibody [HL2370] (Anti-AAV9 antibody [HL2370]ab315820) (1:2000), Anti-AAV9 antibody [HL2374] (Anti-AAV9 antibody [HL2374]ab315823) (1:2000), and Anti-AAV9 antibody [HL2374-IgG1] - BSA and Azide free (Anti-AAV9 antibody [HL2374-IgG1] - BSA and Azide freeab315826) (1:2000) specifically recognize AAV9 capsid.}

^{Anti-AAV8 + AAV3B antibody [HL2383] (ab315827) (1:2000), and Anti-AAV8 + AAV3B antibody [HL2383-IgG1] (Anti-AAV8 + AAV3B antibody [HL2383-IgG1]ab315829) (1:2000) specifically recognize AAV8/3B capsid.}

^{Anti-AAV8 + AAV9 antibody [HL2372] (Anti-AAV8 + AAV9 antibody [HL2372]ab315821) (1:2000) specifically recognizes AAV8/9 capsid.}

^{Anti-AAV5 antibody [HL2476] (Anti-AAV5 antibody [HL2476]ab315831) (1:2000) specifically recognizes AAV5 capsid.}

^{Developed using the ECL technique.}

^{Exposure time: 8min}

Immunoprecipitation

We do not carry out immunoprecipitation (IP) as standard when batch-testing our antibodies; however, we will do this for individual antibodies if requested.

When we validate antibodies for IP, we carry out a standard IP protocol using magnetic beads as we find that these beads give better results in less time. After isolating the protein of interest, we run a western blot consisting of the pulldown sample from the test antibody, the input lysate or supernatant, and a pulldown sample in which an isotype control antibody was used instead of the test antibody to act as a negative. When we do this, we are looking for a clean band at the expected protein size from our IP sample from the test antibody which is enriched compared to the input sample, and no band in the isotype control sample. We are also checking the other lanes of the blot to check for non-specific binding that would appear also in our negative controls (Figure 10).

^{Figure 10: Western blot: antibody description (ab248535) staining at 10,000 dilution, shown in Black. In Western blot, ab248535 was shown to bind specifically to PLCG2. An enriched band was observed at 160 kDa in samples pulled down with ab248535 with no band observed in Isotype control sample. Following IP, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween$®$ 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with a high-sensitivity ECL substrate kit and imaged. Secondary antibodies used was VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution.}

Chromatin immunoprecipitation

ChIP testing is carried out on our histone modification antibodies. After antibody specificity is tested by peptide array, ChIP is used to check that the protein target complexed with DNA can be pulled down using our antibody.

For our ChIP testing, we use chromatin extracted from formaldehyde-cross-linked HeLa cells or mouse NIH/3T3 cells. We carry out ChIP using our standard protocol and check the pulldown's performance via qPCR using a panel of primers for positive and negative control loci known for each histone modification. This gives us a profile of our antibody binding in active and inactive genomic control regions (Figure 11).

^{Figure 11: Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 2 μg of ab4729 (blue), and 20 μl of Protein A/G sepharose beads.}

^{No antibody was added to the beads control (yellow).}

^{The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.}

To pass an antibody, we check that the profile we get from the ChIP-qPCR matches what we expect, ie our acetylated histone modification antibody binds to active regions on the genome (Figure 7). We also test the pulldown strength of our new stock and compare it to previous batches to make sure that the pulldown capability is consistent between batches.

We use several standard controls in every ChIP validation experiment, including an IgG and bead-only negative control. In addition to the positive and negative loci we use to control our q-PCR, we also use a no-template negative control for every validation experiment.

We are currently further developing our ChIP methods and ChIP-validation protocols and are working to validate many more of the antibodies currently in our catalog.

Flow cytometry

When validating antibodies for flow cytometry, we are careful to optimize every step of the staining protocol, including fixation, permeabilization, blocking and washing. Our scientists review the available literature to understand which cell types and conditions are best suited to validate specific antibodies, ensuring compatibility with antibody target and epitope.

We include relevant experimental controls like positive and negative samples, unstained cells, isotype control, viability stain, Fc-receptor blocking, fluorescence minus one (FMO), and single-stain controls. For an FMO control, we stain the sample with all fluorescent conjugates except the one being tested. This shows the contribution of each fluorescent conjugate to the spillover of signal into the channel of interest. Incorporating this control is important to determine the true signal in the channel of interest; in this case, the signal from the conjugate being tested. The extent of non-specific antibody signal can be observed from negative controls included in the experiment. These are generally cellular populations or immortalized cell lines known not to express the antibody target or our selection of KO cell lines when these are available.

Isotype controls allow us to separate background signal from the signal given by specific antibody binding. These controls use primary antibodies matching the isotype of the primary antibody being validated but do not have specificity for the target.

^{Figure 12: Detection of CD20 expression in human PBMCs.Isolated primary human PBMCs were fixed with 4% formaldehyde and permeabilised using saponin. PBMCs were blocked with buffer containing human Fc receptor block and then stained with either isotype control (left plot) or ab322915 (FITC Anti-CD20 antibody [EP459Y]) (right plot). PBMCs were also co-stained for detection of CD19. Events were acquired using the CytoFlex LX flow cytometer and gated on viable lymphocytes. As expected, CD20 expression was detected specifically in CD19+ cells using ab322915.}

Validating antibody specificity with knock-out cell lines

Through our ongoing KO-validation program, we can access a library of validated human KO cell lines to use as a tool for investigating antibody specificity. By employing the CRISPR/Cas9 mechanism for effective gene knockout in a background immortalized cell line, the KO cells provide an excellent model for antibody validation as a negative control with a null expression of the antibody target.
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Genetic KO models are also powerful because they allow us to understand a gene's function by observing its loss-of-function phenotype. We have KO-validated hundreds of antibodies and removed non-specific antibodies from our catalog.

^{Figure 13: IL13RA2 expression in wild-type and knock-out A375 cell line. IHC staining of FFPE a) A375 IL13RA2 +/+ and b)  A375 IL13RA2 -/- cell lines using anti-IL-13 receptor alpha 2 [EPR29921-543]. Positive staining in brown; nuclear hematoxylin counterstain in blue.}

^{Figure 14: Validating antibody specificity in flow cytometry using a human knockout model.Immortalized wild-type (WT) Raji cells or CXCR5 KO Raji cells were blocked with buffer containing human Fc receptor block and stained with isotype control (grey – KO, black – WT) or ab322305 (FITC Anti-CXCR5 antibody [EPR23463-30]) (magenta – KO, green – WT). Events were acquired using the CytoFlex LX flow cytometer. CXCR5 expression was detected only in WT Raji cells (green curve) with no signal (above control) detected in CXCR5 KO Raji cells (magenta curve).}

Consistency testing

To ensure the same, accurate results can be obtained across batches of the same antibody, we perform consistency tests to assess batch-to-batch variation.

In the case of recombinant antibodies, consistency between batches is very high, meaning you are unlikely to need to perform additional optimization procedures (eg titration experiments) between batches. This may not be the case with other non-recombinant hybridoma-produced monoclonal and polyclonal antibodies where the degree of variation and drift is inherently higher.

When available and suitable for assay development, recombinant monoclonal antibodies are favored and provide the best batch-to-batch consistency (Figure 15).​

^{Figure 15}

Biophysical testing enables confirmation of antibody identity at a molecular level, delivering robust, reproducible, and quality results across a large portfolio of products for the best lot-to-lot consistency.​

We use a variety of methods as part of our robust quality control process, including:​

• Liquid chromatography-mass spectrometry (LC-MS) to measure sequence identity and integrity ​
• Dynamic light scattering (DLS) to measure aggregation​
• High-performance liquid chromatography (HPLC) to measure purity