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This page covers what is a primary antibody, differences between primary and secondary antibodies, history of antibody development, and assays that utilize antibodies. Boster's antibodies have been cited over 25,000 times.
Primary antibodies are immunoglobulins that can only be bound to a specific targeted antigen . The quality of a primary antibody is usually defined by its affinity and specificity. Affinity is the strength to which it forms the non-covalent bond with the antigen of interests, while specificity is the amount of binding it has with antigens other than its intended antigen, the higher the binding to unintended antigens, the worse the specificity. A good primary antibody can be used to detect, purify and quantitatively measure the antigen of interests.
In biochemistry, cell biology, and histology and various other academic as well as clinical/IVD areas, scientists have long been utilizing antibodies’ unique ability to detect (in other words, bind to) pathogens and immunogens. By attaching a signal generating molecule to the antibody, once the antibody binds to the antigen, we can then measure, either in a visual or quantitative fashion, the existence of the antigen.
"Excellent Caspase-3 antibody from Boster Bio. Easy to use and reliable."
--Postdoctoral Researcher from Duke University Hospital
"This GFAP antibody works perfectly. It did not need several trials to optimize the protocol. I will purchase it again."
--Postdoc from Yale University
"A good PKC Alpha antibody for IP and WB. Works great without optimization. Good quality and ready for publish Ab."
--Research Scientist from University of New England
Secondary antibody is the antibody that binds to the primary antibody. Secondary antibody usually carry a signal generating molecule, so that we can measure the signal and detect the presence and quantity of the antigen bound.
However, attaching the signal generating molecule can require a significant amount of work. It is easier to conjugate these molecules, sometimes also referred to as tags, labels or conjugates, to an antibody that can bind to other antibodies, so that we can avoid having to conjugate every antibody we use. The first antibody that is specifically binding to the antigen is called primary antibody, the second antibody that binds to the primary antibody is called secondary antibody. Primary antibodies are typically unconjugated because it is inconvenient to conjugate every primary with necessary antigens. For some of the commonly used primary antibodies, there are conjugated primary antibodies, again, for convenience reasons.
Validated for IHC, ICC/IF, WB, ELISA, Flow Cytometry
How are antibodies made, important milestones in the primary antibody history, and some famous scientists.
At their origins, all primary antibodies are made from building blocks from immunesystems of the hosts. These building blocks are called complementarity-determining regions (CDRs). CDR is a complex topic which we will not go into in this article so for more information on CDRs you can visit this Wikipedia page.
Traditionally, scientists made polyclonal primary antibodies by immunizing the host animals, typically rabbit, goat, chicken, mice and variety of mammalian and avian species, with antigens, inciting the immune responses which produce antibodies, and extracting (by a method called immune-affinity purification chromatography) the resulting antibodies from the host’s sera or eggs.
In 1975, Georges Köhler and César Milstein developed the monoclonal antibody technology. In 1985, George P Smith developed the method called phage-display, which marks the start of the antibody engineering era where antibody development moves from hijacking hosts’ adaptive immune systems towards rational design.
However the majority of antibodies used today, in the 2020s, are still made from host animals at some point in its production. A truly animal free primary antibody development solution has yet to be invented, primarily because scientists have not found an animal-free mechanism that can replace the somatic maturation of B-cells. Without such mechanism, it is impossible to achieve the same level of affinity and specificity of naturally matured antibodies. This opinion is from the author of this article and is up for debate, if you believe otherwise you can send your arguments to [email protected] or post on our Facebook page.
With the monoclonal technology, people can now fuse the antibody producing cells (Plasma B-cells) with myeloma cell lines making immortal, antibody secreting hybridoma cells. Monoclonal antibodies are derived from clones of the same plasma B cell thus all the antibodies have the same characteristics, including their specificity. Once a good monoclonal antibody clone is discovered, scientists can keep using it without having to worry about consistency issues.
A brief overview of common immunoassays
The Primary Antibodies plus secondary antibody combination is used as the core components, or active ingredients in experiments and assays, or in consumer terms, clinical tests. These assays are in turn, called immunoassays, for the fact that their principles rely on the antibody’s ability to bind to antigens specifically.
Common immunoassays include: Western Blotting (a proteomics assay), immunohistochemistry and immunofluorescence (histopathology tests), ELISA (short for Enzyme linked immunosorbent assay, an enzyme mediated immunoassay used for lipid biopsy, or quantitative measurement of existence of target molecules in biological samples) and flow cytometry, which measures the amount of certain cell types of interests in biological samples.
Because It allows the flexible configuration of immunoassays using the same primary antibody. The primary antibody may work for multiple applications, such as western blotting, a (IHC), immunofluorescence (IF), flow cytometry (FACS) and ELISA. However these applications require different conjugates to visualize/quantify the proteins of interests. This can easily be achieved by conjugating secondary antibodies to different conjugates so that the antigen bound by a primary antibody can appear fluorescently green in one IF experiment and yellow in a different IF experiment and yet resulting in a brown precipitation in a western blot experiment. This flexibility is especially important in immunofluorescence where multiplexing antigens, in other words, showing different antigens with different colors on the same sample, is very important. The primary-secondary antibody system enables dual (or more) labeling of specimens, which allows you to ask more questions with the specimens you have, and provide you more robust answers/data that are contextually connected.
Sometimes it is not enough to simply bind the secondary antibody to signal molecules. This is because sometimes the level of target protein is low and the surface area of the secondary antibody molecule is limited and only a limited amount of conjugates can be covalently conjugated to the secondary antibody, and the amount is not enough. In these cases, signal amplification systems, which are sometimes called detection systems, are used. The first and mostly widely used detection system is the biotin-avidin complex (ABC). The strongest non-covalent bond between molecules known to humanity is the bond between biotin and avidin. Each avidin binds to 4 biotin while Each biotin can bind to 2 avidin forming a Tetrahedron complex. With avidins conjugated to signal molecules (typically Horseradish Peroxide, HRP), the primary-secondary-ABC system allows researchers to locate the proteins of interest with low background and high specificity. Other detection systems were later developed using poly-saccharides and other organic polymers or complexes. Boster Bio’s Super Vision Detection kits is one of those innovative detection systems.
Here are some highlights of special primary antibody formats. Most primary antibodies are plain and unconjugated antibodies, but the ones below are not most primary antibodies.
|Reactivity||Human, Mouse, Rat|
|Applications||IF, IHC, WB|
|Reactivity||Chicken, Human, Mouse, Rat|
|Applications||Flow Cytometry, IF, IHC-P, IHC-F, WB|
|Reactivity||Human, Mouse, Rat|
|Applications||Flow Cytometry, IF, IHC-P, IHC-F, ICC, WB|
|Reactivity||Human, Mouse, Rat|
|Applications||ELISA, IHC, WB|
|Reactivity||Human, Mouse, Rat|
|Applications||IF, IHC-P, WB|
Boster provides mouse and rabbit monoclonal antibodies that have been validated for several application including IHC, ICC/IF, WB, IP, and Flow cytometryFind out more
Known for their high specifity and affinity, hundreds of rabbit monoclonal antibodies that have been tested for several applications are available from BosterFind out more
Here are some quick facts about bosterbio.com
Boster antibodies and ELISA kits are cited over 23,000+ times in publications around the world. According to the big data research company CiteAb.com, Bosterbio is globally the fastest growing antibody company in terms of the number of academic publication citations. For Picokine® ELISA kits specifically, an estimate of 6000+ publications are available. You can find specific citations for ELISA kits and antibodies on each product page.
What life scientists are saying about Boster ELISA kits
Maria Teresa Dell'Anno
This Antibody Works Perfectly!
I used it for IHC on frozen sections at a dilution of 1:500. It did not need several trials to optimize the protocol. No bad things overall. I will purchase it again.
Immunohistochemistry , Spinal cord , Confocal microscope
Verification Of Antibody On Recombinant Protein
We tested the antibody against recombinant GST-tagged BnaA07g20720D protein. As controls, we used another GST-tagged protein GST-MYB25, and GST alone. The antibody was well capable of detecting specifically BnaA07g20720D. We loaded ~100 ng of either GST:BnaA07g20720D, GST:MYB25, or GST alone, and the antibody was very specifically detecting BnaA07g20720D. It did not cross-react at all with the other two proteins. Highly specific. I would highly recommend the antibody for basic western-blot analysis of purified proteins. Probably one can significantly reduce the dilution to 1:10000 to still get a good result. Unfortunately, we did not have the opportunity with good controls to verify how the antibody works on whole plant extracts or for IP experiments.
Western Blot , Recombinant protein expressed in and purified from E. coli , Chemiluminescence
A Good PKC Alpha Antibody For IP And WB
We used Anti-PKC Alpha Picoband™ Antibody for immunoprecipitation experiment from neuronal cells in order to characterize PKC Alpha expression in different conditions. Strong positive band with right mass. Works great without optimization! Good quality and ready for publish Ab
Western Blot , Dorsal root ganglia (neuronal cells) , CyDye
Best Calbindin Antibody For IHC In Mouse Cerebellum
I tried this antibody for Purkinje cell labeling in mice cerebellum using IHC-P. Antigen retrieval - 20min boiling in EDTA based buffer. Permeabilization - 0.3% triton for 15min. Blocking - 10% goat serum for 1hr. Primary antibody - 1:250 overnight at 4 Degree C. Secondary antibody - Fluorescence conjugated secondary antibody at 1:250 for 2hr at RT. I was amazed to see the beautiful dendrites, soma and axons. In my hand, this is the best labeling of Purkinje cells I have ever seen using IHC-P. Beautiful labeling of Purkinje cell soma, dendrites and axons. Best antibody I have used till now. Highly recommended for IHC-P.
Immunohistochemistry , Mice Cerebellum , Fluorescence
Good Zebrafish Antibody For Nlz2
Zebrafish-specific antibody. Used in whole mount IF on 24hpf zebrafish embryos. The signal is not precisely clean, but we do observe nlz2 expression in the ventral region of the retina as expected from in situ hybridization results. This antibody shows some promising results in whole mount applications using 24hpf embryos. Applications with cryosections may be more precise.
Immunohistochemistry , Zebrafish embryos 24hpf , Confocal Microscopy
Awesome Kip2 Antibody From Boster Bio
We've been using this Ab for several months, and it's been working great. We have already ordered several vials of this Ab for detecting podocytes in mice kidney. We used an autostainer machine for the process. With this Ab dilution, and using EDTA for Ag retrieval, we had the least background. Easy to use, works every time. Worth it.
Immunohistochemistry , Mice kidney , DAB chromogen
Quantification Of BDNF Post-Transfection In Primary Neuronal Cells
The kit was used to quantify amount of BDNF produced after transfection with plasmid DNA encoding human BDNF using non viral vector, liposomes. Provided good reliable quantification of BDNF produced. No interference. Good reproducibility. Reliable estimation, good sensitivity. Reasonable price as well. Would purchase again." (Customer’s Tip: "Don't dilute sample, assay directly.
ELISA , Cell lysate and culture medium
Adiponectin Analysis in Mouse Serum and Culture Media
I used this kit to quantify adiponectin adipokine in cell culture supernatant (3T3-L1 cells) and mouse serum (C57BL6 mice on control/ western diet). The goal was to check if western diet treatment reduced serum and tissue adiponectin levels. The results were reasonable and reproducible. Although we had to use two different standard curves for high and low concentration estimations. Performing a standard curve with each sample helped. Reliable estimation, good supplier, reasonable price. Will keep using it." (Customer’s Tip: "Dilute tissue homogenate 1:100, use serum directly.
ELISA , Serum, tissue homogenate
Good TIMP-1 Antibody for Western Blotting
We study neurodegeneration in our lab and I was looking for an antibody for human TIMP-1. The antibody detects a band at 28kD, which is the predicted molecular weight of TIMP-1. Good antibody for TIMP-1, cleaner than other antibodies I have tested. In summary, this antibody is good for human samples and I highly recommend it.
Western Blot , Human NSC cell lysates , ECL chemiluminescence
Peter J. Thompson
This Antibody Gives Strong Membrane Staining
CD47 is broadly expressed in many cell types. This antibody gives strong membrane staining throughout many of the beta cells (Insulin-positive) in the immune cell infiltrated islets of NOD mice at age 12 weeks.
IHC-Paraffin, IHC-cryo , NOD/ShiLtJ mouse pancreas sections, formalin-fixed paraffin embedded , Zeiss Apotome widefield fluorescence microscope
The Signal Was Very Strong And Clean!
Thirty micrograms of whole cell extract of pancreatic tumor cell MIA PaCa-2 were loaded on a 4-12% Bis-Tris gel. The membrane was blotted with 1xPBS + 0.2% Tween-20 + 10% non-fat dry milk. The membrane was incubated with Anti-HMOX1 Antibody (PB9212) at the concentration of 200 ng/ml at room temperature for 3 hours. The membrane was then blotted with HRP-conjugated anti-rabbit secondary antibodies at 4oC overnight. The band was detected using regular ECL with 1 minute exposure. A major band with 32 kD was detected.
Western Blot , Whole cell extract of pancreatic tumor cell MIA PaACa2 , Enhanced chemiluminescence