Tips To Validate The Antibodies

The antibodies are one of the most widely used tools in research laboratories to identify, isolate or block various molecules. Due to the specificity assumed, it is essential to validate the antibodies for use in each of the techniques in which they are going to be applied, since, as we have seen on other occasions, an antibody can be very specific, for example in an ELISA immunoassay , and yet give a very weak signal in other types of tests such as Western Blot .

Validating the antibodies implies testing the following aspects:

  • Antibody specificity in the selected technique
  • Affinity for antigen
  • Reproducibility between trials

In this post we collect some tips to guide you when validating the antibodies and defining their specificity.

Main Problems With Antibodies, How To Solve Them?

The most common problems that arise from the use of antibodies are cross-reactivity, variability between batches and their application in an incorrect technique.

1.- CROSS REACTIVITY

  • Problem: Antibodies not only bind to the target antigen, but also to other proteins present in the sample.

2.- VARIABILITY BETWEEN LOTS

  • Problem: It is more frequent in the case of polyclonal antibodies, since the same antibody produced in different animals could work slightly differently.

3.- WRONG APPLICATION

  • Problem: The different techniques and experimental conditions in which each one is carried out can modify the folding of the target protein, and consequently, its ability to bind to the antibody.

To avoid any of these problems, it is vitally important to validate the antibodies before use.

But how do you do it? Pay attention to the trials we list below.

Assays To Validate The Antibodies

Antibodies can be validated by a multitude of functional tests, each of which has its advantages and disadvantages:

ELISA

  • Pros:
    • Quantitative test, allows confirmation of sensitivity.
    • High performance assay, a high number of samples can be processed simultaneously.
  • Cons:
    • It does not determine whether the antibody can lead to cross reactivity.

WESTERN BLOT (WB)

  • Pros:
    • Qualitative test, easy and simple to carry out.
    • Ideal for denatured proteins.
  • Cons:
    • Difficult to optimize.
    • It takes a lot of time.
    • Only a few antibodies can be tested at the same time.

IMMUNOHISTOCHEMISTRY (IHC) / IMMUNOCYTOCHEMISTRY (ICC)

  • Pros:
    • Relatively low-cost techniques.
    • Qualitative test.
  • Cons:
    • Difficult to standardize.
    • The accessibility of epitopes can vary in fixed tissues.
    • It does not determine whether the antibody non-specifically recognizes other proteins with the same cell location.

FLOW CYTOMETRY (FC)

  • Pros:
    • High performance assay, a high number of samples can be processed simultaneously.
    • Easy to optimize.
  • Cons:
    • It does not determine whether the antibody can lead to cross reactivity.

KNOCKDOWN siRNA

  • Pros:
    • KnockDown cell lines can be used in all assays: WB, IHC / ICC, FC …
  • Cons:
    • KnockDown is transitory.
    • Difficult to optimize.

KNOCKOUT CELL LINES (KO)

  • Pros:
    • They guarantee that there will be no expression of the target gene, so they are excellent negative controls, guaranteeing the specificity of the antibody.
    • They can be used in all tests: WB, IHC / ICC, FC …
  • Cons:
    • KnockOut cell lines against specific genes are not always viable.

MASS SPECTROMETRY (MS)

  • Pros:
    • High performance assays, a high number of samples can be processed simultaneously.
  • Cons:
    • Requires the use of a mass spectrometer and qualified personnel.
    • Difficult to optimize.

Other Considerations To Take Into Account To Validate The Antibodies

1.- The choice and preparation of cell lines or tissue samples , using the corresponding positive and negative controls, and taking into account whether the antibody recognizes the protein in its native or denatured form.

2.- The protocols , paying special attention and optimizing the incubation times individually (very short periods can affect sensitivity, and on the contrary, excessive incubation times could result in background noise problems), dilutions of work, blocking conditions, etc.

3.- The choice of buffers , determining the optimum in each case.

Ionic Channels And Cancer Immunology

The ion channels are membrane proteins that control the passage of ions therethrough in response to different stimuli. The dysregulation and / or dysfunction of these ion channels is related to a multitude of diseases, including cancer.

Cancer development depends on the effectiveness of the immune system in destroying tumor cells, and the key steps in this process are regulated by various ion channels .

These channels can affect the activation of immune cells and their toxicity against cancer, making them a more than interesting target for research and development of new antitumor therapies.

The Role Of Ion Channels In Cancer Immunology

Tumor cells are recognized by the immune system through the specific antigens they express on their surface, and through joint and integrated action between innate and adaptive immunity mechanisms, these cancer cells are killed. But some of them are capable of developing mechanisms to reduce their immunogenicity, and therefore, not to be recognized by the immune system and thus avoid their eradication.

Strategies to enhance anti-cancer immunity could be directed towards increasing efficiency in antigen presentation, recognition and elimination of tumor cells by the immune system, or inhibition of tumor-induced immunosuppression. Ionic channels play a fundamental role in any of these processes, as the following examples show:

  • Lysis of tumor cells by CD8 + cytotoxic T cells is mediated by Ca2 + Kv1.3 and KCa3.1 ion channels.
  • Lack of functional Ca2 + channels inhibits the development of regulatory T cells.
  • Blocking the KCa3.1 ion channels increases cytotoxicity and lysis of tumor cells by NK cells.

For all these reasons, the network of ion channels present in immune cells that regulate both innate and adaptive immune responses are highly interesting targets in the development of new therapeutic tools against cancer.

Tumor Processes And Ion Channels

Some of the tumor processes in which the active participation of ion channels has been described are the following:

  • Cell cycle progression and proliferation

Ionic transport is an integral part of the regulation of cell cycle progression and proliferation.

  • Apoptosis

Ionic fluxes are also crucial in the regulation of programmed cell death pathways.

  • Metabolism of tumor cells and tumor microenvironment

The cellular microenvironment usually presents different physical-chemical characteristics than most normal tissues and contains different types of cells in addition to tumor cells. Ionic channels are essential in the interaction between tumor cells and their microenvironment.

  • Tumor cell motility and invasiveness

The motility and invasion of tumor cells, the main steps in metastasis, require the functional integration of several processes, almost all of them regulated by ion channels.

Antibodies For Ion Channel Research

Specific antibodies against ion channels are a fundamental research tool for the study of cancerous phenomena of proliferation, migration and metastasis, angiogenesis and resistance to apoptosis.

Here we leave you a selection of antibodies for the investigation of ion channels (IC)  from the Genetex company .

1.- PROLIFERATION

C. I.AntibodiesApplicationsReference
Shaker-like K + channelsKv1.1 antibodyICC / IF, WBGTX54875
Kv1.5 antibodyICC / IF, IHC, IP, WBGTX54837
EAG-related K + channelsKv11.1 antibodyICC / IF, IHC, IP, WBGTX54776
Kv11.1 antibodyICC / IF, IHC, IP, WBGTX54785
Kv11.1 antibodyFACS, ICC / IF, IHC, IP, WBGTX54789
Ca2 + activated K + channelsKCA3.1 / KCNN4 antibodyICC / IF, IHC, IP, WBGTX54786
TRPTRPC6 antibodyFACS, ICC / IF, IHC, IP, WBGTX54757
TRPM7 antibodyICC / IF, IHC, IHC-P, WBGTX54865
TRPM8 antibodyICC / IF, IHC, IHC-Fr, IP, WBGTX54866
P2XP2X7 antibodyICC / IF, IHC, IHC-P, IP, WBGTX54794
SOCsORAl1 antibodyFACS, WBGTX54826

2.- MIGRATION AND METASTASIS

CIAntibodiesApplicationsReference
EAG K + channelsKv10.1 antibodyICC / IF, IHC, WBGTX54788
Kir channelsKir3.1 / GIRK1 antibodyICC / IF, IHC, IP, WBGTX54831
TRPTRPM7 antibodyICC / IF, IHC, IHC-P, WBGTX54865
TRPM8 antibodyICC / IF, IHC, IHC-Fr, IP, WBGTX54866
TRPV1 antibodyICC / IF, IHC, WBGTX54762
P2XP2X7 antibodyICC / IF, IHC, IHC-P, IP, WBGTX54794
SOCsORAl1 antibodyFACS, WBGTX54826

3.- TUMORAL ANGIOGENESIS

CIAntibodiesApplicationsReference
TRPTRPC6 antibodyFACS, ICC / IF, IHC, IP, WBGTX54757
SOCsORAl1 antibodyFACS, WBGTX54826

4.- RESISTANCE TO APOPTOSIS

CIAntibodiesApplicationsReference
TRPTRPA1 antibodyICC / IF, IHC, IP, WBGTX54765
P2XP2X7 antibodyICC / IF, IHC, IHC-P, IP, WBGTX54794
SOCsORAl1 antibodyFACS, WBGTX54826

In addition to GENETEX , other of our represented houses also offer specific antibodies against ion channels . You can check the list of each of them by clicking on the links that we leave you below:

COVALAB (+ info)

  • Antibodies to COVALAB ion channels

LSBIO (+ info)

  • Antibodies to LSBIO ion channels

PROSCI (+ info)

  • Antibodies to PROSCI ion channels

ST. JOHN´S LAB (+ info)

  • Antibodies against ion channels of ST JOHN´s LAB

ORIGIN (+ info)

  • Antibodies to ion channels OriGene
Differential Dynamics of the Maternal Immune System in Healthy Pregnancy and Preeclampsia.

Differential Dynamics of the Maternal Immune System in Healthy Pregnancy and Preeclampsia.

Preeclampsia is one of the most excessive being pregnant points and a primary set off of maternal demise.

However, early prognosis of preeclampsia stays a scientific downside. Alterations in the common immune variations wanted for the repairs of a healthful being pregnant are central choices of preeclampsia.

However, prior analyses primarily centered on the static analysis of select immune cell subsets have provided restricted information for the prediction of preeclampsia. Here, we used a high-dimensional mass cytometry immunoassay to characterize the dynamic changes of over 370 immune cell choices (along with cell distribution and purposeful responses) in maternal blood all through healthful and preeclamptic pregnancies.

We found a set of eight cell-specific immune choices that exactly acknowledged victims successfully sooner than the scientific prognosis of preeclampsia (median area beneath the curve (AUC) 0.91, interquartile differ [0.82-0.92]).

Several choices recapitulated beforehand acknowledged immune dysfunctions in preeclampsia, much like elevated pro-inflammatory innate immune responses early in being pregnant and impaired regulatory T (Treg) cell signaling.

The analysis revealed further novel immune responses that had been strongly associated to, and preceded the onset of preeclampsia, notably irregular STAT5ab signaling dynamics in CD4+T cell subsets (AUC 0.92, p = 8.0E-5).

These outcomes current a world readout of the dynamics of the maternal immune system early in being pregnant and lay the groundwork for determining clinically-relevant immune dysfunctions for the prediction and prevention of preeclampsia.

Differential Dynamics of the Maternal Immune System in Healthy Pregnancy and Preeclampsia.
Differential Dynamics of the Maternal Immune System in Healthy Pregnancy and Preeclampsia.

Impact of the Gastrointestinal Microbiome in Health and Disease: Co-evolution with the Host Immune System.

Microbes inside the gastrointestinal tract speak with each other and with the host, which has profound outcomes on nicely being and sickness enchancment.

Only now, it is turning into apparent that how and as soon as we buy our private distinctive assortment of “gut microbes” and moreover how we choose to take care of them is essential to our nicely being.

Helicobacter pylori is the commonest bacterial an an infection worldwide, colonizing spherical half of the world’s inhabitants, and is the predominant hazard concern for gastric adenocarcinoma. More not too way back, it has moreover been confirmed to have some helpful outcomes in phrases of defending in direction of the enchancment of totally different sicknesses.

Here, we analysis the current knowledge on how H. pylori has fashioned gastrointestinal microbiota colonization and the host immune system with explicit think about the impression of H. pylori on the quite a few microbiome niches of the gastrointestinal tract.

We speak about how the presence of H. pylori influences the physiology of three predominant areas inside the gastrointestinal tract-specifically the oesophagus, stomach and colon.

We pay particular consideration to the place of H. pylori beneath energy inflammatory conditions along with the enchancment of most cancers. With elevated incidence of sicknesses much like eosinophilic oesophagitis, oesophageal adenocarcinoma and squamous cell carcinoma being attributed to the decline in H. pylori, their sickness pathogenesis in delicate of altering H. pylori colonization might be talked about.

Understanding the Holobiont: How Microbial Metabolites Affect Human Health and Shape the Immune System.

Understanding the Holobiont: How Microbial Metabolites Affect Human Health and Shape the Immune System.

The human gastrointestinal tract is populated by a various, extremely mutualistic microbial flora, which is called the microbiome.

Disruptions to the microbiome have been proven to be related to extreme pathologies of the host, together with metabolic illness, most cancers, and inflammatory bowel illness. Mood and habits are additionally inclined to alterations in the intestine microbiota.

A very placing instance of the symbiotic results of the microbiome is the immune system, whose cells rely critically on a various array of microbial metabolites for regular growth and habits.

This contains metabolites which are produced by micro organism from dietary parts, metabolites which are produced by the host and biochemically modified by intestine micro organism, and metabolites which are synthesized de novo by intestine microbes.

In this overview, we spotlight the function of the intestinal microbiome in human metabolic and inflammatory ailments and focus specifically on the molecular mechanisms that govern the gut-immune axis.

Understanding the Holobiont: How Microbial Metabolites Affect Human Health and Shape the Immune System.
Understanding the Holobiont: How Microbial Metabolites Affect Human Health and Shape the Immune System.

A cross-sectional examine on dental surgeons’ immune standing towards hepatitis B virus in the Public Health System.

Hepatitis B is a extreme public well being downside. The foremost world well being facilities have mentioned it because of its pandemic proportion, excessive pathogenicity and infectivity.

This examine aimed to find out the immunization profile of dental surgeons – towards hepatitis B virus (HBV) in the Public Health System (PHS) via the detection of anti-HBsAg antibodies by immunochromatography and associations with dental surgeons’ social and instructional profile, components associated to skilled data and practices.

This is a cross-sectional examine on the prevalence of vaccination and immune standing to HBV in dentists of Sao Paulo State PHS.

Data assortment occurred in three levels: questionnaire software, evaluation of adhesion to the vaccination protocol and anti-HBsAg checks.

Statistical evaluation used the Bivariate Analysis and the Binary Logistic Regression. From the complete of 219 interviewees, 74.9% reported having acquired three doses of the vaccine, however 35.6% weren’t resistant to HBV.

The dependent variable was related to years in the public service (years) (OR [Odds Ratio]=1.04; 95% CI 1.00-1.08); data on the topic (OR=6.93; 95% CI 1.39-34.40); incorrect reply regarding the etiological agent of the illness (OR=2.60; 95% CI 1.30-5.22); ignorance on the variety of vaccine doses which were administered (OR=3.43; 95% CI 1.14-10.30); and lower than three doses of the vaccine in the immunization schedule (OR=8.76; 95% CI 3.50-21.91).

A substantial portion of execs non-immune to the HBV have been discovered. We concluded that data, years of apply and completion of the vaccination schedule (three doses) affected dental surgeons’ immune standing to HBV.

Single-cell transcriptomics to explore the immune system in health and disease.

Single-cell transcriptomics to explore the immune system in health and disease.

The immune system varies in cell sorts, states, and places. The advanced networks, interactions, and responses of immune cells produce various mobile ecosystems composed of a number of cell sorts, accompanied by genetic range in antigen receptors.

Within this ecosystem, innate and adaptive immune cells preserve and defend tissue perform, integrity, and homeostasis upon modifications in useful calls for and various insults. Characterizing this inherent complexity requires research at single-cell decision.

Recent advances resembling massively parallel single-cell RNA sequencing and refined computational strategies are catalyzing a revolution in our understanding of immunology.

Here we offer an summary of the state of single-cell genomics strategies and an outlook on the use of single-cell methods to decipher the adaptive and innate parts of immunity.

Single-cell transcriptomics to explore the immune system in health and disease.
Single-cell transcriptomics to explore the immune system in health and illness.

Interactions between the microbiota, immune and nervous methods in health and illness.

The various assortment of microorganisms that inhabit the gastrointestinal tract, collectively known as the intestine microbiota, profoundly influences many points of host physiology, together with nutrient metabolism, resistance to an infection and immune system improvement.

Studies investigating the gut-brain axis display a crucial function for the intestine microbiota in orchestrating mind improvement and conduct, and the immune system is rising as an essential regulator of those interactions. Intestinal microbes modulate the maturation and perform of tissue-resident immune cells in the CNS.

Microbes additionally affect the activation of peripheral immune cells, which regulate responses to neuroinflammation, mind damage, autoimmunity and neurogenesis.

Accordingly, each the intestine microbiota and immune system are implicated in the etiopathogenesis or manifestation of neurodevelopmental, psychiatric and neurodegenerative ailments, resembling autism spectrum dysfunction, melancholy and Alzheimer’s illness.

In this overview, we talk about the function of CNS-resident and peripheral immune pathways in microbiota-gut-brain communication throughout health and neurological illness.

Coxiella burnetii – how deadly could be the Q fever?

  1. What is this Coxiella pathogen?

Coxiella burnetii is a Gram-negative bacterium that infects animals and humans causing the Q fever (query fever). This is a strictly intracellular pathogen that encodes several basic proteins and buffers to acidic pH the environment of the phagolysosome-like vacuole. The genome of this bacterium includes 4 Na+ exchangers and transporters for molecules that protect the cells from osmotic and oxidative stress.

The Coxiella’s life cycle contains 2 forms of development – large-cell (LCV) or the active replicating form found during the log phase and small-cell variant (SCV) that are typical for the stationary phase. SCVs show a significant resistance to various stressful factors of the environment – mechanical, heat, chemical, changes in the osmotic levels. Many reports describe the survival of the Coxiella cells on wool at room temperature for up to 1 year, in milk for more than 3 years, and in fresh meat for 4 weeks. The Coxiella burnetii is characterized as highly virulent and can be transmitted by aerosols. Due to this, U.S. Centers for Disease Control and Prevention categorized this pathogenic agent as Class B bacteria. The infection is not observed with high death rate, but it can lead to persistent disease and cause disability and serious consequences for the human population health status.

1. SEM image of Coxiella burnetii cell culture

2. How to recognize the Q-fever infection?

The main mammalian sources of Coxiella burnetii are ruminants – goats, sheep, cattle. The disease has been reported in almost all regions, except in New Zealand. The epidemiological levels of Q fever highly vary for the different countries and it depends on the geographic area and endemic profile. Scientists focused on the disease in 1999. Then, in The United States, the spread of the disease had led to 250% more cases of infected people in the period of 2000-2004. In Martigues (France), every 34 citizens from 100 000 were diagnosed with Q fever. More than 4000 cases were reported in The Netherlands between 2007 and 2010. An epidemic situation occurred in French Guiana due to the high virulence and stability of Coxiella burnetii cells. Normally, Q fever is not an acute disease and it is observed with flu-like symptoms. Most people infected with Coxiella burnetii have no symptoms at all.

However, there are cases of patients who get ill years after the infection. This is a result of an acute Q fever that can damage lungs, heart, liver. Q fever can be transmitted from animals to humans through urine, feces, milk and via inhalation of Coxiella burnetii-contaminated barnyard dust particles. The risk from infection is highest for groups like farmers, vets and researchers in field of veterinarian medicine and animal care. Q fever is cured quickly with antibiotic therapy but sometimes the treatment could take up to 1,5 years.

The symptoms occur between 3 and 30 days after exposure to Coxiella burnetii and may include high fever (more than 39 degrees Celsius), vomiting, cough, nausea, chills, fatigue, diarrhea, sensitivity to light.

Along with the location in a farm or near to farms or farming facilities, another risky factor is the time of the year – highest numbers of acute Q fever infected people are reported in April and May in The United States. Some reports show that the men are more likely to develop acute Q fever symptoms than women.

The risk of developing the acute form of Q fever (sometimes with lethal end) is higher for people with heart valve disease, weakened immune system, damaged kidney function, complications. The deadliest complication as a result of a Q fever recurrence is endocarditis. Q fever infected people can also develop pneumonia and acute respiratory distress. Pregnant women with chronic Q fever could have problems like low birth weight, premature birth or miscarriage. Other complications are observed with livers – people could have hepatitis due to Q fever infection, and brain meningitis – an inflammation of the membrane surrounding the brain and spinal cord.

2. X-ray image of a patient with pneumonia as a complication after Q

The prevention from Q fever includes vaccination for people who live in high-risk locations. The vaccine has been developed in Australia, 1989. The infection with Coxiella burnetii can be avoided with a consummation of pasteurized milk and pasteurized milk products only.

3. Which are the techniques used for diagnosis of Q fever?

The reference technique for detection of Coxiella burnetii is Indirect immunofluorescence assay (IFA) but other serology methods as complement fixation test (CFT) and ELISA are also used. Only kits and reagents for these assays are available on the market. ELISA has several advantages because it is easy to perform, the assay can be automated, and it is mainly used for diagnosis of acute and chronic Q fever. The most sensitive technique was IFA for detection IgM antibodies at an early phase of infection and after 12 months of follow-up. The sensitivity of CFT is reported as too low to be used for diagnosis and follow-up of C. burnetii persistent infection.

Several PCR-based analyses have been developed for the detection of Coxiella burnetii in clinical samples. The first standard PCR systems amplified plasmid sequences, the 16S-23S RNA, the superoxide dismutase gene, the com1 gene or the IS1111 repetitive elements in human or animal samples. Real-time PCR/qPCR is reported as faster and more sensitive PCR technique that provides information for the bacterium quantity in the tested clinical samples. Due to this, the qPCR assay is the most frequently used for detection of Coxiella burnetii these days. It targets IS1111 and can detect the bacterium within 2 weeks after exposure. Real-time PCR is also used for detection of persisting Coxiella burnetii infection in patients’ blood samples. The isolation and culturing of Coxiella burnetii is still used in many clinics and it can be achieved through the shell vial technique.

A new and interesting tool for detection of this pathogen is Immuno-PCR technique. It combines the PCR amplification with ELISA’s specificity and versatility which results in higher sensitivity. Many studies report that Immuno-PCR has significantly better sensitivity than ELISA and IFA in serum samples collected during the first 2 weeks after the symptoms’ development.

  Nowadays, the scientists are mainly focused on understanding the factors that could decrease the virulence of Coxiella burnetii. This will allow the development of effective strategies for treatment and prevention of Q fever and avoid the risk from complications such as endocarditis, hepatitis and pneumonia in patients from all over the world.