What are Hormones?

• Understanding Hormones
• Hormone Analysis and Testing: In Vitro Diagnosis
• Common Challenges with Hormone Tests
• 7 Tips to Help Choose an Antibody Provider That Best Meets Your Needs
• Why Choose Medix Biochemica?

Understanding Hormones

Specialized organs, called endocrine glands, produce and release hormones into the bloodstream.² The endocrine glands include:

• hypothalamus
• pituitary gland
• adrenal glands
• gonads (i.e. testes and ovaries)
• thyroid gland
• parathyroid glands
• pancreas²

Hormones are transported through the bloodstream to certain target cells, where they interact with a corresponding receptor to trigger a cascade of biochemical reactions that ultimately modifies the cell’s function or activity.² In order to maintain homeostasis and respond appropriately to changes in the environment, several hormones may regulate each other to control a single bodily function.²

Figure 1: The endocrine system

Selected Important Hormones and their Function in a Healthy Body

Table 1: Glands, hormones and their physiological functions^2,3

What Happens When Hormone Levels are Imbalanced?

Defective endocrine functions and hormone imbalances are causal factors in a number of diseases.⁴ Some of the most common types of endocrine disorders are:⁵

• Diabetes 
• Addison’s disease
• Cushing’s disease
• Graves’ disease
• Hashimoto thyroiditis
• Hyperthyroidism/hypothyroidism
• Prolactinoma
• Cancers of the endocrine glands

Hormone Analysis and Testing: In Vitro Diagnosis

Due to the important role hormones play in human health and disease, monitoring of hormone levels has emerged as a valuable diagnostic tool.⁶ Hormone tests can be used to assess the many physiological changes associated with aging, fertility and reproduction.⁶ They can also give insights into disease risk and the therapeutic effects of various treatments.⁶

The symptoms of defective endocrine functions may begin insidiously and be nonspecific, delaying clinical recognition and diagnosis for months and even years.⁷ For this reason, biochemical diagnosis is an important tool.⁷

Hormone concentrations from various biological fluids can be measured to diagnose disease or predict health outcomes.⁸ However, because of the relatively low levels of hormone present in bodily fluids, sensitive assays are required for those hormones to be reliably quantified.⁹

What Are Immunoassays?

Immunoassays, such as radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs), are widely used  to measure hormones.¹⁰ Immunoassays are rapid and accurate tests which detect the presence or quantity of certain molecules (e.g. a hormone), based on the specific binding of an antibody to the antigen of its target molecule.^11,12 

Because antibodies only bind to the antigen of their specific intended targets, specially developed monoclonal and polyclonal antibodies (see key terms below) can be used as reagents to detect and monitor specific target molecules.¹³

RIAs and ELISAs can measure the circulating concentrations of hormones such as insulin, human chorionic gonadotrophin (hCG), GH, progesterone, thyroxine, triiodothyronine, TSH, gastrin and renin.¹⁴ They can also be used to measure several other tissue and cell products, including blood group antigens, blood clotting factors, interferons and tumor markers.¹⁴

Understanding Immunoassays: Key Terms

Three of the most common types of immunoassays used for testing hormone levels are described below:

1. Sandwich immunoassay:

The analyte to be measured is ‘sandwiched’ between two different antibodies, one of which is labeled with a signal to be measured. The other one, named the ‘capture antibody’, detects and binds the analyte, allowing for the separation from the sample being tested. The higher the analyte present in the sample, the higher the signal detected. ¹⁷

Many hormones such as TSH, FSH, LH, and others are commonly tested using sandwich immunoassays. ^9,17

2. Competitive immunoassay:

The sample is incubated with an antibody able to detect the analyte to be measured. At the same time, a labeled analyte corresponding to the analyte being measured is also added to the sample mixture. The present analyte and added labeled analyte compete for the antibody binding sites of the antibody added to the mixture. After binding the bound antibodies to a solid phase, the signal is measured. Unlike in the sandwich immunoassay, in a competitive immunoassay a lower detected signal indicates a higher concentration of the analyte of interest in the sample. ¹⁷

The competitive immunoassay is often necessary to utilize when the sandwich immunoassay is not possible. For example, when the size of the analyte is too small to bind two antibodies simultaneously as is the case for some hormones, vitamins, drug of abuse analytes, etc. ¹⁷

3. Lateral flow test:

A common immunoassay platform to note, the lateral flow assay, is often used for self-testing.  This is especially true for pregnancy tests.  In all lateral flow tests, a sample is added to a membrane.  The sample passes along the membrane that utilizes immunoassay principles to detect the target of choice.  The specific lateral flow test can be designed as either competitive or as sandwich assays.¹⁹

The placental hormone hCG can be detected in the blood as early as the second week of pregnancy.⁹ The following schematic illustrates how the ‘sandwich’ immunoassay works as a lateral flow test for pregnant women.

Figure 3: The pregnancy test lateral flow 

Free monoclonal antibodies specific to hCG are conjugated to an enzyme that changes the color of a dye.¹⁹ A second set of monoclonal antibodies specific to hCG are immobilized to the dye substrate. If hCG is present in the urine, it will interact with both sets of monoclonal antibodies (forming an antibody ‘sandwich’).¹⁹ When both sets of antibodies are bound to hCG, the enzyme is brought into physical proximity with the dye, changing its color.¹⁹

A third set of monoclonal antibodies will bind any unattached enzyme-linked antibodies, functioning as a control.¹⁹

Common Challenges with Hormone Tests

Although immunoassay tests are a valuable tool in diagnostics, they are not foolproof.²⁰ Factors that can interfere with testing can occur at any phase of the testing process, beginning with specimen collection.²¹ Such interference can be ‘analyte-dependent’ or ‘analyte-independent’ and may increase (positive interference) or decrease (negative interference) the measured result.²²

Interferences That Alter the Measurable Analyte Concentration in the Sample

• Analyte-independent interference (sometimes called pre-analytical factors), such as:
• Hemolysis, when the measurement of hormones is compromised by a delay in plasma separation from normal human blood²³
• Effects of anticoagulants²⁴
• Lipemia, usually caused by inadequate time of blood sampling after a meal or parenteral administration of synthetic lipid emulsions²⁵

• Hormone-binding proteins – such as albumin, sex hormone binding globulin (SHBG), thyroid binding globulin (TBG) and cortisol binding globulin (CBG) – which can remove or block the analyte²²
• Autoanalyte antibodies, which are antibodies that can cause interference for certain analytes. These include thyroid hormones, thyroglobulin, insulin, prolactin and testosterone²²

Interferences That Alter Antibody Binding

• Heterophile antibodies produced against poorly defined antigens.²² These multi-specific antibodies of the early immune response generally show low affinity and weak binding.²² They can therefore bind to the conjugate, enzyme or other parts of the detection system in reagent-limited assays. This causes interference in assays for steroid hormones, thyroid function tests and digoxin²⁰
• Human anti-animal antibodies, which are specific polyclonal antibodies that show strong binding with antigens of a single chemical composition. They are produced in a high titre, such that they compete with the test antigen by cross-reacting with reagent antibodies of the same species to produce a false signal²⁰

• High-dose hook effect, which is the decrease in signal at very high concentration of analyte. This occurs when excessively high concentrations of analyte simultaneously saturate both capture and detector antibodies. It can occur in immunoassays with very large analyte concentration ranges, such as ferritin, growth hormone, hCG, prolactin (PRL), thyroglobulin (Tg) and the tumor markers prostate-specific antigen (PSA), CA19.9 and CA125.²²

7 Tips to Help Choose an Antibody Provider That Best Meets Your Needs

It is important to choose a company that provides the right support for your application as well as the products you need.²⁶

1. Choose a Raw Material Supplier With a Wide Variety to Choose From²⁶

Antibodies can work differently under various conditions. It will save time and money choosing a vendor with multiple sample options for a given analyte so that the process of choosing the right antibody for a given application can be streamlined. 

2. Choose a Provider Who is Willing to Help You Troubleshoot²⁶

If an antibody does not perform as expected, a good provider can help you address the problem. Avoid providers who provide only generic troubleshooting advice as this suggests a lack of technical expertise.

3. Carefully Scrutinize The Provider's Validation Data²⁶

Look for providers who can supply detailed validation protocols and who validate their antibodies using multiple biologically relevant sample types or tissues. Incomplete validation data can be a sign that an antibody is of poor quality and/or that the provider will not be able to help you troubleshoot if the antibody does not perform as expected.

4. Make Sure the Provider Specifies How Much Protein Was Loaded²⁶

If your protein of interest is present in much lower amounts than the provider gives, you may still be able to use the antibody for some applications. This can be done by enriching your protein of interest through fractionation or immunoprecipitation (IP). 

5. Check to Ensure Any Additives are Compatible With Your Application²⁶

Providers often include additives that stabilize and extend the shelf life of antibodies. For most applications this is not problematic, but there are some exceptions.

6. Review Publications²⁶

It is important to carefully scrutinize antibody data and references as you review publications referencing your proposed immunoassay.

7. Always Test Antibodies on Your Sample Yourself²⁶

Regardless of the antibodies’ source and validation state, be sure that they work the way you want them to on your specific sample.

Why Choose Medix Biochemica?

Medix Biochemica is your critical raw materials partner from concept to commercialization.

We have over 30 years of experience in producing premium-quality monoclonal antibodies for detection of hCG, LH, TSH and many other hormones.

At Medix Biochemica, we offer more than just a wide range of high-quality reagents for studying hormones and ready-to-use master mixes; we also offer expert customer service. Our technical support is provided by professionals with extensive experience in the highly specialized manufacturing of in vitro diagnostic (IVD) raw materials and kits.

A full solutions provider for test kit manufacturers, we suggest validated antibody pairs, match antigens, supply control materials and offer in-house expertise and experience.

Medix Biochemica has offices and manufacturing facilities around the world. All our manufacturing sites adhere to strict quality management systems and are ISO 13485 or ISO 9001 certified. Partnering with us means having access to international, industry-leading expertise.

Our emphasis on customer needs and operational excellence ensures consistent product performance, high quality and accurate results. This is what enables us to streamline assay development and validation, shortening our customers’ time to market.