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Category: Immunoassays

Lyme disease is an infectious disease caused by Borrelia bacteria transmitted by tick bites from ticks of the Ixodes genus. Particularly if untreated, serious and long-term symptoms can occur such as neurological problems, facial palsy, lower limb impairment, heart complications, arthritis, encephalomyelitis and psychosis. It is estimated to affect 300,000 people per year in North America and 65,000 in Europe. Transmission can occur across the placenta during pregnancy.

The causative agent of Lyme disease is a group of bacteria called Borrelia burgdorferi sensu lato. The group comprises 21 closely related species but only 4 of them clearly cause Lyme disease:

  • B. mayonii
  • B. burgdorferi sensu stricto
  • B. afzelii
  • B. garinii

The incubation period for Lyme is usually 1 to 2 weeks but can be longer or shorter. Most of those infected did not realise they had been bitten by a tick as the causative ticks are very small in the nymphal stage.

You didn’t want to see a picture of a tick, did you?

Some people infected with Lyme develop a distinctive bullseye-shaped rash known as erythema migrans. These are perhaps the lucky ones as the presence of it aids Lyme diagnosis. However, the rash commonly fails to conform to the notion of what it is supposed to look like and this can result in Lyme being misdiagnosed as spider bites, cellulitis or shingles. The rash can even be absent entirely and this makes it particularly challenging to diagnose since the other early symptoms of Lyme disease (fever, fatigue, etc.) are ones which are also common to a multitude of other ailments.















The classic Lyme erythema migrans rash

Aside from the rash, Lyme diagnosis typically looks for the patient’s immune response. Anti-Lyme IgM antibodies can usually be detected at 2-4 weeks post-infection, while Anti-Lyme IgG antibodies are usually present 4-6 weeks post-infection. IgM antibodies diminish approximately 4-6 months after infection while IgG antibodies can persist for years. Confusing the issue, the IgM antibodies can persist for years in some individuals and Lyme IgG may never be produced in some “seronegative” individuals. A seronegative patient who doesn’t get the classic rash – that’s going to be difficult to diagnose.

The reason for IgM antibodies being present in chronic patients could be the bacterium reactivating and producing novel proteins, recognisable to the immune system, due to genome instability in the bacterium. The host’s immune system therefore may recognise the altered bacterium as a new one it hasn’t encountered before and develop IgM antibodies against it.

The US Centers for Disease Control recommends a two-tier protocol – first test with ELISA or Immunofluorescence Assay to detect anti-Lyme antibodies, and if it is positive or equivocal, a Western Blot should be performed, also detecting anti-Lyme antibodies. Since Lyme disease is caused by numerous strains of bacterial species with inherent genome instability, the Western Blot test looks for antibodies raised by the patient against a large number of host proteins. Multiple bands should be present for the test to be considered positive.

Lyme Western Blot result annotated with protein bands and molecular weights

 

 

 

 

 

 

 

 

 

False positives are rare although possible where anti-Lyme antibodies cross-react with other bacterial antigens present in the patient sample, but false negatives are common particularly early in testing, partly because the anti-Lyme antibodies take some time to develop following the infection.

PCR is not particularly useful for Lyme diagnosis because of low sensitivity in certain sample types and a poor ability to detect Borrelia DNA in patients with neuroborreliosis.

If test sensitivity is the ability to correctly identify those with a disease and test specificity is the ability to correctly identify those without the disease, Lyme tests sadly still have some way to go to reach 100% for either metric.

 

Bulk Lyme IgM plasma is available from Logical Biological.

Congratulations! Anyone reading this far qualifies for extra reading.

Extra Reading: The Accuracy of Diagnostic Tests for Lyme Disease in Humans, A Systematic Review and Meta-Analysis of North American Research.
Waddell. L. et al. PLoS One. 2016, 11 (12).

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December 3rd 2019
Immunoassays Infectious Diseases
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What is HAMA?

HAMA is an acronym for Human Anti-Mouse Antibodies. Some humans produce HAMA and have it present in their blood. Unfortunately for them, and the in vitro diagnostics industry, the presence of Human anti-mouse Antibodies (HAMA) in patient samples can lead to false positive and false negative results in immunoassays.


Murine MC HAMA

Why do some Humans produce HAMA?

Some people work directly with mice while others might inadvertently encounter mouse proteins or immunoglobulins by coming into contact with mouse urine or contaminated food. These people could develop an immune response against mouse immunoglobulins (antibodies) they encounter such that their immune system produces HAMA.

In the past, mouse monoclonal antibodies were used as therapeutics and could elicit an immune response resulting in the presence of HAMA in human individuals. However, these days monoclonal antibody-based pharmaceuticals are “humanised” to avoid this problem.

Presence of HAMA in individuals is rare, but still needs to be accounted for in immunoassay design.

Could I interfere with your immunoassay?

How can HAMA impact Immunoassays?

When a patient is tested for a condition, the test performed is commonly an immunoassay and the sample that is tested is serum or plasma derived from the patient’s blood. Immunoassays are typically developed using a “matched pair” of mouse monoclonal antibodies to bind to and detect the marker of interest. The ‘marker’ of interest depends on the condition being diagnosed but could be, for example, Troponin I which is a marker of acute myocardial infarction, or HIV p24 antigen which is a marker for the presence of HIV virus. The patient sample (e.g. serum or plasma) is applied to the immunoassay and if the marker being tested for is present the mouse monoclonal antibodies within the assay bind to it and a signal is generated.

However, where HAMA is present in the patient sample the HAMA can bind to the mouse monoclonal antibodies used as immunoassay components and can either i) block the mouse monoclonal antibodies from binding to the marker of interest resulting in a false negative result, or ii) form a bridge between the pair of mouse monoclonal antibodies, generating false positive signal.  Where a patient receives an incorrect diagnosis due to the presence of HAMA, the consequences can be devastating. At least 34 cases of hCG false-positive tests in the United States between 1999-2004 resulted in the patients receiving chemotherapy or surgery, including 10 hysterectomies, for assumed cancer1.

How can HAMA interference be prevented in immunoassays?

Assay manufacturers can develop their assays in such a way as to minimise the interference from HAMA, for example by adding excess mouse immunoglobulin to their assay buffers. When this is done, the HAMA present in the patient sample can bind the excess mouse immunoglobulin rather than the reagents being used in the assay.

Immunoassay developers will need to access patient material in order to design an assay that is not affected by HAMA, and also to show that the assay works in the presence of HAMA. Logical Biological is able to provide HAMA-positive serum and plasma.

Even if blockers are used to control HAMA, the heterogeneous nature of its presence in patients mean that it is difficult to rule out its influence entirely, unless it is measured. If HAMA is suspected, the clinical laboratory can perform serial dilutions with an appropriate buffer to demonstrate nonparallelism (higher recovery of the signal than expected).

An alternative solution to controlling for HAMA interference is to use monoclonal antibodies from an alternative species in the immunoassay, in place of mouse monoclonal antibodies. Rabbit and Sheep monoclonal antibodies are candidates here. However, this is not a great solution as some individuals in the population will be producing human anti-rabbit and anti-sheep antibodies. Synthetic peptide binders and/or recombinant antibodies can theoretically avoid the problem of HAMA when used as alternatives to antibodies raised in animals.

Reference

  1. Human Chorionic Gonadotropin (hCG), By Laurence A. Cole, Stephen A. Butler. Elsevier. 2010.

Blog Overview

October 17th 2019
Antibodies Auto-immune Immunoassays Interference
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Toxoplasma gondii is a protozoan parasite present in a third of the world’s human population. People acquire this infection in three ways: i) inadequately cooked infected meat, particularly pork, ii) unwitting ingestion of oocysts passed in cat feces, which could, for example, occur whilst gardening, and iii) pregnant women passing the infection transplacentally to their unborn fetus – known as Congenital Toxoplasmosis.

In most cases the infection is asymptomatic or presents with mild nonspecific symptoms. However, the infection remains in the host indefinitely, latent in the heart, brain, eye, and muscle tissues. It can reactivate in people with weak immune systems, such as patients with advanced HIV disease or those on immunosuppressive therapy, and can result in life-threatening disease.

Fluffy, but could be harbouring Toxoplasma gondii

Congenital Toxoplasmosis  

Women infected prior to conception rarely pass the parasite on to the fetus, although it can happen in individuals where the Toxoplasma has reactivated due to immunosuppression. However, if newly infected with Toxoplasma during pregnancy there is a 20-50% likelihood of the infection being passed to the unborn fetus. The risk of congenital disease is lowest when the maternal infection occurs in the first trimester of pregnancy but the disease is more severe if acquired in the first trimester.

The consequences of being infected as a fetus can be severe, both in infancy and later in life. The list does not make pleasant reading, and includes:

  • Convulsions
  • Deafness
  • Growth impairment
  • Intracranial calcifications
  • Learning disabilities
  • Mental impairment
  • Microcephaly
  • Visual impairment

If an acute infection is diagnosed during pregnancy, treatment can be given which, although not able to influence the likelihood of in utero transmission, can reduce the likelihood and intensity of the manifestations listed above. Drugs include pyrimethamine and sulfadiazine. Despite efforts to encourage prevention, there are sadly around 3000 cases in the United States alone each year.  Abortion may be considered if infection is thought to have occurred before the 16th week of pregnancy or if the fetus shows evidence of hydrocephalus.

Diagnosis

Typically in infections of any type, the host produces IgM antibodies as an initial response to the infection, and later produces IgG antibodies in larger quantities. IgM levels typically drop following the initial infection, whereas IgG antibodies take longer to appear and persist in large quantities.  

Acute toxoplasmosis diagnosis is made tricky by the fact that presence of anti-Toxoplasma IgM antibodies in the host can persist for several years after the infection. Therefore, presence of IgM antibodies in the host cannot lead to the conclusion that an acute infection is present; a study showed that 36% of IgM-positive patients actually had a chronic infection and not an acute infection. False positive test results for Toxoplasma IgM are also an issue.

Furthermore, presence of Toxoplasma IgG, which would typically suggest an older infection, particularly where IgG levels are high, cannot rule out an acute infection; one study found that only 22% of patients positive for Toxoplasma IgM and Toxoplasma IgG actually had an acute infection.

Host antibody response following Toxoplasma gondii infection

So, how is acute Toxoplasma infection diagnosed in pregnant women? Specialised reference laboratories may be able to narrow the time of infection using a wider battery of tests, including tests to IgA, avidity tests and differential agglutination of the AC and HS antigens. Following this, if it is suspected the infection may be acute, the fetus itself may be tested; this typically involves performing PCR on amniotic fluid. Where there is any suspicion that a child may be at risk of Congenital Toxoplasmosis, he/she should be tested for regularly for anti-Toxoplasma antibodies following birth; Toxoplasma IgG antibodies should decrease by approximately a half every month in uninfected individuals but will not disappear by 12 months in infected individuals.

Blog Overview

September 14th 2019
Immunoassays Infectious Diseases
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