Blood test products improve management of chronic kidney disease

Low concentrations of 1,25-dihydroxy vitamin D, a major hormone that regulates calcium and phosphate metabolism, are common in patients with chronic kidney disease due to loss of kidney function. With the 1,25-dihydroxy vitamin D blood tests manufactured by IDS, doctors and patients have an additional tool to diagnose and manage CKD.

Due largely to the prevalence of lifestyle diseases, chronic kidney disease (CKD), or the gradual loss of kidney function, has become an emerging public health issue affecting 8% to 16% of the world’s population.

This translates to at least 560 million people whose kidneys can no longer work properly, resulting in the build-up of fluids, minerals and wastes in the body.

CKD can lead to an increased risk of heart and blood vessel diseases, as well as complications such as hypertension, anaemia, and weak bones. Patients with CKD have a high rate of severe deficiency in 1,25-dihydroxy vitamin D, a steroid hormone that maintains calcium and phosphate homeostasis.

This deficiency is further aggravated by the CKD patients’ reduced kidney functions to turn 25-hydroxy vitamin D from diet and other sources into 1,25-dihydroxy vitamin D, the active form that the body can use.

‘It‘s not something you need to take as a classical vitamin. It has a very specific structure with hydroxyl groups which distinguish it from other steroid hormones,’ says Professor Anne White, a leadingUK endocrinologist from the Faculty of Medical and Human Sciences at the University of Manchester.

She explains: ‘The active form of vitamin D that is measured is 1,25-dihydroxy vitamin D. In order to develop a way of measuring it in the presence of the inactive 25-hydroxy vitamin D, we developed monoclonal antibodies that are very specific to that form of vitamin D. Before this development, it had always been difficult to produce very specific antibodies in sufficient quantities for diagnostic companies to use them in diagnostic kits.

When vitamin D levels in the body get too low, the parathyroid glands, which modulate calcium metabolism, secrete more parathyroid hormone that can lead to secondary hyperparathyroidism (SHPT). Early identification and treatment of SHPT is crucial as SHTP can also cause the parathyroid glands to enlarge, which can result in permanent damage.

To slow the progression of SHPT, active vitamin D (calcitriol) therapeutic regimens are recommended. Doctors order several blood tests–also known as immunoassays–including the 1,25-dihydroxy
vitamin D test. This particular test is of interest to CKD patients because the main enzyme converting 25-hydroxy vitamin D to the active form is located in the kidney. Immunodiagnostic Systems PLC (IDS) manufactures several blood test products to measure the 1,25-dihydroxy vitamin D levels of CKD patients and help them manage their condition. IDS does this using a monoclonal antibody developed by White and the late Professor Barbara Mawer at the University of Manchester.

Three decades of partnership IDS was the first to market the 1,25-dihydroxy vitamin D immunoassay in 1981. The development of IDS’s 1,25-dihydroxy vitamin D immunoassay was carried out at IDS’s own laboratory, after gaining the licence to manufacture and sell diagnostic products incorporating the unique monoclonal antibody based on research by White and Mawer at the university of Manchester. The partnership with the university has continued for more than three decades now.

White recalls: ‘A head of research at IDS, Dr. Roger Duggan, used to come to conferences that we were at and would talk with us about what we were doing.’Unlike others who have approached her and Mawer with unrealistic views, Duggan was ‘very interested and very realistic,’ she says.

White and Mawer agreed to collaborate with IDS to try to develop the kit to measure 1,25-dihydroxy vitamin D. ‘We used to produce all the monoclonal antibodies for the kits in our lab, but then as they sold more and more kits, it became absolutely impossible to do that, and they had to take that on as well,’ White says. She credits their successful collaboration to the fact that IDS was a company that worked in a very specialised area and made decisions quickly.

UMIP, The University of Manchester’s agent for intellectual property commercialization, worked closely with White to negotiate licence terms.

Assisted by the deep understanding of the technology at IDS and White’s commercial insight, the negotiation process served to strengthen the relationship between all parties and provide a solid platform for commercial impact.

Robust and reliable products

The concentration of 1,25-dihydroxy vitamin D in the body is very low, so it is important to ensure that it is actually the hormone being measured in tests.

Avoiding the need for complex specimen purification, as well as the use of tritium, a radioactive component, were the main challenges in developing a robust and reliable blood test product. It was fortuitous that White, who is trained as a cell biologist, had made hybrids between normal and malignant cells for her doctorate. When Georges Kohler and Cesar Milstein came up with a method to make monoclonal antibodies by producing hybrid cells, White was one of the people who knew the technique. At that time, White had a postdoctoral post in a university endocrinology laboratory where they wanted to develop new methods for the diagnostic assays of hormones.

‘I was the cell biologist and I quickly learnt how to measure the hormones,’ White says. She worked alongside clinical biochemists who measured patient samples and told her the problems they
were encountering with the assays they were using. White’s job was to develop monoclonal antibodies that improved their assays for patients. Making monoclonal antibodies, particularly for 1,25 dihydroxy vitamin D, is no mean feat. White explains that to make monoclonal antibodies, a form of the hormone (as an immunogen) must be injected into a mouse. The mouse would then make the antibody producing plasma cells, and these lymphocytes would be taken from the mouse. The plasma cells are then fused with myeloma cells to make hybridomas which are then cloned so they secrete monoclonal antibodies.

‘We had to have an organic chemist who could make the structures that we injected into the mice. These structures would hold the vitamin D molecule in a way that presented it to the lymphocytes in the mice,’ White says. ‘So there were three different specialties: I was knowledgeable in making monoclonal antibodies; we had somebody who was knowledgeable on making these chemical structures to make the immunogens; and then there was an expert on vitamin D assays, Barbara Mawer.’

She explains that the difficulty in producing monoclonal antibodies for vitamin D is that in order to determine the concentration of the active form, the antibodies must only recognise that form, and not the inactive form. A good antibody must also be able to bind very tightly to the hormone. On top of that, the process must be done quickly because the hybrid cells must be cloned repeatedly and stabilised.

‘The one thing that’s quite tricky with making good monoclonal antibodies to steroid hormones is it’s like trying to find a needle in a haystack,’ White says. Using the monoclonal antibodies produced by the University of Manchester, IDS then went on to develop a novel immuno-extraction method that addressed the problem of sample purification. IDS has applied for a patent for the method they developed.

Commercial success

There are currently four products available from IDS. Three of them – 1,25-Dihydroxy Vitamin D RIA, 1,25-Dihydroxy Vitamin D EIA, and the automated IDS-iSYS 1,25-Dihydroxy Vitamin D – are being sold worldwide. Another product, the fully automated IDS iSYS 1,25 VitDXp, is available only in the European Union and Brazil.

Vitamin D has received increased attention in the last 5 to 10 years due to growing knowledge of its importance. In fact, IDS acknowledges that Vitamin D has been a substantial part of its positioning and success as a company. IDS was among the first in the market with 25-hydroxy and 1,25-dixydroxy vitamin D immunoassays. After only a few years, IDS reported a market share of 80% and revenues of some £50M.

Technology has come a long way since the launch of the 1,25-dihydroxy vitamin D immunoassay some 30 years ago. IDS has developed a fully automated instrument for the vitamin D test, called IDS-iSYS, which reduces the pressure of increasing volumes of testing on small and mid-sized laboratories. From an overnight manual assay done by two to three people using a lot of equipment, IDS-iSYS brings that down to 90 minutes’ testing and up to 500 results a day. The goal, IDS says, is to keep improving its products according to its customers’ needs.

In the spotlight

CKD and diabetes
Diabetes is a disease in which the body does not make enough insulin or is unable to properly use normal amounts of insulin, a hormone that regulates the amount of sugar in the blood. It is
one of the two main causes of CKD, the other being hypertension. Around 30% of patients with juvenile onset or Type 1 diabetes and 10% to 40% of people with adult onset or Type 2 diabetes will eventually suffer from kidney failure.

Diabetes causes injury to the blood vessels, leading to diabetic kidney disease, also called diabetic nephropathy. When the blood vessels in the kidneys are damaged, the kidneys lose their
ability to filter the blood, resulting in higher levels of water, minerals and waste in the body. The nerves can also be damaged, causing difficulty in emptying the bladder. The pressure from a full bladder can injure the kidneys.

There are five stages of CKD, mainly based on measured or estimated glomerular filtration rate. The earliest sign of diabetic kidney disease is a higher level of albumin in the urine. Blood urea nitrogen and creatinine levels will increase as the kidneys fail. Patients with Stage 1 CKD have normal kidney function, while those with Stage 5 suffer from end-stage renal failure.

Treatment options for kidney failure are kidney transplant, haemodialysis, and peritoneal dialysis. Diabetic kidney disease is expected to account for about 30% of the projected $1.1 trillion global cost of dialysis in the current decade.