This contribution describes the possibility of applying liquid chromatography coupled to tandem mass spectrometry for analysing sewage in order to track down the use of new psychoactive substances.
by J. Kinyua, Prof. A. Covaci, Prof. A. L. N. van Nuijs
Introduction
Sewage-based epidemiology (SBE) is an alternative method of monitoring population drug use by the analysis of excretion products of drugs in sewage (Fig. 1). SBE has been applied since 2005 as a complementary approach to classical investigation methods, such as interviews with users, medical records, population surveys, and crime statistics for estimating illicit drug use in communities [1–3]. Data obtained from SBE provide information on drug use in a direct, quick and objective way.
New psychoactive substances (NPS) are substances that are not controlled by the 1961 United Nations Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances and that may pose a threat to public health [4, 5]. These compounds mimic effects of illicit drugs like cocaine, cannabis and amphetamines and are produced to evade law enforcement by introducing slight modifications to chemical structures of controlled substances [6]. Currently, more than 450 NPS are being monitored by the European Monitoring Centre for Drug and Drug Addiction (EMCDDA) with 101 new substances reported for the first time in 2014 to EU Early Warning System (EWS). Synthetic cannabinoids and synthetic cathinones are the largest groups in the NPS scene [7]. NPS are easily acquired through online vendors and in smart shops where they are sold with misleading information about their effects and safety [8]. They are considered a growing problem in many communities and are responsible for numerous fatal intoxications [9]. SBE has the potential to be usefully applied for the detection and quantification of NPS to document their occurrence to appropriate authorities. Being an emerging issue, only a few studies have applied SBE for the analysis of NPS [10–13]. In this contribution, the optimization, validation and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC–ESI-MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine, 4-methoxymethamphetamine (PMMA), and 4-methoxyamphetamine (PMA) is described together with a critical evaluation of the methodology.
LC-MS/MS methodology
An LC-MS/MS method was developed and validated using a Phenomenex Luna HILIC (hydrophilic interaction liquid chromatography) 200A (150 x 3 mm, 5 µm) column, with a mobile phase composed of A) 5 mM ammonium acetate in ultrapure water and B) acetonitrile. The mass spectrometer compound dependent parameters, fragmentor voltage and collision energy, were optimized to acquire two multiple reaction monitoring (MRM) transitions (qualifier and quantifier) for each compound, and one MRM for the internal standards (IS). The method was validated, assessing accuracy and precision, using blank sewage (samples collected prior to 2009 in which NPS have not been detected). A linear range with lower limits of quantification (LLOQ) of 0.5 ng/L (MXE and methylone) and 2 ng/L (all other compounds) and upper limits of quantification (ULOQ) of 200 ng/L was achieved for investigated compounds. The limit of detection (LOD) was between 0.02 and 0.2 ng/L for all compounds.
Sample collection and preparation
24-h composite influent sewage samples were collected from different wastewater treatment plants (WWTPs) in Belgium and one WWTP in Zurich. Before sample extraction, 50 mL sewage was filtered through a 0.7 µm glass filter to remove solid particles. After filtration, the samples were brought to pH 2 using a 6 M HCl solution and spiked with deuterated IS at a concentration of 100 ng/L. Thereafter the solid-phase extraction (SPE) procedure was performed using a mixed-mode strong cation exchange sorbent-Oasis MCX (Fig. 2).
Application of the procedure
The method could reliably differentiate the analytes and IS from endogenous components. MXE, methylone and ethylone could be detected. The method revealed the presence of MXE in sewage from five urban centres within two counties in Belgium. Methylone was detected and quantified in only two samples from Switzerland at levels slightly higher than LLOQ (Fig. 3). The compounds that were not detected could be absent in the sewage or present in the form of metabolites which were not targeted in the present study.
Advantages/limitations of the SBE methodology
Phenylethylamine-based compounds (synthetic cathinones and amphetamine-like substances) form a large group of NPS and they are very polar. Hydrophilic interaction was found to be a good and robust LC stationary phase to obtain retention for these high-polarity compounds. Furthermore, we showed for the first time in SBE that the use of a more realistic matrix for method development, such as real sewage, can help in overcoming challenges associated with matrix effects in MS detection. The results from these samples demonstrate the importance of developing highly sensitive analytical methods that can detect and quantify NPS at very low concentrations (<10 ng/L).
Limitations of the present methods
It is difficult to determine if the low drug concentrations in sewage are related to low popularity of the NPS or due to the presence of an unknown form of the parent drug in sewage, urinary metabolites or transformation product from other in-sewer processes. SBE requires a specific, reliable and stable biomarker for the NPS of interest. Further studies on the metabolism and in-sewer transformation processes (which may affect stability of drug residues) of NPS needs thus to be carried out to provide SBE with information regarding additional biomarkers of NPS parent drugs.
Future of SBE in NPS analysis
Concentrations of NPS in sewage may be low depending on the area served by the WWTP and on the prevalence of its use [14]. Therefore, pooled urine analysis would be useful in detecting the occurrence of NPS before dilution into sewage [15]. It would be a valuable approach to combine pooled urine analysis and SBE to track down the actual use of NPS in communities.
Conclusion
In conclusion, SBE can help in revealing the occurrence of NPS within catchment areas of urban centres and showed the need to develop very sensitive analytical methods to detect NPS in sewage.
References
1. Bijlsma L, Sancho JV, Pitarch E, et al. Simultaneous ultra-high-pressure liquid chromatography-tandem mass spectrometry determination of amphetamine and amphetamine-like stimulants, cocaine and its metabolites, and a cannabis metabolite in surface water and urban wastewater. J Chromatogr A 2009; 1216: 3078–3089.
2. Boleda MR, Galceran MT, Ventura F. Trace determination of cannabinoids and opiates in wastewater and surface waters by ultra-performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 2007; 1175: 38–48.
3. Huerta-Fontela M, Galceran MT, Ventura F. Ultraperformance liquid chromatography-tandem mass spectrometry analysis of stimulatory drugs of abuse in wastewater and surface waters. Anal Chem 2007; 79: 3821–3829.
4. United Nations Office on Drugs and Crime (UNODC). Global synthetic drugs assessment. (United Nations publication, Sales No. E.14.XI.6), 2014. http://www.unodc.org/documents/scientific/2014_Global_Synthetic_Drugs_Assessment_web.pdf
5. King LA, Kicman AT. A brief history of ‘new psychoactive substances’. Drug Test Anal. 2011; 3: 401–403.
6. Dargan PI, Wood DM. Novel psychoactive substances classification, pharmacology and toxicology. Elsevier/Academic Press, 2013. ASIN: B00FK8HYY2.
7. European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). New psychoactive substances in Europe. An update from the EU Early Warning System, 2015. http://www.emcdda.europa.eu/publications/2015/new-psychoactive-substances
8. EMCDDA. EMCDDA–Europol 2013 Annual Report on the implementation of Council Decision 2005/387/JHA, 2014. http://www.emcdda.europa.eu/publications/implementation-reports/2013
9. Vevelstad M, Øiestad E.L, Middelkoop G, et al. The PMMA epidemic in Norway: Comparison of fatal and non-fatal intoxications. Forensic Science International 2012; 219: 151–157.
10. Kinyua J, Covaci A, Maho W, et al. Sewage-based epidemiology in monitoring the use of new psychoactive substances: validation and application of an analytical method using LC-MS/MS. Drug testing and analysis 2015 ( In press).
11. Reid M.J, Derry L, Thomas K.V. Analysis of new classes of recreational drugs in sewage: Synthetic cannabinoids and amphetamine-like substances. Drug Test Anal. 2014; 6: 72–79.
12. Van Nuijs ALN, Gheorghe A, Jorens PG, et al. Optimization, validation, and the application of liquid chromatography-tandem mass spectrometry for the analysis of new drugs of abuse in wastewater. Drug Test Anal. 2014; 6: 861–867.
13. Kankaanpää A, Ariniemi K, Heinonen M, et al. Use of illicit stimulant drugs in Finland: a wastewater study in ten major cities. Sci Total Environ. 2014; 487: 696–702.
14. Archer JRH, Dargan PI, Lee HMD, et al. Trend analysis of anonymised pooled urine from portable street urinals in central London identifies variation in the use of novel psychoactive substances. Clinical Toxicol (Phila). 2014; 52: 160–165.
15. Archer JRH, Dargan PI, Hudson S, et al. Analysis of anonymous pooled urine from portable urinals in central London confirms the significant use of novel psychoactive substances. QJM 2013; 106: 147–152.
The authors
Juliet Kinyua MSc, Adrian Covaci PhD, Alexander L.N. van Nuijs* PhD
Toxicological Center, University of Antwerp, Belgium
*Corresponding author
E-mail: alexander.vannuijs@uantwerpen.be
In spite of increased publicity in the Western world about malaria and drives to provide mosquito nets, the disease is still endemic in a large part of the world. This article discusses different methods of malaria diagnosis and the role that point-of-care tests can play in the ultimate goal of malaria elimination.
by Dr Jackie Cook
Finding the balance: over- and under-diagnosing malaria
Malaria remains a huge burden in many parts of the world, particularly in sub-Saharan Africa. Despite increased availability of effective treatments and interventions, malaria elimination is still out of reach for many countries. Whilst availability of effective interventions that reduce contact with infected mosquitoes, such as insecticide treated bed-nets or indoor spraying with insecticide are key to reducing malaria prevalence, case management also plays a key role. Many who need treatment are unable to get it, either through lack of access to healthcare, or because infections remain undiagnosed. Conversely, some studies suggest that many patients are receiving anti-malarials unnecessarily due to a tendency to diagnose based solely on clinical symptoms, many of which are similar to other infections, rather than using a diagnostic. In under-resourced settings, this can result in any child presenting with a fever being prescribed malaria drugs. This simultaneously means non-malaria fevers remain undiagnosed and untreated, as well as a large proportion of unnecessary prescriptions for malaria drugs, which increases healthcare costs and the risk of drug resistance, a very potent threat. In order to counteract this, the last few decades have brought a push from health officials, researchers, donors and governments alike to confirm every suspected case of malaria before prescribing treatment.
Microscopy
For many, malaria diagnosis is performed using microscopy, a procedure that is relatively cheap but requires a skilful operator. Malaria is caused by the plasmodium parasite and it undergoes several developmental and replication stages in the human. These stages can be seen through a microscope when blood is prepared on either a thin or thick film and stained, normally, with Giemsa or Wright’s stains. Experienced microscopists can detect down to 1 parasite per microlitre of blood, although the typically quoted sensitivity for microscopy is approximately 100 parasites per microlitre. In reality, the sensitivity of the test depends greatly on the microscopist. In areas where malaria transmission is declining, microscopists can go months without seeing a positive slide, and as such, skills may begin to decline. In addition, the need for well-maintained microscopes and access to slides and stain can mean microscopy is not always available.
Rapid diagnostic tests
The first malaria rapid diagnostic test (RDT) was developed in 1993 and in the decades since many variations have proliferated on the market. RDTs are typically immunochromatograhic tests that use monoclonal antibodies to detect the presence of plasmodium antigens (proteins produced by the parasite) which are present in the blood of infected, or recently infected, individuals. They are generally stable at a range of temperatures and do not require special storage conditions. RDTs require significantly less training for use than microscopy and a positive infection is easy to identify by visualization of a ‘positive’ line, meaning the results are much less subjective. Most RDTs require 15–20 minutes for development, meaning treatment can be given while patients wait at health facilities.
However, there are a few downsides to the use of RDTs. The presence of parasite antigen doesn’t always equate with a current infection, but can signify a recently cleared infection from within the previous two weeks. In addition, several studies have reported the deletion of certain antigens detected by RDTs in plasmodium parasites, meaning false-negative results may be obtained in areas using these types of RDTs [1]. The World Health Organization (WHO), in collaboration with the Foundation for Innovative New Diagnostics (FIND), has set up an RDT product testing programme, an essential quality assurance component considering the huge influx of RDT brands that have popped up in the past 20 years [2]. The reports from the programme make worrying reading with very low sensitivity for some brands, differences between batches of RDT and a general lower sensitivity for non-falciparum infections for nearly all brands.
The hidden reservoir: asymptomatic, low-density infections
In general, the limited sensitivity of both microscopy and RDT (unreliable detection in infections with a parasite density less than 100 parasites per microlitre) is not an issue for symptomatic malaria infections, the majority of which will consist of high parasite densities. However, asymptomatic infections are numerous, in high and low transmission settings. These asymptomatic infections pose a problem for control programmes. The carriers do not feel unwell so have no reason to present to a health facility for testing and yet, they may be infectious to mosquitoes, meaning they pose a risk for onward transmission. In order to detect and treat these asymptomatic infections, malaria programmes are now taking their diagnostics into the community in a strategy termed Mass Screening and Treatment (MSAT). This involves testing everyone within a community regardless of whether they have symptoms. Many of these infections are asymptomatic and therefore also likely to be low-density; hence which test you use can mean the difference between detecting 10 infections or 100 infections. Whilst RDT is ideal for field conditions, studies have shown that they can miss a large proportion of infections that are present [3].
Molecular tests
Polymerase chain reaction
More sensitive diagnostics are available in the terms of molecular tests. The most commonly used is polymerase chain reaction (PCR). Numerous PCR assays have been developed, many based on amplifying the 18S ribosomal RNA (18SrRNA), first published by Snounou and colleagues in 1993 [4]. PCR detects parasite nucleic acids and can detect much lower parasite densities than RDT or microscopy, with tests reportedly able to detect down to 1 parasite per microlitre of blood, as well as being able to accurately distinguish between plasmodium species. However, the number of assays available has resulted in calls for a standardized test so results can be compared across the world. PCR tests are generally performed on blood collected on filter paper but the equipment required for PCR and the expense of maintaining a sterile lab environment precludes PCR from being available in many health facilities. This means that samples need to be sent away, with an often long wait for results. Although more field-friendly PCR methods are in the pipeline, currently, PCR is not generally considered suitable for a point-of-care test, although it’s use in epidemiological studies is undisputed.
Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) was first developed in 2000, with the aim to amplify DNA in a sensitive, specific and speedy manner (Figs 1, 2). One of the main advantages is the fact it can be performed under isothermal conditions, and thus averting the need for a thermocycler. LAMP can be thought of as a ‘rough-and-ready’ PCR, as it is also less sensitive to inhibitors present in biological samples, and therefore allows the use of simple and cheap DNA extraction methods. The fast time-to-results and the minimal equipment required make LAMP an attractive option for field diagnosis. In order to make this a viable option, FIND and partners Eiken Chemical Ltd, Japan, and the Hospital for Tropical Diseases (HTD), London, UK have developed a field-stable kit with all reagents freeze-dried into the lid of the reaction tube, which means minimal processing is required. Although still in the development and testing stage, current results of the use of the kit are promising, with strong agreement with PCR results and a considerably higher sensitivity than RDT [5-8]. Whilst seemingly the most sensitive of the point-of-care tests available, there are some downsides to LAMP. Results still take considerably longer than RDT, requiring patients to wait at clinics for 2 hours for results, or leaving the health facility staff with the complicated task of contacting and following up any positive patients. In addition, electricity is required for the processing of samples, making it not practical for many places.
Future for point-of-care diagnostics for malaria
These advances in molecular diagnostics mean infections that would previously have remained undetected can now be confirmed, treated and cleared. Identifying and treating all infections becomes a greater priority as transmission reduces and the possibility of elimination comes into focus. This is occurring in areas around the world such as Swaziland and Zanzibar in Africa and in South East Asia, where the need to eliminate has become ever more important with the emergence of drug-resistant parasites. In these areas, identification of every last parasite is the aim and development of a quick, sensitive and reliable diagnostic is key to that.
As more studies reveal the extent of the low-density parasite reservoir, there is a sense of ‘the more we look the more malaria we will find’. But do we need to find all these infections in order to eliminate malaria? It should be noted that these ‘super-sensitive’ tests are a relatively recent phenomena and that countries have succeeded in malaria elimination without them. The role these low-density parasitemias play in transmission is not fully understood but for now the aim remains to clear the last parasite standing.
References
1. Houze S, Hubert V, Le Pessec G, Le Bras J, Clain J. Combined deletions of pfhrp2 and pfhrp3 genes result in Plasmodium falciparum malaria false-negative rapid diagnostic test. J Clin Microbiol. 2011; 49(7): 2694–2696.
2. WHO, FIND, CDC. Malaria rapid diagnostic test performance: Results of WHO product testing of malaria RDTs: Round 5. 2013; http://www.who.int/malaria/publications/atoz/9789241507554/en/.
3. Cook J, Xu W, Msellem M, Vonk M, Bergström B, Gosling R, Al-Mafazy AW, McElroy P, Molteni F, Abass AK, Garimo I, Ramsan M, Ali A, Mårtensson A, Björkman A. Mass screening and treatment on the basis of results of a plasmodium falciparum-specific rapid diagnostic test did not reduce malaria incidence in Zanzibar. J Infect Dis. 2015; 211(9): 1476–1483.
4. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, Thaithong S, Brown KN. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol. 1993; 61(2): 315–320.
5. Hopkins H, González IJ, Polley SD, Angutoko P, Ategeka J, Asiimwe C, Agaba B, Kyabayinze DJ, Sutherland CJ, Perkins MD, Bell D. Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda. J Infect Dis. 2013; 208(4): 645–652.
6. Polley SD, González IJ, Mohamed D, Daly R, Bowers K, Watson J, Mewse E, Armstrong M, Gray C, Perkins MD, Bell D, Kanda H, Tomita N, Kubota Y, Mori Y, Chiodini PL, Sutherland CJ. Clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria. J Infect Dis. 2013; 208(4): 637–644.
7. Aydin-Schmidt B, Xu W, González IJ, Polley SD, Bell D, Shakely D, Msellem MI, Björkman A, Mårtensson A. Loop mediated isothermal amplification (LAMP) accurately detects malaria DNA from filter paper blood samples of low density parasitaemias. PLoS One 2014; 9(8): e103905.
8. Cook J, Aydin-Schmidt B, González IJ, Bell D, Edlund E, Nassor MH, Msellem M, Ali A, Abass AK, Mårtensson A, Björkman A. Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar. Malar J. 2015; 14(1): 43.
The author
Jackie Cook PhD
London School of Hygiene and Tropical Medicine, London, UK
E-mail: Jackie.cook@lshtm.ac.uk
To reduce the window period for HIV-1 infection, a method for detecting trace amounts of HIV-1 p24 in blood is needed. We developed a simple de novo ultrasensitive colorimetric ELISA by adding a thio-NAD cycling solution to the standard ELISA. The limit of detection for p24 was 0.005 IU (i.e. attomoles) per assay by the ultrasensitive colorimetric ELISA.
by Dr A. Nakatsuma, M. Kaneda, H. Kodama, M. Morikawa, S. Watabe, et al.
Background
During the window period between infection with human immunodeficiency virus type 1 (HIV-1) and the appearance of detectable antibodies to HIV-1, the infection cannot be diagnosed. Attempts to shorten this period have been made using a fourth-generation immunoassay that detects both HIV-1/2 IgG/M and HIV-1 p24 antigens [1]. However, most of the commercially available detection systems for fourth-generation immunoassays use chemiluminescent measurement and thus require specialized, highly expensive automated measurement equipment. For this reason, fourth-generation immunoassays are performed only at diagnostics companies and hub hospitals. To overcome this limitation and to test many samples simultaneously, there is need of an immunoassay with increased sensitivity for the HIV-1 p24 antigen that nonetheless uses a common enzyme and does not require any specialized instruments.
In 2010, French health authorities mandated a limit of detection of at least 2 IU/mL of HIV-1 p24 antigen for a Conformité Européenne (CE)-marked HIV antigen/antibody assay [2]. According to this mandate, commercially available assay kits were manufactured to detect p24 antigen with limits of detection ranging from 0.505 to 1.901 IU/mL and from 11.9 to 33.5 pg/mL [2]. Units of pg/mL are used for the Société Française de Tranfusion Sanguine (SFTS) standard (i.e. recombinant proteins), versus IU/mL for the WHO (World Health Organization) standard. As 1 IU/mL is estimated to be equivalent to 10 pg/mL and MW = 24 000 for p24, the best sensitivity in these kits is 0.505 IU/mL, which is ~2 × 10−16 moles/mL.
To date, numerous methods have been proposed for the detection of p24 antigen. However, the limit of detection of p24 antigen is not expected to overcome the sensitivity of 10−17 to 10−18 moles/mL. In addition, we have to note that HIV testing of many samples requires not only ultrasensitive HIV-1 p24 detection but also rapidity, a reasonable cost, and a simple protocol without the requirement of special equipment. In the present review, we introduce a de novo ultrasensitive colorimetric enzyme-linked immunosorbent assay (ELISA) for HIV-1 p24 [3].
Mechanism of ultrasensitive colorimetric ELISA
Watabe and colleagues developed an ultrasensitive ELISA to measure trace amounts of proteins by combining a conventional ELISA with thionicotinamide-adenine dinucleotide (thio-NAD) cycling [4]. Their rationale was that although proteins cannot be amplified by polymerase chain reaction (PCR) in the manner of nucleic acids, a detectable signal for proteins can be amplified. Thus, their ultrasensitive ELISA (Fig. 1) employs a sandwich method using a primary and a secondary antibody for antigens. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α-hydroxysteroid dehydrogenase (3αHSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors.
This method enables the detection of a target protein with ultrasensitivity (10−19 moles/assay) by measuring the cumulative quantity of thio-NADH by a colorimetric method without the use of any special instruments for the measurements of fluorescence, luminescence or radio isotopes [4]. Further, we should note that this ultrasensitive method will allow a technician to detect trace amounts of proteins simply by applying thio-NAD cycling reagents to the conventional ELISA system. We therefore applied this ultrasensitive ELISA to the detection of HIV-1 p24 antigen in blood [3].
Sensitivity and stability of the ultrasensitive colorimetric ELISA for HIV-1 p24
A typical linear calibration curve for HIV-1 p24 antigen provided by the ultrasensitive ELISA coupled with thio-NAD cycling was y = 0.27x + 0.019, R2 = 0.99 in the range of 0.1‒1.0 IU/mL. The limit of detection of p24 was 0.0055 IU/assay (i.e. ~2 × 10−18 moles/assay). These findings indicate that the ultrasensitive colorimetric ELISA succeeds in detecting p24 at the attomole level [3]. Because this measurement system employs a 50 µL solution for each assay, the detection limit corresponded to 0.1 IU/mL, or 10−17 moles/mL. Therefore, even in terms of the concentration per mL, our detection limit is less than one-tenth of that required by the French health authorities [2]. The coefficient of variation was 8% for 1 IU/mL.
Spike-and-recovery test using serum
We attempted to perform spike-and-recovery tests in which the HIV-1 p24 antigen was added to the control serum. Because our results demonstrated that the ratio was about 100% for 0.5 IU/mL of HIV-1 p24, which was less than the value (2 IU/mL) required for a CE-marked HIV antigen/antibody assay (see Background), the ultrasensitive method was judged to sufficiently detect IV-1 p24 antigen in human blood obtained from patients in the very early period after infection.
Detection of HIV-1 p24 in the early stages of infection
It is important to diagnose primary HIV-1 infection and begin antiretroviral treatment as early as possible. Most HIV-1/2 antibody diagnostic tests detect the antibodies for the antigens of HIV-1 gp41 and HIV-2 gp36, which are highly conservative transmembrane proteins. These tests are quick and easy, and thus have been widely used in many clinics and public health centres. However, when only the antibody diagnostic tests are used, there is a long delay (generally a 28-day window period) before diagnosis is possible [5]. Further, HIV-1/2 antibody tests in children younger than 18 months tend to be especially inaccurate as a result of the continued presence of maternal antibodies [6]. To shorten the delay and to validate HIV tests, the HIV-1 p24 antigen, the concentration of which is expected to increase before antibodies emerge, should be detectable in trace amounts. HIV-1 p24 in blood emerges transiently in the very early period after infection, and then its concentration quickly returns to the basal level [5]. An HIV-1 p24 test is, therefore, very useful as a screening test in the early stage of infection.
Closing the gap on PCR-based nucleic acid testing (NAT)
Generally, the gold standard for diagnosing HIV-1 is PCR-based nucleic acid testing (NAT) [7], but this method is expensive and has infrastructure requirements, a long measuring time, and high complexity, thereby limiting its usefulness for large numbers of samples. There is also the issue that much of the world lacks access to reliable NAT, and thus in many geographic regions the policy is to simply wait until symptoms develop. Use of ultrasensitive detection of HIV-1 p24 antigen for early diagnosis would be a simple and reasonable alternative to NAT, such as for monitoring HIV treatment and protecting the blood supply. Accordingly, it is time to reconsider whether NAT should be the gold standard for diagnosing HIV-1. Barletta et al. claimed that the target protein (i.e. HIV-1 p24 antigen) is present in the virion in much higher numbers than viral RNA copies (approximately 3000 HIV-1 p24 antigen molecules versus 2 RNA copies per virion) [8]. The 10−18 moles/assay value in our present results corresponds to 106 protein molecules/assay, or ~103 RNA copies/assay. Although under laboratory conditions a real-time PCR (i.e. NAT) can detect on the order of 101 RNA copies/assay, the limitation of detection is usually in the order of 102 RNA copies/assay [9]. Hence, the ultrasensitive ELISA coupled with thio-NAD cycling for HIV-1 p24 is closing in on the detection limit obtained by NAT, with a margin of difference of only one order of magnitude.
Conclusion
The ultrasensitive ELISA coupled with thio-NAD cycling is a very convenient method for the early testing of HIV-1 infection because it requires only the addition of a thio-NAD cycling solution to the usual ELISA without the use of any specialized measuring equipment. Consequently, the present method could be widely used as a powerful tool to test many samples simultaneously.
References
1. George CRR, Robertson PW, Lusk MJ, Whybin R, Rawlinson W. Prolonged second diagnostic window for human immunodeficiency virus type 1 in a fourth-generation immunoassay: Are alternative testing strategies required? J Clin Microbiol. 2014; 52: 4105–4108.
2. Ly TD, Plantier JC, Leballais L, Gonzalo S, Lemée V, Laperche S. The variable sensitivity of HIV Ag/Ab combination assays in the detection of p24Ag according to genotype could compromise the diagnosis of early HIV infection. J Clin Virol. 2012; 55: 121–127.
3. Nakatsuma A, Kaneda M, Kodama H, Morikawa M, Watabe S, Nakaishi K, Yamashita M, Yoshimura T, Miura T, Ninomiya M, Ito E. Detection of HIV-1 p24 at attomole level by ultrasensitive ELISA with thio-NAD cycling. PLoS One 2015; 10: e0131319.
4. Watabe S, Kodama H, Kaneda M, Morikawa M, Nakaishi K, Yoshimura T. Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method. BIOPHYSICS. 2014; 10: 49–54.
5. World Health Organization (WHO). HIV/AIDS Fact sheet No 360. WHO 2015; http://www.who.int/mediacentre/factsheets/fs360/en/
6. Zijenah LS, Tobaiwa O, Rusakaniko S, Nathoo KJ, Nhembe M, Matibe P, Katzenstein DA. Signal-boosted qualitative ultrasensitive p24 antigen assay for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years. J Acquir Immune Defic Syndr. 2005; 39: 391–394.
7. Patel P, Mackellar D, Simmons P, Uniyal A, Gallagher K, Bennett B, Sullivan TJ, Kowalski A, Parker MM, LaLota M, Kerndt P, Sullivan PS; Centers for Disease Control and Prevention Acute HIV Infection Study Group. Detecting acute human immunodeficiency virus infection using 3 different screening immunoassays and nucleic acid amplification testing for human immunodeficiency virus RNA, 2006-2008. Arch Intern Med. 2010; 170: 66–74.
8. Barletta JM, Edelman DC, Constantine NT. Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen. Am J Clin Pathol. 2004; 122: 20–27.
9. Wagatsuma A, Sadamoto H, Kitahashi T, Lukowiak K, Urano A, Ito E. Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR. J Exp Biol. 2005; 208: 2389–2398.
The authors
Akira Nakatsuma1 PhD, PhC; Mugiho Kaneda1 BAgr; Hiromi Kodama1 MAgr; Mika Morikawa1,2 BASc; Satoshi Watabe3 BPha; Kazunari Nakaishi2; Masakane Yamashita4 PhD; Teruki Yoshimura5 PhD, PhC; Toshiaki Miura6 PhD, PhC; Masaki Ninomiya1 PhD, PhC; Etsuro Ito*1 PhD
1 Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Japan
2 TAUNS Laboratories, Inc., Izunokuni, Japan
3 BL Co., Ltd., Numazu, Japan
4 Faculty of Science, Hokkaido University, Sapporo, Japan
5 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan
6 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
*Corresponding author
E-mail: eito@kph.bunri-u.ac.jp
by Dr Petraki Munujos The anti-parietal cell antibodies show one of the most distinctive fluorescent patterns in the autoantibody screening by indirect immunofluorescence. Although these antibodies react with a well known target antigen (H+/K+ ATPase) solely present in the parietal cells of the gastric gland, the use of combined tissue sections in the same reaction […]
Mass spectrometry-based methods hold great promise for addressing protein heterogeneity. As a result of post-translational processing, proteins can exist in vivo as multiple proteoforms. The added information contained in the protein profile can be important in physiological and pathological states. Presented here is an overview of a mass spectrometric immunoassay (MSIA) for quantitative determination of the chemokine RANTES proteoforms. MSIA offers protein quantification and profiling in a high-throughput and time-efficient manner. Across a cohort of ~300 human plasma samples, a total of 11 different RANTES proteoforms were quantified in less than 3 hours.
by Dr O. Trenchevska, N. D. Sherma, Dr P. D. Reaven, Dr R. W. Nelson and Dr D. Nedelkov
The role of mass spectrometry in protein analyses
Mass spectrometry (MS) has proven successful in the clinical laboratory for the analysis of small molecules, but is on the rise as an emerging methodology for peptides and proteins [1]. Currently, a handful of MS-based protein assays have been adapted in the routine clinical analyses and used for in vitro diagnostic (IVD) testing [2, 3]. MS-based methodologies are the assays of choice because they can overcome the limitations of immunoassays (i.e. nonspecific binding, cross-reactivity of analytes, etc.). In order to be clinically applicable, all MS-based assays should comply with the well-established ‘fit-for-purpose’ approach and be fully validated and characterized [4]. Also, working protocols must be practical (in terms of sample preparation), as well as cost efficient, so they are price-competitive with current immunoassays. Although overcoming these requirements is still a challenge, one inevitable advantage that makes MS-based protein assays indispensable, is their unique ability to address protein heterogeneity.
The majority of clinically adapted MS-based methodologies for protein profiling are the single/multiple reaction monitoring liquid chromatography MS (SRM/MRM LC-MS) assays [5, 6] and mass spectrometric immunoassays (MSIA) [7, 8]. MRM assays are ‘bottom-up’ assays and use isotopically labelled peptides as internal reference standards for surrogate protein quantification via chosen, enzymatically generated peptides. Because SRM/MRM LC-MS assays detect only specific peptides, important information about novel proteoforms or post-translational modifications with potential clinical implications can be overlooked. MSIAs, on the other hand, follow a ‘top-down’ approach, having intact proteins as primary targets. As a result of the immunoaffinity capture of a targeted protein(s), and the ‘soft’ ionization in MALDI-TOF (matrix-assisted laser desorption/ionization–time of flight) MS, MSIA enable for detection of post-translationally modified proteoforms as well as other changes in protein structure without the harsh enzyme digestion. Literature data show that post-translationally modified proteins have the potential to be used as biomarkers [9]. Having that in mind, the proteoform detection adds a whole new dimension to the way we look at proteins.
Mass spectrometric immuno-assay for analysis of RANTES proteoforms
Here we review a mass spectrometric immunoassay (MSIA) for quantification of the chemokine RANTES proteoforms in human plasma samples. RANTES (Regulated on Activation, Normal, T-cell Expressed and Secreted), is a member of the CC chemokine family (hence its alternative name – CCL5) and is essential in the initiation and maintenance of inflammation [10]. RANTES has been studied extensively in clinical context, in association with autoimmune diseases, arthritis, diabetes, obesity and metabolic syndrome, some types of cancer and viral infections [11–13]. In addition, RANTES proteoforms have been associated with atherosclerosis and cardiovascular diseases [14].
There are several types of commercially available, as well as in-house developed immunoassays for total RANTES quantification [15]. These assays, however, are not tailored for detecting and quantifying the numerous proteoforms associated with RANTES. In previous work, we have addressed RANTES heterogeneity by qualitative and quantitative MSIA [16, 17]. In developing the quantitative MSIA for RANTES, we took on the approach of using RANTES standard and a homologous RANTES derivative – met-RANTES as an internal reference standard (IRS) for quantification. Met-RANTES is a recombinant derivative of RANTES (therefore not found in humans) and has a molecular weight (MW) of 7979.2 Da, which is in close proximity to that of full-length human RANTES (MW=7847.9 Da). Another advantage of using the RANTES/met-RANTES pair was the ability of a single anti-RANTES antibody to capture both proteins from the biological samples.
The immobilization of the anti-RANTES antibody was onto activated surfaces of affinity pipettes as previously described [17]. The quantity of the anti-RANTES antibody (7.5 µg Ab/tip) was optimized to be enough that variable RANTES concentrations in the samples could be truly quantified with the assay. Due to low plasma RANTES physiological concentration (in the ng/mL level), undiluted plasma was used for the analyses. In the analytical samples, met-RANTES was spiked at a constant concentration (V=250 µL at c=50 ng/mL), in order to produce a constant signal in the mass spectra. Following sample preparation and affinity pipette derivatization, the antibody-coated pipettes were mounted onto the head of an automated 96-channel pipettor and initially rinsed with PBS/0.1% Tween buffer. Next, the pipettes were immersed into a microplate containing the analytical samples and 500 aspirations and dispense cycles were performed (100 μl volumes each) allowing for affinity capture of RANTES proteoforms and met-RANTES. The pipettes were then rinsed with assay buffer water to remove non-specifically bounded proteins. Captured proteins were eluted directly on a 96-well formatted MALDI target using sinapic acid. Five-thousand laser shots of mass spectra were acquired from each sample spot on a Bruker’s Ultraflex III MALDI-TOF/TOF mass spectrometer. The mass spectra were externally and internally calibrated with protein standard mix and the singly and doubly charged met-RANTES signals before analysis.
In the mass spectra, several RANTES proteoforms can be detected. As shown in Figure 1, most abundant are signals representing full-length, native RANTES (1-68) and met-RANTES, along with the N-terminally cleaved RANTES proteoforms (3-68) [MW=7,663.7; missing the ‘SP’ N-terminal dipeptide, product of dipeptidyl peptidase IV (DPP IV) enzyme cleavage] and (4-68) (MW=7,500.6; missing ‘SPY’ N-terminal tripeptide). RANTES proteoforms missing N-terminal tripeptide and C-terminal dipeptide, (4-66) (MW=7,282.3) completed the dominant signals (Figure 1, top right inlet). Additional RANTES proteoforms were identified, in lower abundance and frequency: (7-66) (MW=6993.1; missing six N-terminal and two C-terminal amino acids), (4-64) (MW=7040.1; missing three N-terminal and four C-terminal amino acids), (4-65) (MW=7153.2; missing three N- and three C-terminal amino acids) and (3-66) (MW=7445.5; missing two N- and two C-terminal amino acids). The signal labelled M-RANTES with MW=7413.5 has multiple N- and C-terminal truncation possibilities, and has not been specifically assigned. The assignation of these signals was done using the observed m/z values and the program Paws, and was in accordance with previously published qualitative results [16].
All identified RANTES proteoforms were quantified using an eight-point standard curve, in the range from 1.56 to 200 ng/mL. The standard curve was constructed from the ratio of the peak intensities of the RANTES standard and the met-RANTES IRS (y-axis) versus the RANTES standard concentration (x-axis). For the analytical samples, first, the RANTES/met-RANTES peak intensity ratios for each proteoform were determined and summed up. Using the generated standard curve equation, these ratios were used to determine the total RANTES concentration in the analysed plasma sample. Then, the concentration of the individual RANTES proteoforms was calculated based on their percentage of the total RANTES. The assay was validated through several standard procedures. The intra- and inter-assay precision experiments yielded coefficients of variation of <10%. Linearity and spiking-recovery experiments produced results between 92 and 112% (observed vs expected concentration). In a final test, the results of the RANTES MSIA were compared with those obtained with commercially available ELISA using Altman–Bland plot. A good correlation, with slight positive bias (11.3%) was obtained with the native RANTES [17].
The developed MSIA for RANTES proteoforms was applied to a cohort of 297 human plasma samples. The analyses were performed on an automated platform, which enabled for a high-throughput analysis of 96 samples in a single run. Among the samples, we were able to determine the concentration and frequency of 11 RANTES proteoforms (Figure 2). The total average concentration of RANTES was found to be 44.9 ng/ml (2.15–163 ng/mL). In majority of samples, the main proteoform was the full-length, native RANTES [c(RANTES(1-68))avg=37.4 ng/mL; 1.92–132 ng/mL], followed by RANTES (3-68), [c(RANTES(3-68))avg =6.64 ng/mL; 0.138–34.4 ng/mL]. The other truncated RANTES proteoforms were present in variable frequencies in the samples, albeit at much lower concentrations (<10% of the total RANTES). Figure 2 summarizes the distribution and frequency of all 11 RANTES proteoforms. Even though majority of RANTES proteoforms were detected in only a handful of samples and in low quantities, they should be given full attention. Cleaved proteoforms have the potential to be used as indicators of an enzymatic activity, and, in turn, of changes in the metabolic homeostasis [18]. The information that this MSIA provides puts a new perspective of RANTES quantitative analysis and can be a good starting point for looking at RANTES heterogeneity in clinical context.
Concluding remarks
The assay described above uses MALDI-TOF-MS to fully quantify RANTES proteoforms, and it is one of just a handful of such MALDI-based assays in existence today. The assay’s two-step approach is similar to that of well-established immunoassays, with the added benefit of MS detection as an enabling factor in differentiating the multiple proteoforms. The MALDI target is designed to accept the eluates from 96 tips at the same time, therefore making it high-throughput and time efficient (total time for RANTES assay is ~1 hour). The assay is performed on an automated platform, which limits the errors that can occur during assay execution. In review of previous and ongoing work, MSIA for RANTES performs well and introduces a new prospect and capacity for potential clinical applications in the field of biomarker discovery/rediscovery and diagnostics.
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The authors
Olgica Trenchevska*1, Nisha D. Sherma1, Peter D. Reaven2, Randall W. Nelson1, Dobrin Nedelkov1
1Molecular Biomarkers, The Biodesign Institute at Arizona State University, Tempe, AZ, USA
2Phoenix Veterans Affairs Health Care System, Phoenix, AZ, USA
*Corresponding author
E-mail:
olgica.trenchevska@asu.edu
November 2025
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