Why is western blotting used to confirm positive ELISA HIV tests?

Why is western blotting used to confirm positive ELISA HIV tests?

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I know that when an ELISA test indicates a positive result for HIV, a western blot is done to confirm. When I search online for the reason why, all that I see is that western blotting is "more specific" than ELISA. If they both use antibodies, shouldn't these procedures have similarly accurate results?

My only guess would be that the combination of Ab-Ag binding in addition to expecting your antigen to be a certain size is sort of a "double yes" and thus western blot results would possibly be taken as more accurate.

One reason a Western blot is more specific than an ELISA - even one using the same set of antibodies - is background.

Antibody-linked colorimetric reactions aren't completely on/off. There's always some background reaction. The antibodies are a little bit cross reactive, or non-specifically sticky, or the colorimetric coupling enzymes aren't completely washed away, etc. There's a number of reasons you'll get a small amount of signal even in the abscence of the desired reagent.

So imagine how this non-specificity looks in an ELISA assay. You're only getting a single readout. Any non-specificity/background in the assay adds noise to your output level. Hopefully for any decent assay the noise is normally well below the threshold for crossing over into a "positive" readout, but if the noise is Normally distributed, there's always a small chance that the noise will exceed that threshold. (There's also those non-normal failure modes, where you mess up a wash step, or you get a bad tube of antibody, etc. Negative controls help, but only so much.)

In contrast, think about how non-specificity/background looks in a Western blot. If you get non-protein linked background, the presence of this background will be across the entirety of the blot. It's very obvious that something went wrong with the assay in those cases. Even if you get random spots and not overall background, the chance you'll get a random spot at just the correct molecular weight is very, very unlikely.

An additional benefit is that Western blots first separate the proteins prior to doing antibody-linked detection. This means if there's some protein in the sample which cross-reacts with the antibody, it's very likely it will migrate at a different molecular weight and not be counted as a false positive. (As opposed to ELISAs, where every signal counts toward the total.)

These effects mean that if you're looking for low false positives (as you would with an HIV test) a well-done Western blot is likely to give a more confident signal than a well-done ELISA would. (At the cost of time and effort.)

A final issue is that combining the two will give you more confidence in a positive result than either one alone, especially if you use different antibodies for each. The final confidence in a positive result is not just the confidence from the Western blot, but the confidence that both the Western and the ELISA report positive results, so there's a benefit of doing both, even if they were equally specific.

Why is western blotting used to confirm positive ELISA HIV tests? - Biology

There are no reference materials for modified proteins except for glycated hemoglobin.

Commercial non-enzymatically modified proteins exist, but they are rarely used.

Non-enzymatically modified protein standards are mostly prepared “in house”.

There is no official proficiency testing scheme, except for glycated hemoglobin.

There are no criteria relating quantity of the modified protein and health assessment.

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University of Denver, Denver, CO, United States

E-mail address for correspondence: [email protected]

Keywords: quality of care, accountability, performance measures

The quality and safety of today's medical care are quite variable and there is growing concern about runaway health care costs. Studies have demonstrated poor adherence to guidelines, indefensible variations in care, high cost and waste. As a result, there is growing distrust of the medical profession and calls for greater accountability by consumers, payers and regulators. A variety of methods are used to achieve accountability including competition, transparency, differential payment, accreditation, regulation and litigation. All of these approaches rely on performance measures even though measuring quality of care is in its infancy. Today's emphasis on information technology to assure accountability has created high societal expectations that can only be met if greater emphasis and resources are applied to measure quality of care.

Common measures of health care quality include: (1) how health care is organized (structure) (2) what is done (process) and (3) what happens to the patient (outcome). Because outcomes are the most difficult to obtain, many systems rely heavily on structure and process measures to hold physicians accountable. Successful approaches require system thinking, a culture of process improvement, and the development of performance data that truly reflect cost-effective patient care. Barriers that have to be overcome include: insufficient evidence, the cost of information technology, a perceived threat to physician autonomy, a traditional silo approach to health care delivery, and competing priorities.

Increasingly, academic medicine must support fundamental research in quality, safety and efficiency. Successful models for quality assessment and improvement will be presented and include creating and implementing data registries, developing and promoting cross department collaboration and fostering a no-blame culture of accountability. Future medical practice must extend beyond the quest for medical innovation to incorporate systems and contexts for using medical discoveries cost-effectively.



1 California Pacific Medical Center, San Francisco, CA, United States, 2 University of Texas Health Science Center, San Antonio, TX, United States, 3 University of Kentucky, Lexington, KY, United States, 4 Louisana State University Health Sciences Center, New Orleans, LA, United States, 5 University of Alberta, Edmonton, AB, Canada, 6 Neurological Institute(NI-9), New York, NY, United States, 7 UCSF Fresno, Freson, CA, United States, 8 London Health Sciences Center, London, ON, Canada, 9 American Academy of Neurology, St. Paul, MN, United States

E-mail address for correspondence: [email protected]

Keywords: evidence-based medicine, management of ALS, practice parameters

Background: The American Academy of Neurology (AAN) issued an evidence-based report on managing patients with amyotrophic lateral sclerosis (ALS) in 1999.

Objective: To systematically review evidence bearing on the management of patients with ALS and update the 1999 AAN practice parameter.

Methods: The authors completed a systematic literature review from 1998 to 2008. Topics included breaking the news, symptom management, slowing disease progression, nutrition, respiratory management, palliative care, cognitive and behavioral impairment, multidisciplinary clinics, and communication for patients with ALS.

Results: The authors identified 10 Class I studies, 13 Class II studies, and 73 Class III studies in ALS. More studies are clearly needed to examine the best tests of respiratory function in ALS, the optimal time for starting PEG, the impact of PEG on quality of life and survival, the effect of vitamins and supplements, symptomatic therapies and palliative care. The following recommendations are made based on the studies analyzed: Riluzole should be offered to slow disease progression (Level A). Percutaneous endoscopic gastrostomy (PEG) should be considered to stabilize weight and to prolong survival (Level B). Noninvasive ventilation (NIV) should be considered to treat respiratory insufficiency in order to lengthen survival (Level B), and may be considered to slow the decline of forced vital capacity (Level C) and improve quality of life (Level C). Early initiation of NIV may increase compliance (Level C), and insufflation/exsufflation may be considered to help clear secretions (Level C). Multidisciplinary clinic referral should be considered to optimize health care delivery and prolong survival (Level B) and may be considered to enhance quality of life (Level C). For the treatment of refractory sialorrhea, botulinum toxin B should be considered (Level B) and low-dose radiation therapy to the salivary glands may be considered (Level C). For treatment of pseudobulbar affect, the combination therapy of dextromethorphan with quinidine should be considered, though side effects are not uncommon and the treatment is currently not approved by the U.S. Food and Drug Administration (Level B). For patients who develop fatigue while taking riluzole, withholding the drug may be considered (Level C). Because many patients with ALS demonstrate cognitive impairment, which in some cases meets criteria for dementia, screening for cognitive and behavioral impairment should be considered in patients with ALS (Level B). Other management strategies all lack strong evidence.

Discussion: There are many treatments available for patients with ALS that can alleviate suffering. NIV, PEG, riluzole, and multidisciplinary clinics are the most important and have the best evidence.

Conclusions: More high-quality, controlled studies are needed to guide management and to assess outcomes in patients with ALS.



1 Wisdom Hospice, Rochester, United Kingdom, 2 St Catherine's Hospice, Scarborough, United Kingdom, 3 Marymount Hospice, Cork, Ireland, 4 Weldmar Hospice, Dorchester, United Kingdom, 5 St Christopher's Hospice, London, United Kingdom, 6 Cynthia Spencer Hospice, Northampton, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: opioids, palliative care, end of life

Background: Specialist palliative care providers (SPC) are often involved in the care of people with ALS/MND in the UK, particularly in the management symptoms at the end of life. Despite this experience in the use of medication in the management at the end of life many patients and their families and professionals fear the last days of life with ALS/MND and the use of medication at this time.

Objectives: This study aimed to show the medication used in the last 72 hours of life within six specialist palliative care units in the UK and Ireland.

Methods: Six SPC units provided details of the last 10 patients who had died under their care. Patient information was collected together with the details of medication used in the last 72 hours before death.

Results: 60 patient records were audited −63% male and 37% female with a mean age of 67 years. The mean time from first symptom to death was 32 months. The majority of patients received medication in the last 72 hours of life, primarily:

Morphine-23 patients in the last 24 hours, commonly (38%) by subcutaneous infusion with a mean dose of 80mg (oral equivalent) over 24 hours

Midazolam-35 patients, commonly by subcutaneous infusion with a mean dose of 31mg/24 hours

Anticholinergic medication-as glycopyrronium bromide or hyoscine hydrobromide- 35 patients

All patients were reported as dying peacefully, without distress.

Discussion and Conclusion: This study showed that medication is commonly given within SPC units for the management of symptoms at the end of life. The doses used are similar to those in other studies and in surveys of cancer patients-for ALS/MND the studies showed a mean oral equivalent dose of morphine of 98mg/24hours (1) and 90mg/24 hours (2), and for cancer patients a mean dose of 166mg/24 hours (3).

The results show that professionals can feel secure in the administration of medication at the end of life and the doses used are not large and similar or less than for other terminal care groups. Patient and families can also be reassured that with good symptom management and the best use of medication dying form ALS/MND is peaceful.




1 University of Ulm, Ulm, Germany, 2 Eberhard-Karls University, Tübingen, Germany, 3 University of Liége, Liége, Belgium, 4 University of Würzburg, Würzburg, Germany

E-mail address for correspondence: [email protected]

Keywords: end-of-life decision, depression, cognitive processes

Background: Over the course of the disease, ALS patients have to make decisions with regards to life-sustaining treatment (non-invasive ventilation, NIV percutaneous endoscopic gastrostomy, PEG invasive ventilation, IV). Depression and quality of life were found to be independent of the progression of the disease but to be predictors of the wish to die and physician-assisted suicide (PAS). Data on the processes underlying these end-of life decisions are sparse.

Objectives: Our investigation aimed to identify factors underlying the end-of-life decision by a longitudinal approach. The course of depression, subjective quality of life, attitudes toward life sustaining treatment and hastened death, as well as the development of the decisional process itself were investigated.

Methods: Patients with definite ALS and no cognitive impairment were eligible to participate in the study. The time between interviews was 6 months. Depression, subjective quality of life and attitudes toward hastened death were assessed by standardized instruments. Attitudes and other cognitive factors regarding the end-of-life decision were assessed by the Life Sustaining Treatment Questionnaire (Häcker, 2008, unpublished).

Results: T1: The sample consisted of 61 patients. Depression was neither related to demographic or disease related variables (time since diagnosis, bulbar symptoms, pain, ventilatory status) nor associated with the wish to die. Level of depression was related to quality of life (r = − 0.513), fear of death (r = 0.324), and the wish for legalizing PAS (r = 0.542). Quality of life was highest in IV-patients and was not associated with disease progression or bulbar symptoms. Low quality of life was related to stronger approval of the legalisation of PAS (r = − 0.438) and a stronger wish to die. About half of the sample had not made a decision regarding life sustaining treatment yet. Individuals reporting a positive decision toward life sustaining treatment and those who had not made a decision yet, reported a low wish to die. The highest wish to die was found in patients who decided against life sustaining treatment.

T2: Present results suggest that depression, quality of life and the wish to die do not change over time. Only the fear of death decreased significantly.

Discussion/conclusion: Most strikingly, attitudes toward life sustaining/shortening treatment might not be related to the disease itself. Rather, they seem to be mediated by psychological processes that could be subject to intervention. During all stages of the disease, a high quality of life and low levels of depression are possible. The results can contribute to the general debate over the legalisation of PAS. Focus of this debate should lie on the necessity to ensure every possible support to ALS patients to improve their well being towards the end-stage of the disease.



Preston MND Care and Research Centre, Preston, Lancashire, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: preferred priorities of care (PPC), choices end of life care, place of death

Objectives: The Preferred Priorities of Care document (PPC) gives the terminally ill patient an opportunity to think, talk about and write down preferences and priorities for end of life care. Our aim was to establish if patients had specified a choice of where their care would be received at their end of life, that this had been achieved and if not what had happened to prevent this.

Methods: Comparisons were made between three groups of patients with ALS, those who completed a PPC, those who had had discussions with the MND specialist nurse with regard to their PPC but had not formally recorded their wishes and those who did not wish or had not had the opportunity to complete a PPC. Data recorded included their preferred place of terminal care

Results: Of the 44 people who chose to complete preferred priorities of care, thirty-nine stated they would prefer to die at home. Twenty-five of these achieved their death at home, three in a hospice, nine in hospital, two in nursing homes. Two patients wished to die in a hospice as their second choice and this was achieved. Two chose to die in hospital and one in a nursing home.

Of the 45 patients who did not complete a PPC eighteen died at home, sixteen in hospital, eight in a nursing home and two in hospice. Fourteen patients had had discussions about PPC but had not made decisions on place of terminal care. Six of these died in hospital, five at home, two in a nursing home and one in hospice.

The main reasons for not achieving their desired PPC included carers being unable to cope and sudden change in medical condition.

Discussion: It is suggested that in the UK between about 80–90% of those with a terminal illness expressed a preference for death at home but 60% die in hospital. The results of our audit suggest that patients have a higher chance of achieving their preferred place of end of life care if their wishes have been recorded on a PPC. Further investigations should be made to establish causes of breakdown in care leading to hospital admission in the last few days or hours of life and how these admissions can be prevented



1 Penn State Hershey Medical Center, Hershey, Pennsylvania, United States, 2 ALS Association, Greater Philadelphia Chapter, Harrisburg, Pennsylvania, United States, 3 Penn State University, Harrisburg, Pennsylvania, United States,

E-mail address for correspondence: [email protected]

Keywords: caregiving, quality of life, evidence based practice

Background: Information on how to support caregivers of ALS patients is sparse. An Evidenced Based Practice (EBP) approach was used to identify potential interventions to support caregivers. EBP is the process of systematically searching for the best available evidence to support a clinical decision or clinical intervention.

Objectives: To report on the process by which we used EBP to develop a questionnaire which can be used to design interventions to support ALS caregivers.

Methods: A multidisciplinary team used EBP to gather and analyze available information on what can be done to support ALS caregivers. Information on caregiving was obtained from three sources: 1) published literature 2) expert opinions 3) caregiver focus groups. The information was reviewed for potential intervention development.

Results: Published literature: 70 relevant articles were retrieved, 21 of which were applicable to intervention development: Level 1 (systematic review or multicenter controlled studies)-5 articles Level II (single-center controlled studies)-4 articles Level III (case-control studies)-9 articles Level IV (qualitative reviews or studies)-3 articles. The study population in the articles included 393 ALS caregivers, 4154 Alzheimer's caregivers, and 2062 other caregivers. The literature revealed the following: early interventions are most effective high risk-factors and protective functions for caregivers can be identified interventions should be multifocal strategies exist to decrease caregiver perceptions of burden extra supports aid well-being. Expert opinions generated by clinicians from an interdisciplinary ALS clinic supplemented the literature: educate about all aspects of the disease at 3 month intervals identify sources of stress and coping identify available support systems caregiving experiences differ depending on the relationship to the patient caregivers need encouragement to call the ALS team for assistance. Caregiver focus groups reinforced services that are helpful (hospice repeating information about services emotional/social supports from friends/community) and identified areas in which more attention is needed (finding good in-home care providing information and options about equipment removing physical signs after the loved one dies). The three sources of information were reviewed by the EBP team, who determined that the design and administration of a Caregiver Assessment Form in ALS clinic would potentially be a useful intervention. The assessment form includes demographics, assessment for caregiver risks (concern with tasks, health and well-being, stress), and protective functions (optimism, confidence, spirituality).

Discussion and Conclusions: EBP can be used to foster collaboration between clinicians and researchers to design and construct an instrument for the assessment of ALS caregivers. Such a tool can form the basis for interventions to maximize the quality of life of caregivers of patients with ALS.

Lipase Medicina Laboratorial

A water molecule then donates a proton to the histidine, creating a reactive hydroxyl anion. The hydroxyl anion can then attack the carbonyl carbon of the lipid, forming another negatively charged tetrahedral intermediate which is stabilized in the oxyanion hole (Reaction 3).

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Se precisar de aconselhamento de um especialista, marque uma consulta online.

Stability in human samples is 1 week at room temperature or 4C and 1 year at -20C (per package insert).

LPL gene encodes lipoprotein lipase, which is expressed in the heart, muscle, and adipose tissue. Through catalysis, VLDL is converted to IDL and then to LDL. Severe mutations that cause LPL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LPL are linked to many disorders of lipoprotein metabolism.[28]

The primary function of lipase is to help the body process and absorb fat, but science is starting to show that there are other benefits that may come from it. One of the most essential is the role it plays in managing triglycerides. This is a form of fat that is needed for energy, and you should have some levels of triglycerides in the body but higher levels can lead to heart issues.4

Hyperlipasemia may be described as an excess of the pancreatic enzyme, lipase, in the blood. High levels may indicate a problem related to your pancreas. Digestion of your dietary intake of carbohydrates and fat, rely on the action of the pancreatic enzyme amylase, found in the saliva, to begin the digestion of starches. At the same time, lipase from your gastric secretions breaks down the fats. The salivary and gastric lipases work to stabilize fats from food while the food is being digested in the stomach. Blood amylase and lipase levels are most frequently drawn to diagnose pancreatitis.

There are many reasons why you might be experiencing abdominal pain or other symptoms. Amylase and lipase tests are just pieces of the puzzle. Your doctor will first take a medical and family history, perform a physical examination, and ask if youre taking any medications.

jmolSetTarget(1)jmolLink(delete clickGreenLinkEcho refreshsetL = setLoading() javascript setL script /wiki/extensions/Proteopedia/spt/wipeFullLoadButton.spt isosurface DELETE scn = load(/wiki/scripts/Lipase/Lipase_colipase_inhibitor/1.spt) scn = scn.replace(\_setSelectionState, \_setSelectionState message Scene_finished) script inline scn,Methoxyundecylphosphinic acid (MUP),Methoxyundecylphosphinic acid (MUP)) (purple), a C11 alkyl phosphonate, is a competitive inhibitor of pancreatic lipase. It binds directly in the active site pocket. There are also five B-octylglucoside (gray and red) molecules which associate with lipase. MUP forms hydrogen bonds with jmolSetTarget(1)jmolLink(delete clickGreenLinkEcho refreshsetL = setLoading() javascript setL script /wiki/extensions/Proteopedia/spt/wipeFullLoadButton.spt isosurface DELETE scn = load(/wiki/scripts/Lipase/Inhibitor_interaction/1.spt) scn = scn.replace(\_setSelectionState, \_setSelectionState message Scene_finished) script inline scn,four residues,four residues): Ser 152 and His 263, which are part of the catalytic triad, and Phe 77 and Leu 153 which are the stabilizing residues located in the oxyanion hole [20].spt isosurface DELETE scn = load(/wiki/scripts/Lipase/C11p_bound_h_phobics/2.spt) scn = scn.replace(\_setSelectionState, \_setSelectionState message Scene_finished) script inline scn,further stabilized,further stabilized) by van der Waals contacts with hydrophobic side chains Ala 178, Phe 215, Pro l80, Tyr ll4, Leu 213 (shown in blue).

The concentration of LPL displayed on endothelial cell surface cannot be regulated by endothelial cells, as they neither synthesize nor degrade LPL. Instead, this regulation occurs by managing the flux of LPL arriving at the lipolytic site and by regulating the activity of LPL present on the endothelium. A key protein involved in controlling the activity of LPL is ANGPTL4, which serves as a local inhibitor of LPL. Induction of ANGPTL4 accounts for the inhibition of LPL activity in white adipose tissue during fasting. Growing evidence implicates ANGPTL4 in the physiological regulation of LPL activity in a variety of tissues

Recently, Birrane et al. (19) solved the structure of a lipid-free LPL:GPIHBP1 complex by X-ray crystallography. Two LPL molecules were present in the crystallographic unit, and they interacted in a reciprocal fashion at a single site, between the hydrophobic Trp-rich motif in the carboxyl-terminal domain of one LPL molecule (sequences that mediate lipoprotein binding) (5, 6) and the hydrophobic catalytic pocket in the amino-terminal domain of the other LPL molecule (sequences that hydrolyze triglycerides). The same orientation was observed for LPL in solution by small-angle X-ray scattering (SAXS) analyses (19). The conformation observed by X-ray crystallography and SAXS would appear to support the long-held assumption that LPL is a homodimer. Again, however, there are caveats. One caveat is that the LPL homodimers observed by X-ray crystallography and SAXS occurred in the setting of high concentrations of LPL (0.715 mg/mL). It is likely that high protein concentrations favor homodimer formation. If catalytically active homodimers exist physiologically, then it seems likely that they would exhibit a conformation distinct from that observed in the crystal structure.

Easy availability, high sensitivity, and technical simplicity are touted as the advantages of amylase testing, but its greatest disadvantage is its low specificity. Serum lipase is a more reliable diagnostic marker of acute pancreatitis than serum amylase. [6] The major benefit of lipase is in patients with delayed presentation, [7, 8] since its activity remains increased for longer periods (up to 8-14 days), and an increased sensitivity in acute alcoholic pancreatitis. [4, 5] Nonspecific elevations of lipase levels have been reported in almost as many disorders as amylase.

Lipoprotein lipase has been shown to interact with LRP1.[51][52][53] It is also a ligand for 2M, GP330, and VLDL receptors.[23] LPL has been shown to be a ligand for LRP2, albeit at a lower affinity than for other receptors however, most of the LPL-dependent VLDL degradation can be attributed to the LRP2 pathway.[23] In each case, LPL serves as a bridge between receptor and lipoprotein.While LPL is activated by ApoC-II, it is inhibited by ApoCIII

Two molecules of ApoC-II can attach to each LPL dimer.[25] It is estimated that up to forty LPL dimers may act simultaneously on a single lipoprotein.[5] In regard to kinetics, it is believed that release of product into circulation is the rate-limiting step in the reaction

In the presence of colipase, the enzyme is activated which moves the jmolSetTarget(1)jmolLink(delete clickGreenLinkEcho refreshsetL = setLoading() javascript setL script /wiki/extensions/Proteopedia/spt/wipeFullLoadButton.spt isosurface DELETE scn = load(/wiki/scripts/Lipase/N-terminal_flap/1.spt) scn = scn.replace(\_setSelectionState, \_setSelectionState message Scene_finished) script inline scn,N-terminal flap,N-terminal flap)(shown in red, active site in green) which is composed of amino acids 216-239. The N-terminal flap moves in a concerted fashion along with the C-terminal domain to reveal the active site (green), allowing it to bind with a substrate. It is hypothesized that this flexibility may have significance in binding the colipase-lipase complex with the water-lipid interface.[16]


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