Enzyme-Linked Immunosorbent Assay: Types and Applications.

Enzyme-linked immunosorbent assay (ELISA) is an immunological assay widely used in basic science research, clinical application studies, and diagnostics. The ELISA technique relies on the interaction between the antigen (i.e., the target protein) versus the primary antibody against the antigen of interest. The presence of the antigen is confirmed through the enzyme-linked antibody catalysis of the added substrate, the products of which are either qualitatively detected by visual inspection or quantitatively using readouts from either a luminometer or a spectrophotometer. ELISA techniques are broadly classified into direct, indirect, sandwich, and competitive ELISA-all of which vary based on the antigens, antibodies, substrates, and experimental conditions. Direct ELISA relies on the binding of the enzyme-conjugated primary antibodies to the antigen-coated plates. Indirect ELISA introduces enzyme-linked secondary antibodies specific to the primary antibodies bound to the antigen-coated plates. Competitive ELISA involves a competition between the sample antigen and the plate-coated antigen for the primary antibody, followed by the binding of enzyme-linked secondary antibodies. Sandwich ELISA technique includes a sample antigen introduced to the antibody-precoated plate, followed by sequential binding of detection and enzyme-linked secondary antibodies to the recognition sites on the antigen. This review describes ELISA methodology, the types of ELISA, their advantages and disadvantages, and a listing of some multifaceted applications both in clinical and research settings, including screening for drug use, pregnancy testing, diagnosing disease, detecting biomarkers, blood typing, and detecting SARS-CoV-2 that causes coronavirus disease 2019 (COVID-19).

Reduced alternative insulin dosing in hyperkalemia: A meta-analysis of effects on hypoglycemia and potassium reduction.

STUDY OBJECTIVE: Recent studies have identified that reduced alternative intravenous insulin doses, such as 5 units or 0.1 units/kg, may reduce the risk of hypoglycemia compared to standard doses of 10 units in patients treated for hyperkalemia. However, some studies suggest that these alternative doses may reduce the ability to lower serum potassium. This study was performed to determine the impact of alternative insulin dosing on hypoglycemia and potassium reduction in patients with hyperkalemia. DESIGN: Meta-analysis. DATA SOURCE: PubMed/MEDLINE, CENTRAL, Ovid, and ClinicalTrials.gov were searched from inception through November 2020. PATIENTS: Patients treated with standard (10 units) or alternative (<10 units) insulin dosing strategies for hyperkalemia. Only studies that evaluated hypoglycemia (serum glucose <70 mg/dl), severe hypoglycemia (serum glucose <50 mg/dl), and potassium reduction post-treatment were included in the meta-analysis. All articles were assessed for bias using the Cochrane Risk of Bias Assessment Tool and Newcastle-Ottawa scales for randomized prospective trials and retrospective trials, respectively. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Ten retrospective cohort studies (n = 3437) were included and had low- or moderate-risk of bias. Alternative insulin dosing strategies included 5 units, 0.1 units/kg, and <10 units. Alternative dosing had lower pooled odds of hypoglycemia (odds ratio [OR] 0.55, 95% confidence interval [CI] 0.43-0.69, I2  = 8%) and severe hypoglycemia (OR 0.41, 95% CI 0.27-0.64, I2  = 0%). No difference in potassium reduction was detected (mean difference -0.02 mmol/L, 95% CI -0.11-0.07, I2  = 53%). CONCLUSIONS: Alternative insulin dosing strategies for hyperkalemia management resulted in less hypoglycemia and severe hypoglycemia without compromising potassium reduction compared to standard dose. Prospective studies are needed to confirm these findings.

Analysis of Student Perceptions of Just-In-Time Teaching Pedagogy in PharmD Microbiology and Immunology Courses.

Just-In-Time Teaching (JiTT) active learning pedagogy is utilized by various disciplines, but its value in a professional pharmacy curriculum has not yet been demonstrated. The purpose of our research study is to implement and evaluate JiTT in a Doctor of Pharmacy (PharmD) program. The impetus in implementing JiTT into a PharmD curriculum was to provide students with an out-of-classroom learning opportunity to enhance knowledge-based skills. The current study summarizes the implementation of JiTT in four distinct instances: two iterations of the required courses "Integrated Microbiology and Virology" (Fall 2016 and Fall 2017) and "Integrated Immunology" (Winter 2016-2017 and Winter 2017-2018). JiTT included knowledge-based questions in multiple-choice format, integrated case studies, and student responses prior to the actual lecture session. After the conclusion of each course, students were asked to provide feedback on the utilization of JiTT by way of an anonymous survey. Following the Fall 2016 iteration of the Microbiology & Virology course, students found the integrated case studies to be beneficial (mean = 3.27 out of a maximum of 4, SD = 0.62), and their overall endorsement of JiTT was high (mean = 3.61 out of 4, SD = 0.50). For the other three courses included in this study, the primary dependent variable was the student's average rating of JiTT, rated on a five-point scale. Aggregating the scores from the Fall 2017 iteration of the Integrated Microbiology & Virology course and both instances of the Immunology course, students rated JiTT very favorably (mean = 4.17 out of a maximum of 5, SD = 0.77). Students' performances in JiTT-based courses were compared against non-JiTT-based courses. Analysis of assessment data for student's performance on knowledge-based questions showed JiTT was helpful for student learning and JiTT-based courses had more consistent exam scores compared to non-JiTT-based courses. The current results are a promising initial step in validating the usefulness of JiTT in a pharmacy program and lays the foundation for future studies aimed at a direct comparison between a traditional lecture style and JiTT pedagogy implemented into PharmD curricula.

An updated systematic review and meta-analysis on the efficacy and tolerability of dipeptidyl peptidase-4 inhibitors in patients with type 2 diabetes with moderate to severe chronic kidney disease.

OBJECTIVE: This updated meta-analysis determines the effect of dipeptidyl peptidase-4 inhibitors on glycemic and tolerability outcomes in patients with type 2 diabetes mellitus and chronic kidney disease with glomerular filtration rate of ⩽60 mL/min or on dialysis. METHODS: In all, 14 citations were identified from multiple databases. Qualitative assessments and quantitative analyses were performed. RESULTS: There were 2261 participants, 49-79 years of age, 49% men and 44% Caucasians. In seven placebo-comparator studies, reduction in hemoglobin A1c at weeks 12-24 was 0.55% (95% confidence interval: -0.68 to -0.43), P < 0.00001). In three sulfonylurea-comparator studies, dipeptidyl peptidase-4 inhibitors did not significantly reduce hemoglobin A1c at weeks 52-54 (-0.15% (95% confidence interval: -0.32 to 0.02)). In one sitagliptin versus albiglutide study, albiglutide significantly reduced hemoglobin A1c in patients with moderate renal impairment (-0.51%). A similar reduction in hemoglobin A1c was seen with sitagliptin versus vildagliptin (-0.56% vs -0.54%). Compared with placebo or sulfonylurea, dipeptidyl peptidase-4 inhibitors did not significantly reduce hemoglobin A1c after 12 and 54 weeks in patients on dialysis. Hypoglycemia was reported by ~30% of patients in both dipeptidyl peptidase-4 inhibitors and placebo groups over 24-52 weeks. While hypoglycemia was more common with a sulfonylurea at 52-54 weeks (risk ratio: 0.46 (95% confidence interval: 0.18 to 1.18)), there was significant heterogeneity (I (2) = 87%). Limitations included high drop-out rate from most studies and small number of active-comparator studies. CONCLUSIONS: Dipeptidyl peptidase-4 inhibitors in patients with chronic kidney disease caused a modest reduction in hemoglobin A1c versus placebo, but not when compared with sulfonylureas or albiglutide, or when used in patients on dialysis. Additional active-comparator studies are needed to further elucidate the role of dipeptidyl peptidase-4 inhibitors in patients with chronic kidney disease stages 3-5 or on dialysis.

Influence of Cyclooxygenase-2 (COX-2) gene promoter-1195 and allograft inflammatory factor-1 (AIF-1) polymorphisms on allograft outcome in Hispanic kidney transplant recipients.

Cyclooxygenase-2 (COX-2) alleles have been associated with allograft outcomes in kidney transplant recipients; however, these alleles may be in linkage with other genes. Human allograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein and is produced by macrophages. Its synthesis is regulated by several cytokines, including interferon gamma. We investigated whether polymorphisms of gene encoding COX-2 and AIF-1 were associated with allograft outcomes among Hispanic renal transplant recipients (RTRs). A total of 527 de novo RTRs of Hispanic ethnicity were included in this study transplanted at St. Vincent Medical Center (SVMC) during 2000-2009. Patients were genotyped for the following: COX-2 (-1195C>T rs689466, intron 6 rs2066826) and AlF1 (rs2269475). Analysis of the results showed that COX-2-1195 CC genotype (OR=1.92, CI%=1.00-3.67, p=0.04) were more frequent, but COX-2-1195 CT genotype was less frequent in kidney allograft acute rejection in comparison with control group (OR=0.59, CI%=0.38-0.91, p=0.017). The genetic variant TT/CT of the AIF-1 gene was associated with a lower risk of rejection (OR=0.63, CI%=0.41-0.98, p=0.038). No association of COX-2 (rs2066826) was observed with allograft rejection. We are unable to find statistically significant association between COX-2 and AIF-1 gene polymorphisms and allograft survival. The -1195C>T in the COX-2 promoter and AIF-1 gene polymorphisms could be a potential predictor of allograft rejection in our Hispanic kidney transplant recipients.

Genetic polymorphisms of UGT1A8, UGT1A9 and HNF-1α and gastrointestinal symptoms in renal transplant recipients taking mycophenolic acid.

Mycophenolic acid (MPA), a widely used immunosuppressant, has a complex metabolism that involves a number of enzymes. Some of its metabolites are thought to be the cause of gastrointestinal (GI) side effects. In this study, we investigated whether polymorphisms of UDP-glucuronosyltransferases (UGT1) A8, 1A9, and hepatocyte nuclear factor (HNF1α) genes or pharmacokinetic parameters of mycophenolic acid (MPA) were associated with the severity of GI symptoms in patients receiving MPA therapy. A total of 109 kidney transplant patients taking mycophenolic acid (MPA) derivatives were genotyped for UGT1A8, 1A9 and HNF1α genes. Among these, a total of 15 patients were participants in the pharmacokinetic study. Severity of GI symptoms was assessed using a validated Gastrointestinal Symptom Rating Scale (GSRS). The overall and subscale GSRS scores were measured at 1 week (baseline), 2 weeks, 3 months and 6 months post-transplantation. In the case of the pharmacokinetic study, EC-MPS was administered and a total of nine blood samples were obtained at -1, 0, 0.5, 1, 2, 4, 6, 8, and 12h. Genotypes of UGT1A8 were significantly associated with the overall GSRS scores at week 1 (p=0.02) and week 2 (p=0.036). Subscales were only statistically significant for constipation at week 1 (p=0.002) and indigestion at week 2 (p=0.02), while UGT1A9 was only significant for the constipation at week 1 (p=0.04). HNF1α genotypes were significantly different at week 1 in the overall GSRS (p=0.004), and for abdominal pain (p=0.04), acid reflux (p=0.036) and constipation subscales (p=0.04). In addition, abdominal pain was statistically significantly different at 3 months and 6 months after transplantation (p=0.03 and 0.02, respectively). In the case of the pharmacokinetic study, we have found some correlations between MPAC0 and constipation (p=0.02) where MPAAUC was correlated with acid reflux (p=0.02) and constipation (p=0.012), MPAGCL/F was correlated to acid reflux, indigestion, constipation and the sum of the GSRS scores (p=0.037, p=0.032, p=0.033 and p=0.04, respectively). Multinomial regression analysis for MPAGCL/F showed a statistical significance for the subscale indigestion and the sum of the GSRS (p=0.033 and p=0.037, respectively). Our data suggests that among patients receiving MPA the UGT1A9 alleles might play a role in determining the severity of early GI side effects, while the HNF1α allele appears to be associated with a later effect as well as early side effects. Our data also showed that some kinetic parameters might predict MPA side effects.

Association of vitamin D binding protein polymorphism with long-term kidney allograft survival in Hispanic kidney transplant recipients.

Polymorphism of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (VDBP), have been widely explored due to the complex role played by vitamin D in renal transplant outcomes. In this study, we investigated whether polymorphisms of genes encoding VDR and VDBP were associated with allograft survival or acute rejection (AR) among a Hispanic kidney transplant population. A total of 502 Hispanic renal allograft recipients at the St. Vincent Medical Center between 2001 and 2010 were genotyped for four different single nucleotide polymorphisms of VDR: FokI C>T (rs2228570), BsmI G>A (rs1544410), ApaI T>G (rs7975232), and TaqI T>C (rs731236). We also performed genotyping for one common polymorphism in the VDBP gene (rs4588). Survival was significantly improved for patients who were homozygous GG for the rs4588 G>T allele in the VDBP gene (GG vs. GT + TT, OR = 0.63, p = 0.02) while GT genotype was associated with a higher risk of graft loss (GT vs. GG + TT, OR = 1.67, p = 0.01). We found no association for polymorphic markers in VDR with allograft survival and AR. The frequency of the haplotype GTCG (in the order of VDR FokI C>T, BsmI G>A, ApaI T>G, and TaqI T>C), was significantly different in the patients with graft rejection compared to the control (p = 0.007) while ACCA haplotype was found to be associated with graft loss (p = 0.02). Hence, the VDBP G>T polymorphism (rs4588) and two haplotypes (GTCG and ACCA) of VDR appear to be associated with renal allograft outcomes among Hispanic allograft recipients.

Impact of NF-κB gene polymorphism on allograft outcome in Hispanic renal transplant recipients.

BACKGROUND: The dimeric NF-κB transcription factors play critical roles in diverse cellular processes including adaptive and innate immunity, cell differentiation, proliferation and apoptosis. It regulates the expression of numerous genes that play a key role in the inflammatory response during kidney allograft rejection. This study aims to determine the association of NF-κB gene polymorphisms with allograft outcomes in the Hispanic renal transplant recipients. METHODS: A total of 607 Hispanic renal transplant recipients at St. Vincent Medical Center between 2001 and 2010 were included in this study. The NF-κB genotypes were studied along with clinical data. In the case of NF-κB genotypes, the following single nucleotide polymorphisms (SNPs) were included: NF-κB1 (rs3774959, rs3774932, rs3774937, rs230526, rs230519), NF-κB2 (rs1056890, rs7897947, rs12769316) and NF-κB inducing kinase (NIK) (rs9908330, rs7222094). The association of each genotype with renal allograft survival and acute rejection was evaluated. RESULTS: NF-κB1 (rs3774937) CC genotype showed protective association with allograft rejection (OR=0.66, 95% CI=0.44-0.99, p=0.04). There was a significant increase in allograft survival time associated with the NF-κB1 (rs3774959) A allele (OR=0.76, 95% CI=0.60-0.98, p=0.03) while GG genotype was associated with a higher risk of graft failure (OR=1.51, 95% CI=1.02-2.21, p=0.03). There were no associations between polymorphic markers in NF-κB2 and NIK genes with allograft survival or acute rejection. Among non-genetic factors, we found that the use of tacrolimus, a deceased donor, delayed graft function and acute rejection were associated with allograft failure. CONCLUSION: The result of present study suggests that NF-κB1 gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.

The pharmacokinetics of enteric-coated mycophenolate sodium and its gastrointestinal side effects in de novo renal transplant recipients of Hispanic ethnicity.

OBJECTIVE: The aim of the study is to characterize the pharmacokinetics and the gastrointestinal side effect profiles of enteric-coated mycophenolate sodium (EC-MPS) in de novo kidney transplant patients of Hispanic ethnicity. MATERIALS AND METHODS: The pharmacokinetic study of EC-MPS was conducted in 11 de novo kidney transplant patients of Hispanic ethnicity. Eight blood samples were obtained after EC-MPS was given at the steady state. Blood concentrations of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) were measured. RESULTS: The mean age (± standard deviation) was 39.4 (± 12.3) years. The mean daily dose of EC-MPS at the time of the pharmacokinetic study was 1408 ± 108 mg. For MPA and MPAG, the time to peak concentration was 2.5 ± 1.3 hours and 4.6 ± 3.1 hours, respectively; the peak concentration (Cmax) was 19.3 ± 17.2 mg/L and 109.4 ± 49.2 mg/L; and the area under the curve from 6 to 12 hours (AUC6-12) was 32.2 ± 19.3 mg·hr/L and 373.7 ± 235.8 mg·hr/L, respectively, which represents 41.3% and 43.0% of AUC0-12. The AUC0-12 for MPA measured 77.8 ± 53.1 mg·hr/L and for MPAG 869.2 ± 388.8 mg·hr/L. Seven patients (64%) exhibited a second peak at approximately 8.3 hours after the dose at a mean concentration (± standard deviation) of 10.3 ± 7.6 mg/L. The Cmax or AUC of MPA does not correlate with overall Gastrointestinal Symptom Rating Scale scores or subscale scores, but the Cmax of MPAG correlates with indigestion subscale (P = 0.022), diarrhea (P = 0.032), and overall scores (P = 0.028). The AUC of MPAG also correlates with acid reflux (P = 0.024) and indigestion (P = 0.032). DISCUSSION AND CONCLUSION: The pharmacokinetics of EC-MPS has a high variability in de novo kidney transplant patients of the Hispanic ethnicity, which was similar to other ethnic groups. The MPA exposure expressed by the AUC appears to be higher in Hispanic patients than those reported in other ethnic groups, which may be the result of various factors such as difference of the uridine diphosphate glucuronosyltransferase enzyme genotypes, but gastrointestinal side effects were acceptable and the Cmax or AUC of MPAG showed correlations with gastrointestinal symptoms.