Monoclonal Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the Cronobacter sakazakii. A pair of monoclonal antibodies (mAbs) selected from mAbs produced by 11 murine hybridomas was selected for the sandwich ELISA procedure. Targets of two mAbs were 100 kDa and 42 kDa protein extracted from the bacteria, respectively, which were proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. The limit of detection of this method was established as 1 × 104 cfu/mL, and the linear range from 1 × 105 to 1 × 108 cfu/mL. In the real sample test, 1 cfu/g C. sakazakii was detected in artificially contaminated powdered infant formula with 4 h enrichment.

Obesity is associated with decreasing levels of the circulating soluble leptin receptor in humans.

Leptin plays a major role in the regulation of body weight. It circulates in both free and bound form. One of the leptin receptor isoforms exists in a circulating soluble form that can bind leptin. In the present study, we measured the soluble leptin receptor (SLR) levels in lean and obese humans. We investigated the relationship between plasma SLR levels, plasma leptin levels and the degree of obesity. We also examined whether SLR concentrations could be modulated by fat mass loss induced by a 3 month weight-reducing diet. A total of 112 obese (age 18-50 y; body mass index (BMI) 30-44 kg/m2; 23 men and 89 women), 38 overweight (age 19-48 y; BMI 25-29 kg/m2; 10 men and 28 women) and 63 lean (age 18-50 y; BMI 17-24 kg/m2; 16 men and 47 women) humans. A direct double monoclonal sandwich enzyme-linked immunosorbent assay (ELISA) was used for the quantitative measurement of the soluble human leptin receptor. Leptin was measured by radioimmunoassay (RIA). Body composition was assessed by biphotonic absorptiometry DEXA (dual energy X-ray absorptiometry). We observed that the SLR is present in human plasma (range 10-100 ng/ml). SLR levels were lower in obese and overweight than lean subjects (28.7+/-8.8, 40.2+/-14.9, 51.2+/-12.5 ng/ml, respectively) and were inversely correlated to leptin and percentage of body fat (r=-0.74 and r=-0.76; respectively; P<0.0001). The ratio of circulating leptin to SLR was strongly related to the percentage of body fat (r=0.91; P<0.0001). Interestingly a gender difference was observed in SLR levels, which were higher in obese and overweight men than in obese and overweight women. In obese subjects after a 3 month low-calorie diet, SLR levels increased in proportion to the decrease in fat mass. In the gel filtration profile, SLR coeluted exactly with the bound leptin fractions. Obesity, in humans is associated with decreasing levels of the circulating soluble leptin receptor (SLR). The relationship of SLR with the degree of adiposity suggests that high SLR levels may enhance leptin action in lean subjects more than in obese subjects.

Determination of Endogenous Growth Factors in Human Wound Fluid: Temporal Presence and Profiles of Secretion

Growth factors are important substances in the central control of wound healing during the exudative phase. Although these peptides have been applied frequently to chronic wounds in clinical studies, little is known about the naturally occurring levels at the wound site in correlation to healing in superficial wounds. We have therefore investigated the presence of these cytokines in partial thickness wounds. In 16 patients undergoing reconstructive surgery, split-thickness skin wounds were enclosed in cutaneous vinyl chambers filled with 2.5 ml of saline. Chambers placed over unwounded skin served as controls. After 24 hours, the accumulated wound fluid was harvested and replaced by 2.5 ml of saline until the wounds were healed. Wound fluid was centrifuged, aliquoted, and frozen at -70 degrees C. Samples were analyzed for protein and growth factors (insulin-like growth factor-1, epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor-AB, interleukin-1alpha, and transforming growth factor-beta1 and -beta2) and insulin-like growth factor-binding proteins 1 and 3 using a monoclonal Sandwich enzyme-linked immunosorbent assay and radioimmunoassay. All wounds healed in the liquid environment within 7 days (macroscopically) and 11 days (barrier function), respectively. In wound fluid, protein concentrations dropped from 5 mg/ml on day 1 to a baseline of 0.1 mg (unwounded skin), indicating a return of the barrier function. All growth factors could be measured already after 24 hours postwounding. However, the concentrations measured varied from 10 to more than 10,000 pg/ml between the different factors. The highest range was found for insulin-like growth factor-1 (21,000 to 41,000 pg/ml), the lowest for epidermal growth factor (3 to 63 and 3 to 88 pg/ml, respectively). Two different patterns of kinetics were distinguished: (1) a high initial peak decreasing to baseline values or below serum levels by the time of healing (insulin-like growth factor-1, insulin-like growth factor binding protein-1, -3, basic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor-AB, transforming growth factor-beta1) and (2) a low initial concentration followed by an increase to a maximum at the time of epithelialization (interleukin-1alpha, transforming growth factor-beta2). Comparing the growth factor levels measured to serum baseline values, it was found that four of the growth factors appeared in wound fluid at above serum concentrations (interleukin-1alpha, transforming growth factor-beta2, basic fibroblast growth factor, epidermal growth factor); the other factors never reached serum values in wound fluid (insulin-like growth factor, transforming growth factor-beta1, platelet-derived growth factor-AB). It is concluded that the different profiles of secretion might reflect different functions of polypeptide growth factors such as stimulation of epithelialization (epidermal growth factor, insulin-like growth factor-1), matrix synthesis (transforming growth factor-beta), and inflammatory stimulation (interleukin-1alpha). The concentrations determined could serve as guidelines for adapted administration of growth factors once correlations to healing disorders such as overhealing and ulceration are established.