A Rapid Method for Antigen-Specific Hybridoma Clone Isolation.

Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating >2-fold more hybridoma clones in <25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.

Anaphylactic shock due to patent blue: case report and review of literature.

Intraoperative search for the sentinal node using patent blue is considered a non risk procedure. We emphasize the highly exceptional nature of this adverse effect previously observed in other disciplines using this coloring agent. We present a case of allergic reaction to patent blue in a patient who underwent left mastectomy with sentinel lymph node. About 25 min after the dye injection, the patient developed increased heart frequency and allergic skin reaction. The patient was treated successfully with decreased inspired fraction of inhaled anesthetic and fluid replacement. The patient recovered uneventfully and was discharged from the PACU 3h after the end of surgery without skin changes and was discharged from hospital on the morning after surgery. Allergic reactions with the use of patent blue are far superior to the hypersensitivity reactions seen with anesthetic and adjuvant drugs. Therefore, the anesthesiologist must be aware of cardiovascular instability associated with skin changes during the use of patent blue, for early diagnosis and appropriate treatment of this hypersensitivity reaction to this dye. Skin tests done later confirmed allergy to patent blue dye; the tests induced a small syndrome reaction. Surgical personnel who use patent blue dye should be made aware of the risk of allergic reactions, sometimes severe, to this dye.

Biomimetic ELISA detection of malachite green based on molecularly imprinted polymer film.

A highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of malachite green (MG) using a molecularly imprinted polymer (MIP) film as bionic antibody. The MIP film, based on the self-polymerization of dopamine, was fabricated on the surfaces of a 96-well microplate. It showed specific recognition for MG in aqueous solution. A direct competitive ELISA method was established with the sensitivity reaching 10.31µgL-1 and the detection limit being 0.3µgL-1. The cross-reactivity of two structural analogues to MG was less than 10%. The average recovery tested by MG standard spiking was 88.8% for bass and 90.4% for water, and the relative standard deviations were less than 3.6%. All the above results indicated that the developed method could be used to detect MG in fish and water samples rapidly, specifically and accurately.

Immune reactivity to food coloring.

Artificial food dyes are made from petroleum and have been approved by the US Food and Drug Administration (FDA) for the enhancement of the color of processed foods. They are widely used in the food and pharmaceutical industries to increase the appeal and acceptability of their products. Synthetic food colorants can achieve hues not possible for natural colorants and are cheaper, more easily available, and last longer. However, since the use of artificial food coloring has become widespread, many allergic and other immune reactive disorders have increasingly been reported. During the past 50 y, the amount of synthetic dye used in foods has increased by 500%. Simultaneously, an alarming rise has occurred in behavioral problems in children, such as aggression, attention deficit disorder (ADD), and attention-deficit/hyperactivity disorder (ADHD). The ingestion of food delivers the greatest foreign antigenic load that challenges the immune system. Artificial colors can also be absorbed via the skin through cosmetic and pharmaceutical products. The molecules of synthetic colorants are small, and the immune system finds it difficult to defend the body against them. They can also bond to food or body proteins and, thus, are able to act in stealth mode to circumvent and disrupt the immune system. The consumption of synthetic food colors, and their ability to bind with body proteins, can have significant immunological consequences. This consumption can activate the inflammatory cascade, can result in the induction of intestinal permeability to large antigenic molecules, and could lead to cross-reactivities, autoimmunities, and even neurobehavioral disorders. The Centers for Disease Control (CDC) recently found a 41% increase in diagnoses of ADHD in boys of high-school age during the past decade. More shocking is the legal amount of artificial colorants allowed by the FDA in the foods, drugs, and cosmetics that we consume and use every day. The consuming public is largely unaware of the perilous truth behind the deceptive allure of artificial color.

Enhanced sensitive immunoassay: noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green.

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG-mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R(2)LMG = 0.9841; R(2)MG = 0.993; R(2)Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

[A woman with a blue coloured skin]. / Een vrouw met een blauwe huid.

A 66-year-old woman with carcinoma of the breast developed a blue-grey colouring of the skin, after injection of patentblue for sentinel node procedure. This is a rare hypersensitivity reaction on patentblue, with the risk of depression of oxygen saturation or anaphylactic shock.

Investigation of anaphylactic reaction after patent blue V dye injection.

BACKGROUND: Patent blue dye V (PBV) is in widespread use for sentinel node biopsy in breast cancer and melanoma. At present, the best diagnostic approach in investigating possible anaphylaxis due to PBV is not defined. METHOD: We reviewed our experience of patients and the cases reported in the literature that developed an anaphylactic reaction after injection of PBV and suggest a diagnostic protocol. From May 2006 to April 2009 six patients were known to the Cardiff anaesthetics department to have suffered a severe anaphylactic reaction after injection of PBV. We amalgamated the results of the investigations of our patients with those of 42 case reports published in the literature during the last 10 years. RESULTS: Of 40 patients with a documented allergy history 31 patients did not have a past medical history of allergy. The median interval between PBV administration and allergic reaction was 15 min (range 1 min-180 min). Of 20 patients with hypotension 18 received inotropes. 4 patients had a fall in blood pressure as their sole symptom. 23 patients had urticaria or other allergic skin manifestations, 8 had blue wheals. 5 patients had bronchospasm. 2 patients had a cardiac arrest. They were successfully resuscitated. The median dose of PBV was 2 ml (range 0.5 ml-5 ml). Tryptase levels were elevated in 14 of 26 tested patients. Skin prick testing was positive in 24 of 30 tested patients. Intradermal testing was positive in all 13 tested patients. CONCLUSION: Most patients experiencing a severe allergic reaction to PBV have no past medical history of allergy. The value of formal allergy skin testing for PBV-related allergy lies in excluding other agents as the causative factor to avoid their exposure in the future.

[Anaphylactic shock due to patent blue: four case report and review of literature]. / Choc anaphylactique et bleu patente: à propos de quatre cas et revue de la littérature.

OBJECTIVES: Description of four cases of anaphylactic shock with blue dye injection (bleu patenté V Guerbet 2.5%) during sentinel node biopsy for breast cancer. PATIENTS AND METHODS: Women with breast carcinoma and combined approach with radioactive tracer and blue patent injection. RESULTS: Four cases were observed, which means an incidence of 0.57%. We report one case of grade II anaphylactic reaction and three cases of grade III requiring active reanimation and adrenaline perfusion. One patient developed a pulmonary embolism during the postoperative period, but no death was observed. DISCUSSION AND CONCLUSION: We find an incidence of 1.06% of allergy but only 0.25% of anaphylactic shock (grade III). These rare but serious cases must weigh up the benefits and risks of using blue dye. We suggest using blue dye injection only in case of radioisotope detection failure. Of course, this approach must be evaluated.

Anaphylaxis to Patent Blue V. II. A unique IgE-mediated reaction.

BACKGROUND: Patent Blue V (PBV) is injected in order to map sentinel nodes during cancer staging procedures. Anaphylactic reactions, allegedly IgE antibody mediated, have been reported. The aim of the study was to explore the immunological mechanism of anaphylaxis to PBV. METHODS: PBV allergen threshold basophil sensitivity, CD-sens, was performed on cells from nine patients diagnosed as having had adverse reactions to PBV. The mechanisms of the CD-sens were studied by immunological and immuno-chemical methods. RESULTS: Five of the nine patients had a positive CD-sens to PBV which was completely eliminated by washing the cells in phosphate buffered saline before allergen challenge. However, the positive CD-sens was completely reconstituted by incubating the cells in plasma or serum of that patient or the other PBV-anaphylactic patients for 15 min at room temperature. In some patients the factor mediating CD-sens was completely or partially destroyed by heating at +56 degrees C for 30 min or being exposed to the low pH used for elution from anti-Ig columns. A 1000-fold excess of monoclonal IgE blocked the reconstitution by approximately 50%. CONCLUSION: Anaphylactic reactions to PBV are mediated by IgE antibodies giving a classical CD-sens reaction. However, the allergenic configuration seems to constitute a structure completely dependent on PBV, as a hapten, linked to a, so far, unknown carrier that seems to be unique for patients having experienced a PBV-induced reaction. Further studies are needed to characterize the postulated carrier.

Anaphylaxis to patent blue V: a case series and proposed diagnostic protocol.

Patent blue V is widely used in the identification of sentinel lymph nodes in patients with breast cancer and other malignancies. Individual case reports of allergy to patent blue V have been described in the medical literature since the 1960s. However, there is little data on clinical features and little experience of which allergy tests are appropriate or useful. We gathered all cases of patent blue V allergy that had been seen and diagnosed in the Department of Allergy, Addenbrooke's Hospital over a 3-year period. We collected clinical details of each case including skin test results. For comparison we recruited 12 healthy control subjects who then underwent skin testing to patent blue V. Six cases of patent blue V allergy were identified, all occurring during sentinel lymph node identification for breast carcinoma. All 6 had positive skin prick tests to neat patent blue V (25 mg/ml). Skin prick testing with a 1 : 10 dilution (2.5 mg/ml) produced positive results in 3 of 4 patients tested, and intradermal testing at a 1 : 100 dilution was (0.25 mg/ml) was positive in all patients tested. Of 12 control subjects, 11 had negative skin prick tests to both neat and 1 : 10 patent blue V with one subject showing a positive reaction to the higher concentration only. On the basis of our experience of patent blue V allergy, we propose a diagnostic protocol that can be safely and reliably utilised in centres equipped for allergy testing.

Anaphylaxis to dyes during the perioperative period: reports of 14 clinical cases.

BACKGROUND: Vital dyes are widely used for lymphatic mapping and sentinel lymph node biopsy in patients with malignant tumors, and reports of anaphylactic reactions are becoming more frequent. OBJECTIVE: Our aims were to describe specific clinical features of hypersensitivity reactions to Patent Blue (Guerbet, Roissy, France), results of the allergy workup, and their consequences for patient management. METHODS: We report a series of 14 clinical cases of dye-induced anaphylaxis recorded between 2004 and 2006 in 4 member centers of a network of French allergoanesthesia outpatient clinics. RESULTS: Reactions appeared to be relatively severe (6/14 grade III reactions). An average 30 +/- 6-minute delay was observed between dye injection and symptom onset. In 9 (65%) patients reactions were sustained for several hours, requiring prolonged continuous epinephrine infusion and transfer to an intensive care unit. Prick test results were positive in 8 patients. In 5 patients prick test results were negative, whereas intradermal test results were positive. CONCLUSION: Anesthesiologists and allergologists must be aware of this specific risk and of the clinical characteristics of these reactions, which are usually delayed and long lasting.

Production of antibodies for selective detection of malachite green and the related triphenylmethane dyes in fish and fishpond water.

This study provides a practical method for production of the antibodies against malachite green (MG) and its primary metabolite leucomalachite green (LMG). Two ELISA kits are constructed with the MG and LMG antibodies for detection of the residual MG and LMG in fish muscle and fishpond water. The detection limit is established at the level of 0.05 microg/L for both MG and LMG. Our ELISA kits show the advantages of good specificity, high sensitivity, and convenience in rapid screening of MG and LMG residues. The sample of fishpond water, without extraction or prior preparation, is directly assayed by the ELISA kit. More then 80 fish samples can be simultaneously tested in a kit. The toxic crystal violet and its metabolite leucocrystal violet of illegal use in aquaculture are detected by our prepared MG and LMG antibodies, whereas the antibodies do not cross-react with common antibiotics, sulfonamides, and benzene derivatives.