Direct ELISA Method Research Articles

Fast detection of atrazine in corn using thermometric biosensors

Fast detection is important in screening large-scale samples. This study establishes a direct competitive ELISA method (dcTELISA) based on an enzyme thermistor for fast atrazine (ATZ) detection. ATZ competes with β-lactamase-labeled ATZ (ATZ-E) for the binding sites on anti-ATZ monoclonal antibody (mAb). The mAb are covalently bound to Controlled Pore Glass (CPG) in an immunoreactor to form immunocomplexes with ATZ and ATZ-E. Several parameters of biosensor performance were optimized, such as the ATZ-E concentration, concentration and nature of the substrate, flow rate, and effect of temperature on the sensor response. After optimization, the assay time for a single sample was 12 min. The work process and result were compared with those of high-performance liquid chromatography (HPLC). The detection results exhibited a recovery rate of 88% to 107% in ATZ-spiked fresh cut corn stalks and silage samples. The results obtained via dcTELISA had good correlation with that of HPLC, and the biosensor response was reproducible and stable even when used continuously for over 4 months. All these properties suggested that the fast detection method, dcTELISA, may be used to detect pesticide residue in large-scale samples.

Serum and urinary neutrophil gelatinase-associated lipocalin as a predictor of rat kidney histopathology in an early ischemia-reperfusion model

Background: The severity of ischemia-reperfusion (I/R) kidney injury is highly correlated with mortality and morbidity rate. Research on human and animal prove that NGAL predicts kidney injury at early phase. The objective of this study is to prove that the increase in serum and urinary NGAL are correlated with kidney tubular epithelial damage, and this increase has occurred in initiation phase, indicated by rat kidney histopathology in an early I/R model. Methods: Twenty eight male Sprague-Dawley rats were divided into 4 groups: 4 hour sham (Sham 4), 8 hour sham (Sham 8), 10 minute ischemia 4 hour reperfusion (I/R 4) and 10 minute ischemia 8 hour reperfusion (I/R 8). Blood, urine and kidney samples were collected. Serum creatinine level was analyzed with Jaffe method, while serum and urinary NGAL level were analyzed with direct sandwich ELISA method. Evaluation of kidney damage were measured semi quantitatively in tissue stained with HE. Further evaluation to confirm cellular changes on kidney was performed by electron microscope and immunohistochemistry. Results: Serum NGAL was found significantly correlated with degree of kidney tissue damage (ρSpearman NGAL serum = 0.701, p < 0.001), also urinary NGAL (ρSpearman = 0.689, p < 0.001). NGAL expression differs significantly between I/R group and sham (t-test, t = -26635.056, p < 0.001), also kidney damage (t-test, t = -5.028, p < 0.001), and serum and urinary NGAL levels (Mann-Whitney, U = 0, p < 0.001). With cutoff points of 136.95 ng/mL and 58.69 ng/mL subsequently for serum and urinary NGAL , it is found that sensitivity = 1, specificity = 1. Conclusion: Elevation of serum and urinary NGAL are significantly correlated with epithelial tubular kidney damage on rat undergoing early ischaemia reperfusion. (Med J Indones. 2012;21:208-13)

Generation of anti-NAG-2 mAb from patients' memory B cells: implications for a novel therapeutic strategy in systemic sclerosis

We have previously reported that antibodies directed against the cytomegalovirus-derived protein UL94 cross react with the cell surface tetraspanin transmembrane 4 superfamily member 7 (TM4SF7 or NAG-2) molecule inducing apoptosis of endothelial cells and activation of fibroblasts in patients with systemic sclerosis (SSc). We aimed at generating a non-functional mAb directed against NAG-2 from patients’ memory B cells. Direct and competitive ELISA methods have been used to evaluate the binding of antibodies from scleroderma patients’ and controls’ sera to the NAG-2 peptide. IgG memory B cells were sorted, EBV transformed and cloned to obtain NAG-2-specific mAbs. Endothelial cells and fibroblasts were cultured under standard conditions and used for functional assays. Anti-NAG-2-purified antibodies obtained from patients’ Ig induce endothelial cell apoptosis and fibroblast proliferation. Patients’ Igs depleted of the anti-NAG-2 fraction do not exert such functional activity. Therefore, the NAG-2 molecule represents a potential novel candidate for therapeutic intervention in SSc. Here, we describe the generation of a human mAb directed against the NAG-2 molecule. Such mAb does not retain any functional property and is able to block the effect of serum pathogenetic anti-NAG-2 antibodies. The majority of SSc patients present antibodies directed against tetraspanin NAG-2 and mediate both endothelial cell apoptosis and fibroblast proliferation, features of the disease. The anti-NAG-2 human mAb we have obtained blocks signal transduction and therefore may be a potential candidate for a new treatment in SSc, a disease where the current biological therapies have little or no efficacy.

Zearalenone (ZEN) detection by a single chain fragment variable (scFv) antibody

Zearalenone (ZEN) is a mycotoxin produced by members of the genus Fusarium. ZEN detection by ELISA using single chain fragment variable (scFv) represents a novel means of mycotoxin detection and diagnosis of fungal infection. To develop immunoassay of ZEN based on scFv, ZEN was conjugated to horseradish peroxidase (HRP). ELISA and SDS-PAGE analysis showed that the covalent attachment of ZEN to HRP was successful. Anti-ZEN scFv-E-tag fusion protein was used to detect ZEN by competitive direct-ELISA (CD-ELISA) method, and the results indicated that this scFv was appropriate in ZEN detection, meaning that scFv against fungal toxin could have an application in mycotoxin detection and diagnosis of fungal infection.

IMMUNE RESPONSIVENESS ASSOCIATED WITH EXPERIMENTAL ENCEPHALITOZOON INTESTINALIS INFECTION IN IMMUNOCOMPETENT RATS

Purpose: Microsporidial infections have been recognized as an increasingly important infection in immuncompromised patients, particularly those infected with HIV/AIDS. This study was designed to study immune responses associated with experimental Encephalitozoon intestinalis infection in immunocompetent rats. Materials and Methods: Thirty-four Rats in 3 groups, A (Control), B (Intraperitoneal) and C (Oral) were given injections of 0.5 ml of 2 x 106of purified spores of Encephalitotozoon intestinalis spores and were observed for serum specific IgG for 21 days using both direct and indirect ELISA. Results: In indirect ELISA, specific lgG were detected on days 7, 14 and 21 for the group B rats and on day 21 for group C and in direct ELISA method, specific lgG were detected in-group B rats on days 7 and 21, for group C rats on day 21 only, while in the control rats, specific lgG were not detected. There was no significant difference between the direct and indirect methods (df=1, X2, P>0.05). E. intestinalis was observed in stool samples of rats in 1/12 (08.33%) on days 14 and 21 in group B, and in 4/10 (33.33%), 3/10 (25.00%) and 2/10 (16.67%) on days 7, 14 and 21 respectively in group C. In group A, which is the control rats, no microsporidia were observed on days 0, 7, 14 and 21. Conclusions: There were no changes in the T-lymphocyte counts of rats prior to and after inoculation with spores. Extensive lesions were observed along the intestinal walls especially on the middle and lower sections of group C rats only.

Biomarkers to disclose recent intake of alcohol: potential of 5-hydroxytryptophol glucuronide testing using new direct UPLC-tandem MS and ELISA methods

This study compared two new methods for direct determination of 5-hydroxytryptophol glucuronide (GTOL) in urine, a biomarker for detection of recent alcohol consumption. Urine samples were collected from ten alcoholic patients during recovery from intoxication. A direct injection ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for measurement of the urinary GTOL to 5-hydroxyindoleacetic acid (5-HIAA) ratio, and an ELISA assay for direct measurement of GTOL, were used. Comparison was made with the urinary ethanol and ethyl glucuronide (EtG) concentrations. The breath ethanol concentration on admission ranged between 1.0-3.1 g/l. The UPLC-MS/MS method showed a median detection time of 39 h for an elevated urinary GTOL/5-HIAA ratio, while EtG was detected for a median of 65 h. Determination of GTOL by the ELISA assay showed 87% sensitivity in detecting positive samples at a 44% specificity, as compared with the UPLC-MS/MS method. The lower sensitivity of the urinary GTOL/5-HIAA ratio compared with EtG for recent drinking may be clinically useful, in cases where the EtG test provides an unwanted high sensitivity for intake of only small amounts of alcohol or unintentional ethanol exposure.

Immune responsiveness associated with experimental <i>Encephalitozoon intestinalis</i> infection in immunocompetent rats

Microsporidial infections have been recognized as an increasingly important infection in immunocompromized patients, particularly those infected with HIV/AIDS. This study was designed to study immune responses associated with experimental Encephalitozoon intestinalis infection in immunecompetent rats. Thirty-four rats in 3 groups, A (Control), B (Intraperitoneal) and C (Oral) were given injections of 0.5 ml of 2 x 10(6) of purified spores of Encephalitotozoon intestinalis spores and were observed for serum specific IgG for 21 days using both Direct and Indirect ELISA. In indirect ELISA, specific lgG were detected on days 7, 14 and 21 for the group B rats and on day 21 for group C and in direct ELISA method, specific lgG were detected in-group B rats on days 7 and 21, for group C rats on day 21 only, while in the control rats, specific lgG were not detected. There was no significant difference between the direct and indirect methods (df=1, X(2), P>0.05). E. intestinalis was observed in stool samples of rats in 1/12 (08.33%) on days 14 and 21 in group B and in 4/10 (33.33%), 3/10 (25.00%) and 2/10 (16.67%) on days 7, 14 and 21 respectively in group C. In-group, A which is the control rats, no microsporidia were observed on days 0, 7, 14 and 21. There were no changes in the T-lymphocyte counts of rats prior to and after inoculation with spores. Extensive lesions were observed along the intestinal walls especially on the middle and lower sections of group C rats only.

Stability of Major Allergen Proteins in Extract Mixes Diluted in Human Serum Albumin (HSA), Normal Saline (NSP), or Glycerin (GLY)

RATIONALE: The stability of allergen proteins in extract mixes serially diluted in NSP, HSA, or GLY and stored refrigerated for 1 year or 1 month at room temperature was investigated. Major allergen levels in grass (Gr 5), mite (Group 1 and 2), cat (Fel d 1), birch (Bet v 1), olive (Ole e 1), English plantain (Pla l 1), mugwort/sage (Art v 1), and Bermuda (Cyn d 1) were measured.METHODS: Five mixes of extracts from ALK-Abelló were made and diluted 5 fold. Cockroach, Aspergillus, or mixed mold extract was added to a portion of the 1:5v/v dilution. Some vials were stored at room temperature. Major allergens were measured using very sensitive direct binding ELISA methods.RESULTS: Most allergens had at least 50% of their activity after 3 months in all diluents. At 6 months olive, English plantain, Bermuda, birch and mite allergens began to lose activity in dilution 1:125v/v in NSP but lost almost all major allergen in dilution 1:625v/v in NSP or GLY. All allergens except Bermuda were stable at 6 months in HSA and all except Bermuda, English plantain and birch were stable at 12 months. Cockroach extract degraded all the allergens except for mite proteins. Molds degraded the grass, olive and plantain allergens significantly. Allergens maintained >40% of their activity after 1 month at room temperature.CONCLUSIONS: Allergen proteins have different stability profiles depending on the protein, the diluent, and the concentration. Some proteins in dilute solutions without added albumin protein lost ELISA binding activity in glycerinated and aqueous diluents. RATIONALE: The stability of allergen proteins in extract mixes serially diluted in NSP, HSA, or GLY and stored refrigerated for 1 year or 1 month at room temperature was investigated. Major allergen levels in grass (Gr 5), mite (Group 1 and 2), cat (Fel d 1), birch (Bet v 1), olive (Ole e 1), English plantain (Pla l 1), mugwort/sage (Art v 1), and Bermuda (Cyn d 1) were measured. METHODS: Five mixes of extracts from ALK-Abelló were made and diluted 5 fold. Cockroach, Aspergillus, or mixed mold extract was added to a portion of the 1:5v/v dilution. Some vials were stored at room temperature. Major allergens were measured using very sensitive direct binding ELISA methods. RESULTS: Most allergens had at least 50% of their activity after 3 months in all diluents. At 6 months olive, English plantain, Bermuda, birch and mite allergens began to lose activity in dilution 1:125v/v in NSP but lost almost all major allergen in dilution 1:625v/v in NSP or GLY. All allergens except Bermuda were stable at 6 months in HSA and all except Bermuda, English plantain and birch were stable at 12 months. Cockroach extract degraded all the allergens except for mite proteins. Molds degraded the grass, olive and plantain allergens significantly. Allergens maintained >40% of their activity after 1 month at room temperature. CONCLUSIONS: Allergen proteins have different stability profiles depending on the protein, the diluent, and the concentration. Some proteins in dilute solutions without added albumin protein lost ELISA binding activity in glycerinated and aqueous diluents.

Development of Competitive Direct Enzyme-linked Immunosorbent Assay for the Detection of Gentamicin Residues in the Plasma of Live Animals

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities (CR50) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/B0 ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/B0 ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life (t1/2) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 10 : 1498-1504)

Development of ELISA and immunochromatographic assay for the detection of neomycin

Background Reliable analytical methods are required to monitor neomycin residue levels in the livestock products. In particular, a more simple and rapid detection method is required in the veterinary fields. Methods Competitive direct ELISA and immunochromatographic assay were developed using monoclonal antibody to detect neomycin in the animal plasma and milk. Results No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 6.85 ng/ml in PBS, 3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively. Recoveries of neomycin from spiked plasma and milk at levels of 50–200 ng/ml ranged from 87% to 108%. Concentration of intramuscularly injected neomycin was successfully monitored in the rabbit plasma through competitive direct ELISA. Immunochromatographic method was also developed using colloidal gold-conjugated monoclonal antibody. Through this method, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk. Conclusions Immunochromatographic assay developed in this study is suitable for the simple screening of neomycin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA.

Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated testosterone excretion in urine

Background Several methods are now available to estimate urinary testosterone levels that can only be performed in established big laboratories using GC/MS techniques. In clinical practice or for research projects, an inexpensive method that does not require skilled technicians would be useful. A simple, rapid and accurate ELISA method has been developed and applied in our laboratory to measure urinary non-conjugated and total testosterone. Methods High affinity anti-testosterone antibody and HRP-Donkey anti-sheep IgG (Horse Radish Peroxidase) as enzyme tracer were used to develop the ELISA method. The assay was evaluated for specificity, sensitivity, parallelism, accuracy and imprecision by the established methods on samples obtained from healthy volunteers. The results from the direct ELISA were compared to those after enzyme hydrolysis plus solvent extraction and HPLC or commercial kits. Results A satisfactory standard curve for the direct testosterone ELISA has been developed with good sensitivity. Cross-reactivity values of anti-testosterone antibody with major interfering steroids were minimal except for testosterone-3-glucuronide (58.8%). The validity of urinary testosterone assay was confirmed by the good correlation between the results obtained by the direct ELISA and those after enzyme hydrolysis and solvent extraction ( Y = 0.987 X + 0.398, R 2 = 0.97). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Urinary testosterone excretion values obtained by our direct ELISA from healthy volunteers were generally in agreement with those published by other workers. Male urinary total testosterone excretion (non-conjugated and testosterone glucuronide) ranged from 177.9 to 865.3 nmol/day, which was about 3–6 times more than the range for women urinary testosterone excretion (34.5–308.8 nmol/day). Conclusion A direct, reliable, easy to perform, sensitive and highly specific ELISA type assay for the measurement of total testosterone in urine samples (conjugated and non-conjugated) has been developed. The novel features of the assay are that it does not require an initial extraction step or involve time consuming procedures such as chromatography. A simple method has also been developed to measure non-conjugated urinary testosterone excretion after solvent extraction alone.

Single slice method for quantification of hemorrhagic transformation using direct ELISA

An important area of experimental stroke research addresses the development of brain hemorrhage after cerebral ischemia. Investigations of hemorrhagic transformation, however, have been compromised by the absence of sensitive methods for quantification of hemorrhagic transformation. We have developed a direct ELISA method that is sensitive, reproducible and can be conducted on tissue treated with triphenyltetrazolium chloride, a stain commonly used for infarct size determination. We have also localized the slices containing the most hemoglobin to facilitate a single slice analysis. This allows two essential variables in experimental stroke research to be assessed on the same slice, leaving the rest of the brain available for other analysis.

Detection of native and recombinant Bacillus macerans cyclodextrin glycosyltransferase using microtiter elisa techniques

Direct, sandwich, and competitive ELISA methods were developed to quantify Bacillus macerans cyclodextrin glycosyltransferase (CGTase) and its thioredoxin fusion molecule using rabbit polyclonal antibodies. The direct ELISA method was used to demonstrate equivalent molar response of both native CGTase and the thioredoxin-CGTase fusion protein to antibody binding. Sandwich ELISA showed the most sensitive detection range (0.2 ∼ 50 μg ml −1) against CGTase. We improved a competitive ELISA method by coating the microtiter plate with denatured CGTase in 6 m guanidine-HCI solution, resulting in enhanced, rapid response. This competitive ELISA method was specific, precise, and rapid when the method was used to quantify CGTase and monitor production of CGTase and its thioredoxin fusion protein in the recombinant E. coli. These methods are specifically useful when detecting and quantifying recombinant CGTase proteins in which mutations may have reduced or eliminated enzymatic activity.

Direct ELISA Method for the Specific Determination of Prothymosin Alpha in Human Specimens

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProTα) was developed using an antibody against the synthetic C-terminal peptide ProTα[101–109] and isolated bovine ProTα for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProTα, the naturally occuring and partially homologous peptide parathymosin alpha (ParaTα) and the peptide thymosin α1 (Tα1), whose sequence is identical to the [1–28] sequence of ProTα, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProTα concentration in human serum and tissue extracts, without any pretreatment of the samples. ProTα levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 μg/ml (mean value 1.27 ± 0.49 μg/ml) and from 0.47 to 1.74 μg/ml (mean value 1.02 ± 0.29 μg/ml), respectively. ProTα levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.

An enzyme immune assay for serum anti-acetaldehyde adduct antibody using low-density lipoprotein adduct and its significance in alcoholic liver injury.

An acetaldehyde (AcH) adduct was prepared using rabbit low-density lipoprotein as carrier proteins. An antibody against this adduct was raised in Watanabe heritable hyperlipidemic rabbits and cross-reacted with human low-density lipoprotein and bovine serum albumin adducts. Using this antibody, serum anti-AcH-adduct antibody levels were measured by a direct ELISA method in 56 Japanese adults (healthy adults and patients with nonalcoholic gastrointestinal diseases, alcoholic liver injury, or alcoholic pancreatitis). The antibody level (mean +/- SD) was 22 +/- 10 microg/ml in healthy adults, 22 +/- 11 microg/ml in nonalcoholic gastrointestinal diseases, and 16 +/- 13 microg/ml in alcoholic pancreatitis. These antibody levels tended to increase with the progression of alcoholic liver injury, starting from fatty liver via hepatitis to cirrhosis, 29 +/- 24 microg/ml in fatty liver, 35 +/- 29 microg/ml in alcoholic hepatitis, and 46 +/- 54 microg/ml in alcoholic cirrhosis. The antibody level in patients taking 100 g or more of ethanol per day tended to be higher, compared with those in people taking less ethanol. A follow-up observation revealed that alcohol abstinence after hospitalization raised serum anti-AcH-adduct antibody level in some patients and kept it constantly low in other patients. The immunohistochemical study using the anti-AcH-adduct antibody revealed the presence of adduct-like substance in hepatocytes of liver biopsy specimens obtained from patients with alcoholic liver disease. The results indicate that the anti-AcH-adduct antibody may be associated with the progress of alcoholic liver diseases.

The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle

Antigens that were specific to Fasciola gigantica were obtained from the whole worm homogenate of the parasite by immunoaffinity chromatography in cyanogen bromide-activated sepharose 4B columns and used for the production of monoclonal antibodies. The F. gigantica-specific monoclonal antibody was labelled with horseradish peroxidase and used for the detection of circulating antigen by the direct ELISA method in the sera of cattle experimentally infected with the parasite. Circulating antigens were detectable in the sera of the animals as from the third week after infection while negative absorbance values were obtained 2 weeks after the termination of the infection by chemotherapy with oxyclozanide. This immunodiagnostic method offers an attractive alternative as a supplement to the conventional coprological diagnosis of fasciolosis.

Development of a monoclonal antibody for ELISA of CYP1A in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes

Induction of cytochrome P4501A CYP1A in cultured cells can be used to determine the induction potencies of xenobiotics or complex environmental samples. This report describes the development of an enzyme linked immunosorbent assay ELISA for measurement of CYP1A expression in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes. Juvenile rainbow trout were injected with naphthoflavone BNF 25 mg kg-1 body weight to induce the synthesis of CYP1A. The CYP1A isoenzyme was purified, characterized by immunological cross reactivity and N terminal sequencing and used to prepare a monoclonal antibody in Balb C mice. The specificity of the antibody for CYP1A was proved by Western blotting of samples from control and BNF injected fish. Two ELISA methods, a direct and a competitive one, were evaluated, with both methods being of comparable sensitivity. Rainbow trout hepatocytes, maintained as monolayers in serum free, chemically defined medium, were exposed to naphthoflavone, and the induction response was measured both by 7 ethoxyresorufin O deethylase EROD activity and the direct ELISA method. Comparison between EROD activity and immunodetectable CYP1A protein can provide information on the catalytic efficiency of CYP1A.

Fatty acid chain is a critical epitope for antiphospholipid antibody

To explore the role of phospholipid fatty acids in binding of antiphospholipid antibody (aPL) in ELISA, we tested aPL binding to phospholipids containing fatty acids of varying chain length and degree of saturation using direct ELISA and inhibition methods. Polyclonal IgG and IgM human aPL's bind to C18:1 phosphatidylglycerol (PG) better than to C18:0 PG or C18:2 PG. Binding is greater to C18 than to C14:0 or C16:0 PGs; aPL's do not bind to C12:0 PG. aPL binding is not inhibited by C18:1 diacylglycerol, glycerol-3-phosphate, myoinositol, or myoinositol phosphate. The fatty acid chains are critical determinants for antigen recognition and, by projection, biological activity of aPL.

5 MUCOSAL MAST CELLS IN THE BABY RAT JEJUNUM

Mucosal mast cells (MMC) are found mainly within the jejunal submucosa. MMC increase in intestinal graft-versus-host reaction, and their degranulation is observed In parasitic enteropathy. Weaning is associated with morphological changes resembling those seen in mild cell mediated intestinal injury. We studied MMC in the young rat jejunum in relation lo weaning changes. Jejunal morphology, crypl cell production rate, intraepithelial lymphocytes (IEL) and goblet cells were studied, and MMC were counted in rat mast cell protease II antibody (anti-RMCP II) and toluidine blue stained sections. Serum RMCP II was determined with a direct ELISA method. In the next few days following weaning the jejunal villi shortened by about 30%. crypts elongated by the same amount, crypt cell production rate more than doubled, and the numbers of IEL showed a marked increase. The MMC counts showed a steady increase from birth to the time of weaning, sharp fall on the two days immediately after weaning, and further increase thereafter. Serum RMCP II level displayed a sudden rise at the time of weaning, with gradual decline thereafter. Mucosal mast cell degranulation, obviously associated with the event of weaning, may be responsible for the observed morphological changes through epithelial cell damage. Weaning is a period of temporary loss of oral tolerance in rats; this might be a result of jejunal injury caused by mucosal masl cell protease.

CHARACTERIZATION OF SPECIFIC ANTIBODY TO CHLAMYDIA TRACHOMATIS IN HUMAN BREAST MILK (HBM), SERUM (S) AND CERVICAL SECRETIONS (CS) AND ITS ROLE IN DISEASE OF MOTHERS AND INFANTS

There is limited information on the protective role of antibody to C. trachomatis infection in human breast milk and other body fluids and secretions. A total of 46 patients was analyzed for the presence and characteristics of C. trachomatis antibody in HBM, S and CS. Antibody to C. trachomatis was detected by a direct ELISA method using partially purified elementary bodies of strain LGV-2 as antigen. Antibody specificity was confirmed using the appropriate absorption and blocking controls. Evidence of infection was indicated by a positive culture or presence of specific antigen. In an unprecedented finding, chlamydial antibody, primarily secretory IgA (SIgA), was detected in HBM in 7 of 33 normal women. SIgA was detected with a geometric mean titer (GMT) of 28.8. The other antibody isotypes were the following: IgG 4.79, IgM 2.4, and IgA 9.7. Serum IgG GMT in patients with recent infection were 128.8±1.8 in comparison to those with no recent infection at 6.3±8.9. Serum IgM was present, GMT 6.8±3.7, in patients with infection in contrast to those with no infection. In CS of patients with recent infection, IgG (7.9±5.4) and SIgA (15.9± 1.8) were present, and surprisingly in patients having no evidence of recent infection there was a persistence of SIgA at a titer of 8.6±3.7, suggesting repeated past exposure to C. trachomatis or the presence of antigen below the threshold of assay sensitivity.