Comparison between two chromogenic substrates for revealing an immunoperoxidase reaction of human metaphase chromosomes

To study the binding of an antiadenosine serum to human chromosome DNA, two types of chromogenic reagents were compared. The procedure is as follows: lymphocytes with metaphase chromosomes were spread on slides; some slides were irradiated by UV; all slides were then incubated with antiadenosine rabbit serum and then with antirabbit sheep serum labelled with peroxidase; the latter was revealed in the classical manner by 3,3'-diaminobenzidine (DAB), or, alternatively, by p-phenylenediamine plus pyrocatechol (PPD-PC). The present study shows that the results obtained with PPD-PC were equivalent, if not superior, to those obtained with DAB. In the case of weaker reactions, results with PPD-PC were superior. Furthermore, this reagent has the major advantage of being noncarcinogenic.     

Facilitated ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase in peripheral nerve

p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.     

Histamine content of peritoneal and tissue mast cells of growing rats

Summary p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.Supported by NIH Grants DE 04730, DE 02668 and DE 00288 from the National Institute for Dental Research, NIH Grant RR 0533 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation     

A comparison of eight different chromogen protocols for the demonstration of immunoreactive neurofilaments or glial filaments in rat cerebellum using the peroxidase-antiperoxidase method and monoclonal antibodies

Eight different previously described chromogen protocols were evaluated with respect to their sensitivity for the visualization of horseradish peroxidase (HRP) in a peroxidase-antiperoxidase (PAP) complex used with the unlabeled antibody method for immunohistochemistry. The protocols were evaluated in a test system that involved the demonstration of immunoreactive neurofilaments (NF) or glial filaments (GF) in paraffin-embedded sections of rat cerebellum using anti-NF or anti-GF monoclonal antibodies (MA). The chromogens included: amino-ethylcarbazole (AEC), diaminobenzidine (DAB), O-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and tetramethylbenzidine (TMB). The incubation medium using DAB as the chromogen was employed at neutral pH, at pH 5.1, or at neutral pH with the addition of either cobalt chloride or imidazole to intensify the reaction product. The relative sensitivity of the chromogen protocols was quantitated by comparing the dilution of the anti-NF or anti-GF MA at which NF or GF immunoreactivity was extinguished using each protocol. The results obtained with both the anti-NF and anti-GF MA indicated that DAB with imidazole was the most sensitive chromogen protocol.     

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry

Summary Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3-diaminobenzidine (DAB),p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co²⁺ and Ni²⁺ ions. The results indicate that DAB-imidazole and DAB-Co²⁺ and Ni²⁺ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.     

Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods were able to demonstrate both retrograde labeling of motor neurons and transganglionic anterograde transport into the dorsal horn. However, there were differences in the appearance of the various reaction products under the electron microscope. In addition, differences in the distribution of the reaction products were observed by both light and electron microscopy. The largest distribution of reaction product was observed with TMB. BDHC and o-tolidine were next, followed by the DAB procedures and PPD-PC. The TMB, BDHC, and o-tolidine reaction products were all found to be suitable for electron microscopy. The TMB reaction product was electron dense and had a very distinctive crystalloid appearance that made identification of HRP-labeled neuronal profiles easy and unequivocal.     

Diaminobenzidine as a Myelin Stain in Semithin Plastic Sections

A diaminobenzidine (DAB) stain for myelin in glutaraldehyde fixed, osmicated, semithin epoxy sections is described. One or 1.5 μm sections, dried onto slides, are first etched with a 1:2 dilution of saturated sodium ethox-ide:absolute ethanol, then incubated in 0.05% aqueous DAB with 0.01% hydrogen peroxide. DAB specifically stains osmium fixed myelinated nerve fibers. This permits high resolution light microscopic study of myelinated nerve fibers in semithin sections of tissues that also can be studied by electron microscopy.     

The bioanalysis of trastuzumab in human serum using precipitate-enhanced ellipsometry

For the bioanalysis of therapeutic monoclonal antibodies in biological matrices, immunoassays—especially enzyme-linked immunosorbent assays (ELISAs)—are the most widely used techniques. Although ELISAs are very sensitive, the obtained sensitivity is not always sufficient. In this study, we have investigated the possibilities of performing a precipitate-enhanced immunoassay (PEIA) with ellipsometric detection for the bioanalysis of the therapeutic monoclonal antibody trastuzumab. Hydrophobic silicon slides were coated with anti-idiotype trastuzumab antibodies. Trastuzumab in serum samples could bind to this primary catcher, and biotinylated anti-idiotype antibodies were used for detection. After binding of streptavidin-poly-horseradish peroxidase (HRP), the precipitating substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) was added. Precipitation speed was analyzed using a novel prototype eight-cell ellipsometer, and calibration curves were obtained by plotting this speed versus the trastuzumab concentration. Results demonstrate that the PEIA is at least four times more sensitive than the same ELISA using the chromogenic substrate 3,5,3′,5′-tetramethylbenzidine (TMB) instead of precipitating DAB. The calibration range of the assay is 11 to 700 pg/ml. Serum samples are diluted 10 times prior to incubation corresponding to 110 to 7000 pg/ml in undiluted serum. Validation results demonstrate that these low concentrations can be analyzed accurately and precisely. In addition, samples of a patient treated with trastuzumab were analyzed with both the PEIA and the ELISA. Results demonstrate excellent correlation (r = 0.984) between the methods. Thus, when more sensitivity is required than in a conventional immunoassay, a PEIA with ellipsometric detection may be a useful alternative. The prototype ellipsometer is still in development, and from the data obtained in this study, improvements will be implemented.     

Differential identification of mouse granulocyte (CFU-g) and macrophage (CFU-m) precursors in plasma clots

Because benzidine and its derivatives have possible carcinogenic activity, a safe method is needed to demonstrate endogenous peroxidase activity. Colonies derived from mouse bone marrow cells in plasma clot culture were classified as granulocyte (CFU-g) or macrophage (CFU-m) precursors by peroxidase and naphthol AS acetate (NASA) esterase staining, respectively. Endogenous peroxidase activity was measured using benzidine or p-phenylenediazine-pyrocatechol (PPD-PC). The effectiveness of peroxidase staining with both reagents was evaluated under several conditions, and the enzyme property was confirmed by inactivation with a variety of inhibitors. The level of peroxidase activity did not differ significantly between PPD-PC and benzidine. Colony number and number of cultured cells were strongly correlated (P greater than 0.983). We conclude that PPD-PC safely demonstrates peroxidase activity in cultured cells and is as accurate, reliable, and efficient as benzidine.     

Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.     

Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach

Monoclonal antiadenosine receptor antibodies have been raised by an auto-anti-idiotypic approach. BALB/c mice were immunized with adenosine 6-aminocaproyl-bovine serum albumin. Hybridoma cell lines were raised and lines that secreted antibodies that bound to rabbit antiadenosine antibodies were obtained. Two such monoclonal antibodies, AA18 and AA21, were studied in detail and found to be directed at adenosine receptors by the following criteria. They inhibited the binding of [3H] adenosine to rabbit antiadenosine antibodies that had binding characteristics similar to those of adenosine receptors. They bound to rat brain membranes and binding could be inhibited by N6-cyclohexyladenosine and L-N6-phenylisopropyladenosine, both adenosine receptor agonists. They also inhibited the binding of [3H]L-N6-phenylisopropyladenosine to rat brain membranes. In functional assays, they inhibited adenylate cyclase of rat brain membranes, but had no effect on adenylate cyclase of rat hepatic membranes, indicating that they mimic agonists of the A1 receptor, therefore, carrying an "internal image" of the adenosine molecule. When adenosine receptors of rat brain membranes were solubilized with 1% cholic acid, partially purified on an adenosine 6-aminocaproyl AH-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, both AA18 and AA21 recognized a 62,000 band under nonreducing conditions, and a major band of 36,000 under reducing conditions. We conclude that the auto-anti-idiotypic route has yielded specific antibodies that recognize the A1 adenosine receptor.     

Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support

Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity, specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>or=fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.     

Sensitivity studies of AutoPap System Location-Guided Screening of cervical-vaginal cytologic smears.

OBJECTIVE: To present the results of a study that assessed the efficacy of a cervical cytology screening method utilizing the AutoPap System with Location-Guided Screening (AutoPap LGS) software for detecting abnormal Papanicolaou smear slides. STUDY DESIGN: Two hundred cases of abnormal cervical and vaginal smears were selected from the recent archives of the Taipei Institute of Pathology. For each abnormal slide, a matched "within normal limit" slide was included in the study. The slides were processed on the AutoPap Primary Screening System to select slides for Review or No Review and identify areas of the Review slides for human review and diagnosis (AutoPap LGS). The effectiveness of AutoPap LGS for detecting abnormal Papanicolaou smear slides was evaluated at multiple No Review rates. RESULTS: The AutoPap LGS demonstrated statistically superior sensitivity over current laboratory practice for the identification of abnormal slides. Assessing the potential benefit of the AutoPap LGS using a projection method, it is expected that the AutoPap LGS would detect an additional 52 low grade squamous intraepithelial lesion and 13 high grade squamous intraepithelial lesion cases missed by current laboratory practice in a population of 2,860 cases. CONCLUSION: The effectiveness of AutoPap LGS was demonstrated by its statistically superior performance in the detection of missed abnormal slides as compared to current laboratory practice at the Taipei Institute of Pathology.     

Rehydration of air-dried smears. An alternative method for cytologic analysis of exfoliative cells

OBJECTIVE: To compare the cytologic characteristics of gastric mucosal cell smears prepared by air drying and rehydration prior to alcohol fixing with cells wet fixed in alcohol. STUDY DESIGN: Gastric mucosal cells were obtained from 55 consecutive patients undergoing gastroscopy. Paired smears were made, one immediately fixed in 95% ethanol for 20 minutes (wet fixed [WF]) and the other air dried for at least 20 minutes prior to rehydration with normal saline for 30 seconds and fixation in 95% ethanol for 20 minutes (air dried/rehydrated/fixed [ARF]). Both slides were stained by the Papanicolaou method. Coded slides were examined blind and graded 1 (superior), 2 (satisfactory) or 3 (poor) with respect to staining of chromatin, nuclear membrane, nucleoli, cytoplasm/cell border and group morphology. Histology confirmed a benign disease process or normal mucosa. RESULTS: Comparing grade 1 versus grades 2 and 3, ARF slides were significantly better than WF slides for all cytologic features (P < .05). Comparing grade 1 and 2 versus grade 3, there was no significant difference between ARF and WF slides (P > .05) (chi 2 analysis). CONCLUSION: The cytologic features of ARF smears of gastric cells were equal or superior to those of WF smears. This method of preparing smears is simpler and avoids some of the problems of ethanol fixation of wet smears.     

Light microscopic triple-colored immunohistochemical staining on the same vibratome section using the avidin-biotin-peroxidase complex technique

Summary The sequential application of the avidin-biotinperoxidase complex technique was used to localize multiple tissue antigens on a single free floating section of rat brain. sequential visualization of individual antigens was achieved by the silver-gold-intensified diaminobenzidine (DAB) in the first step, nickel-intensified DAB in the second step, and the DAB alone in the third step of the immunostain procedure. For the demonstration of this method, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF), and vasopressin (VAS) antisera were used. Sections from the hypothalamic paraventricular nucleus (PVN) of rats pretreated with colchicine were stained. Black TH containing cell bodies were clearly distinguished from blue stained CRF cells and from yellow stained VAS-containing cell bodies in the PVN on the 25–30 m thick vibratome sections. The sequential immunostaining procedure presented here results in superior staining of multiple antigens as compared to that achieved by the sequential application of the peroxidase-antiperoxidase (PAP) technique.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Light microscopic triple-colored immunohistochemical staining on the same vibratome section using the avidin-biotin-peroxidase complex technique

The sequential application of the avidin-biotin-peroxidase complex technique was used to localize multiple tissue antigens on a single free floating section of rat brain. Sequential visualization of individual antigens was achieved by the silver-gold-intensified diaminobenzidine (DAB) in the first step, nickel-intensified DAB in the second step, and the DAB alone in the third step of the immunostain procedure. For the demonstration of this method, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF), and vasopressin (VAS) antisera were used. Sections from the hypothalamic paraventricular nucleus (PVN) of rats pretreated with colchicine were stained. Black TH containing cell bodies were clearly distinguished from blue stained CRF cells and from yellow stained VAS-containing cell bodies in the PVN on the 25-30 micron thick vibratome sections. The sequential immunostaining procedure presented here results in superior staining of multiple antigens as compared to that achieved by the sequential application of the peroxidase-antiperoxidase (PAP) technique.     

Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative study of nine methods.

Nine currently available methods for HRP neurohistochemistry have been compared with each other on matching tissue sections from four rats and four rhesus monkeys. The nine methods investigated in this report are the diaminobenzidine (DAB) procedures of LaVail JH and LaVail MM (J Comp Neurol 157:303, 1974), of Adams JC (Neuroscience 2:141, 1977) and of Streit P and Reubi JC (Brain Res 126:530, 1977); the benzidine dihydrochloride (BDHC) procedures of Mesulam M-M (J Histochem Cytochem 24:1273, 1976) and of De Olmos J and Heimer L (Neurosci Lett 6:107, 1977); the o-dianisidine (O-D) procedure of De Olmos J (Exp Brain Res 29:541, 1977); the p-phenylenediamine dihydrochloride and pyrocatechol (PPD-PC) procedure of Hanker JS et al., (Histochem J 9:789, 1977) and the tetramethyl benzidine (TMB) procedures of Mesulam M-M (J Histochem Cytochem 26:106, 1978) and of De Olmos J et al. (J Comp Neurol 181:213, 1978). Quantitative comparisons were based on counts of retrogradely labeled perikarya. The extent of anterograde transport and the size of the injection site were also compared at a more qualitative level. The results indicate that one TMB procedure (Mesulam M-M, J Histochem Cytochem 26:106, 1978) is distinctly superior to each of the other eight procedures in the number of labeled perikarya that it can demonstrate. Furthermore, these differences are statistically significant at better than the 0.05 level of confidence. Differences in sensitivity are most evident when the perikarya contain small quantities of transported HRP. The same TMB method also demonstrates more anterograde transport and a larger injection site than all the other procedures. If less sensitive procedures are employed, afferent or efferent connections that are clearly demonstrated by this TMB procedure are either underestimated or completely overlooked. It is suggested that sensitivity in HRP neurohistochemistry is determined by multiple factors which include the method of fixation, post-fixation storage, the choice of chromogen, the incubation parameters, the type of HRP enzyme that is administered, and the postreaction treatment.     

Induction of Isoperoxidases in Resistant and Susceptible Tomato Cultivars by Meloidogyne incognita

Isoperoxidases were detected in resistant Rossol and susceptible Roma VF tomato roots uninfected and infected by Meloidogyne incognita. Syringaldazine, guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), and indoleacetic acid (IAA) were used as substrates, and the corresponding peroxidative activities were detected either in cytoplasmic or in cell wall fractions, except for IAA oxidase, which was measured in soluble and microsomal fractions. Isoperoxidase activities and cellular locations were induced differently in resistant and susceptible cultivars by nematodes. Nematode infestation markedly enhanced syringaldazine oxidase activity in cell walls of the resistant cultivar. This isoperoxidase is involved in the last step of lignin deposition in plants. Conversely, the susceptible cultivar reacted to M. incognita infection with an increase in cytoplasmic PPD-PC oxidase activity, which presumedly is involved in ethylene production; no changes in cell wall isoperoxidases were observed. IAA oxidase was inhibited in susceptible plants after nematode inoculation, whereas in resistant plants this activity increased in the soluble fraction and decreased in the microsomal fraction.     

Peroxisomes in regenerating human skeletal myofibers

Peroxisomes of the human regenerating skeletal myofibers were studied qualitatively and quantitatively by electron cytochemistry and were compared with those of the mature normal human skeletal muscle fibers. Peroxisomes visualized by electron cytochemistry with 3,3'-diaminobenzidine (DAB) were small round or oval bodies delimited by a single membrane and contained the electron-opaque, coarsely granular matrix. Muscle grafts of the regenerating normal human quadriceps obtained from 4 orthopedic patients were analyzed 2 weeks after transplantation into nude mice; they contained peroxisomes with a mean diameter of 0.25 microns, ranging from 0.12 to 0.67 microns. The group mean density of peroxisomes per 100 microns2 was 2.0 +/- 0.4 (SE), while that of histochemically normal mature human quadriceps femoris myofibers was 0. The cytochemical controls without DAB or with the presence of 3-amino 1,2,4-triazole in the solution containing DAB lacked the electron-opaque reaction, indicating that these reactions were on an enzymatic basis. The results of this study showed clearly that the regenerating normal human skeletal myofibers contained numerous peroxisomes differing from the mature normal human muscle fibers in which the peroxisomes were not observed.     

Major histocompatibility complex class II DAB alleles associated with intestinal parasite load in the vulnerable Chinese egret (Egretta eulophotes)

The maintenance of major histocompatibility complex (MHC) polymorphism has been hypothesized to result from many mechanisms such as rare‐allele advantage, heterozygote advantage, and allele counting. In the study reported herein, 224 vulnerable Chinese egrets (Egretta eulophotes) were used to examine these hypotheses as empirical results derived from bird studies are rare. Parasite survey showed that 147 (65.63%) individuals were infected with 1–3 helminths, and 82.31% of these infected individuals carried Ascaridia sp. Using asymmetric polymerase chain reaction technique, 10 DAB1, twelve DAB2, and three DAB3 exon 2 alleles were identified at each single locus. A significant association of the rare allele Egeu‐DAB2*05 (allele frequency: 0.022) with helminth resistance was found for all helminths, as well as for the most abundant morphotype Ascaridia sp. in the separate analyses. Egeu‐DAB2*05 occurred frequently in uninfected individuals, and individuals carrying Egeu‐DAB2*05 had significantly lower helminth morphotypes per individual (HMI) (the number of HMI) and the fecal egg count values. Further, the parasite infection measurements were consistently lower in individuals with an intermediate number of different alleles in the duplicated DAB loci. Significantly, heterozygosity within each DAB locus was not correlated with any parasite infection measurements. These results indicate that the diversity in MHC Egeu‐DAB gene is associated with intestinal parasite load and maintained by pathogen‐driven selection that probably operate through both the rare‐allele advantage and the allele counting strategy, and suggest that Egeu‐DAB2*05 might be a valuable indicator of better resistance to helminth diseases in the vulnerable Chinese egret.