Comparison of immunogenic properties of native and inactivated Mannheimia haemolytica Lkt in calves

The aim of this study was to compare the immunostimulatory properties of Lkt of M. haemolytica inactivated by formaldehyde and glutaraldehyde and to evaluate the neutralizing properties of anti-Lkt antibodies. The experiment was conducted on 20 Black-and-White Lowland calves of 100 kg body weight, assigned to 4 experimental groups. The animals were given subcutaneous vaccine injections with native Lkt, Lkt inactivated by formaldehyde or Lkt inactivated by glutaraldehyde. The anti-Lkt antibody titres were measured using an enzyme-linked immunosorbent assay (ELISA), based on absorbance of the sera obtained from the animals immunized with the different forms of Lkt. The protective effects of the antibodies present in the sera isolated from the vaccinated animals were estimated using an MTT assay. Analysis of the ELISA absorbance values in the sera from calves in the vaccinated groups did not show any significant differences between the groups. The highest increase in absorbance of sera was observed in calves from the group that received formaldehyde-inactivated Lkt. In the case of calves immunized with native Lkt, the absorbance values were lower than in the group immunized with Lkt inactivated by formaldehyde. The lowest absorbance values were observed in sera obtained from calves vaccinated with Lkt inactivated by glutaraldehyde. Analysis of the MTT assay results revealed the greatest Lkt-neutralizing properties of antibodies in the sera of calves immunized with two doses of a vaccine containing native Lkt and Lkt inactivated with formaldehyde.     

Detection of antibodies to infectious bronchitis of fowl using the ELISA, hemagglutination-inhibition test and agar-gel precipitation test]

Chicks of a conventional poultry flock, Shaver Starcross 288 hybrid, were vaccinated with infectious bronchitis (IB) virus H 120 at the age of 21 days. Three weeks later, the chicks were divided into three groups and separate groups were infected with infectious bronchitis viruses M 41 and D 274 or revaccinated with virus H 120. The content of specific antibodies to antigens prepared from homologous and heterologous viruses of infectious bronchitis used for chick vaccination and infection was investigated at regular intervals in the separate groups of chicks by means of an ELISA technique and haemagglutination-inhibition test (HIT). Serotype specificity of haemagglutination-inhibition test was documented by the results; the specificity was obvious mainly after the first vaccination and two weeks after infection, or after chick revaccination (Fig. 1). The dynamics of postinfective or postvaccinal antibodies, recorded by the ELISA technique, had analogical patterns in the separate groups of chicks, and there were no larger differences in the values determined on the basis of different antigens during the investigation (Fig. 2). A total of 52 group samples of fowl serum was examined by the ELISA technique and agar-gel precipitin test (AGPT) in another part of this study. Ten serums of identical origin represented the separate groups. The result of this examination was evaluated from the percentage of samples with precipitin activity in the group, or from the average value of ELISA. Mutual comparison of the mentioned values indicated that the precipitin activity was limited by the positivity degree of ELISA reaction (Fig. 3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis

A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation can be derived that allows demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).     

Single dilution ELISAs using soluble piroplasm, cellular schizont and soluble schizont antigens for the detection of antibodies against Theileria annulata

Single dilution ELISAs were standardised for the determination of antibody titres against Theileria annulata using three antigens namely soluble piroplasm, cellular schizont or soluble schizont antigens. Antibody titres of 20 cattle serum samples of known identity were determined by multi-dilution ELISA using the three antigens. The ratio of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as positive/negative (P/N) ratios. Coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of known sera and their log10 antibody titres by multi-dilution ELISA. The value of "r" was the highest at the dilution of 1:400. From the log10 antibody titres of known sera and their P/N ratios at the dilution of 1:400, regression equations (Y = a + bX, where Y = predicted log10 titre, X = the P/N ratio at 1:400 dilution) were calculated separately for the three antigens. Thus, the equations Y = 1.63 + 1.35X for soluble piroplasm, Y = 2.67 + 0.547X for cellular schizont and Y = 1.817 + 0.663X for soluble schizont antigens were derived. Test sera were diluted to 1:400 and their OD were read in duplicate wells and converted to P/N ratios. The antibody titres were predicted from the P/N ratios using the above mentioned regression equations. Twenty randomly selected sera tested by single and multidilution ELISAs showed non-significant differences (P < 0.01) between antibody titres. Antibody titres of 90 unknown field sera of cattle were determined by single dilution ELISA. The piroplasm antigen detected higher antibody titres followed by cellular schizont and soluble schizont antigens. The study revealed that a single dilution ELISA could be successfully used for field epidemiological studies of tropical theileriosis.     

猪瘟野毒感染抗体阻断ELISA检测方法的建立与初步评价

以猪瘟野毒E2蛋白为包被抗原、辣根过氧化物酶标记的猪瘟野毒单抗作为酶标抗体,建立检测猪瘟野毒抗体的阻断ELISA方法.猪瘟野毒E2最适包被浓度为0.03 μg/mL,待检血清最适稀释度为1∶4,酶标猪瘟野毒单抗稀释度为1∶1 000.用建立的阻断ELISA方法检测369份临床阴性血清,计算阻断率,确定临界值,阻断率＞4...     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.     

Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A

The susceptibility of Pasteurella multocida to killing by serum and the ability of protective vaccines to stimulate this mechanism of immunity in mice were investigated. P. multocida type of bovine origin was used to prepare a vaccine incorporating heat killed organisms and for homologous infection of mice. Bactericidal capacity and ELISA antibody titres were determined for individual mouse sera. Protection was clearly associated with bactericidal antibodies raised by vaccination. The bactericidal assay may be useful as a rapid, simple screening test of vaccinated mice for functional protective antibody levels.     

Lactogenic immunity to transmissible gastroenteritis (TGE) of swine induced by the attenuated Nouzilly strain of TGE virus: passive protection of piglets and detection of serum and milk antibody classes by ELISA

Piglets of eight sows vaccinated by different routes with the attenuated TGE mutant coronavirus, Nouzilly (N) strain, and piglets from two field seropositive sows were challenged with a virulent TGE strain. On the day of challenge and 10 days after challenge, milk and serum samples from sows were analysed for their level of neutralizing antibodies, total immunoglobulin classes and TGE antibody classes by an ELISA. No direct relationship was seen between the level of protection of the litters and the titres of the different antibody classes on the day of challenge. However, an inverse correlation was seen 10 days after challenge between protection and the level of TGE antibodies.     

Establishment and Optimization of INH Epitope Peptide Vaccine for Detection of Antibody

In this study, an indirect ELISA method was established to detect inhibin hormone (INH) epitope peptide vaccine antibody, it would provide oretical reference for the determination of Fine-wool sheep after active immune body INH epitope peptide vaccine antibody. On the basis of the predecessors, using indirect ELISA method to determinate serum INH epitope peptide antibody levels of sheep, and through control different experimental conditions to look for the best experimental conditions. Through explorating the experimental conditions, finally, the testing experiment conditions were determined, which was blocked solution with skimmed milk powder, INH and GnIH synthetic peptides dilution degrees for 20 000 times, the optimum reaction time was 60 min, the best color action time was 15 min. In this experiment, a kind of method to detection antibody in the body after INH active immune sheep was built, it would provide a reference for future research.     

INH表位多肽疫苗抗体间接ELISA测定方法的建立及优化

本试验在间接ELISA的基础上建立了一种检测抑制素（inhibit hormone, INH）表位多肽疫苗抗体的方法并对ELISA试剂盒进行优化,为今后测定主动免疫后细毛羊体内INH表位多肽疫苗抗体提供理论参考。在前人研究的基础上,采用间接ELISA法测定血清中INH表位多肽疫苗抗体的含量,通过对不同试验条件的控制摸索出最佳试验条件,即以脱脂奶粉为封闭液,INH及GnIH以表位多肽疫苗抗体稀释度为20 000倍,最佳反应时间60 min,最佳显色时间为15 min。本试验建立了一种检测细毛羊体内INH及GnIH抗体的方法,为今后对INH表位多肽疫苗抗体的研究提供了参考。     

Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4)

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.     

Clinical evaluation of the efficacy of inoculating cattle with a vaccine containing Tritrichomonas foetus.

To test the efficacy of a polyvalent Tritrichomonas foetus vaccine, 130 nulliparous heifers were randomly assigned to either receive the test T foetus vaccine or to serve as nonvaccinated controls. The polyvalent test vaccine consisted of a Campylobacter fetus/Leptospira canicola-grippotyphosa-hardjo-icterohaemorrhagiae-pamona bacterine containing 5 x 10(7) killed T foetus/dose. The polyvalent control vaccine consisted of the aforementioned formulation without T foetus. Heifers were administered 2 doses of control or experimental vaccine at 3-week intervals. Heifers were bred to T foetus-infected bulls and their conception and pregnancy rates were determined throughout gestation. In addition, serum samples were analyzed to determine induced concentrations of antitrichomonal antibodies and vaginal secretions were sampled to determine T foetus infection rates in control and vaccinated animals. One week after each of the 15-day breeding periods, 60% (6 of 10) of tested vaccinates and 80% (8 of 10) of tested control animals were T foetus culture-positive. The mean duration of infection of vaccinates was 3.8 weeks (+/- 7.5 days), compared with 5.4 weeks (+/- 7.5 days) of infection for control heifers. All vaccinates developed increased immunofluorescence and serum neutralizing antibody titers following the first immunization, and had additional increases of at least fourfold in response to the second injection. In contrast, no consistent increase in immunofluorescence or serum neutralizing antibodies was observed in control animals. Conception rates were 89.2% for vaccinates and 85.9% for control animals 30 days after breeding and 80 to 90% of these remained pregnant 60 days after breeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization of cattle against Aujeszky's disease with inactivated adjuvant vaccine

The experiments with sheep and young cattle were carried out to test the immunizing efficacy of inactivated adjuvant vaccine against Aujeszky's disease. The vaccine application at doses of 1 ml and 2 ml to lambs at the age of eight to ten months caused the neutralizing antibody production with a significant rise of titres after revaccination. A survival of infection induced with a dose of 10(5.5) TKID50 of virulent virus was recorded in 62.5% of once vaccinated animals and in 87.5% of twice vaccinated animals. When applying different doses of vaccines (from 1 to 10 ml) to young cattle, the antibody reaction level was directly dependent on the inoculum quantity. The double inoculation of animals with vaccines of 2 ml and 5 ml caused the neutralizing antibody production at titres of 1:35, or 1:46. The animals, immunized with the live or inactivated IBR-vaccine possessing high antibody titres against IBR-virus, reacted upon the vaccination with inactivated Aujeszky's vaccine anamnestically, by early production of antibodies in high titres. Metaphylactic vaccination (2 ml of vaccine) of cattle in herds with an acute course days, however earlier during five days from the revaccination when it was carried out in seven days following the first vaccination.     

Antibody responses against avian reovirus nonstructural protein sigmaNS in experimentally virus-infected chickens monitored by a monoclonal antibody capture enzyme-linked immunosorbent assay

Crude antigen preparations from avian reovirus (ARV)-infected chicken embryo fibroblasts (sigmaNS) or from bacterially expressed protein sigmaNS (esigmaNS) were captured by monoclonal antibody 1E1(MAb 1E1) against ARV nonstructural protein sigmaNS immobilized on the ELISA plates and were used as the MAb capture ELISA for antibody detection. Sixty one-week-old specific pathogenic free (SPF) chickens were divided into six groups and were vaccinated with live or inactivated ARV vaccine preparations in different combinations or inoculated with a virulent ARV strain. Sera collected from the birds were tested for their antibody responses to ARV nonstructural protein sigmaNS. Using the MAb capture ELISAs, the level of nonspecific binding reactions was tested on the serum samples obtained weekly from mock-infected SPF chickens from 1 to 25 weeks and compared to the results tested by the conventional ELISA. The results indicated that both MAb capture ELISAs had lower nonspecific bindings than those in the conventional ELISA, even in older birds. Antibody responses against ARV sigmaNS of the birds which received the inactivated vaccine twice (group I), inactivated vaccine followed by a live vaccine (group II), or a live vaccine followed by boosting with an inactivated vaccine (group III) were detected by MAb captured ELISA with sigmaNS crude antigens. The absorbance values increased rapidly at 1-2 weeks after boosting, approximated a peak at 5-6 weeks of age, and maintained this throughout the length of the experiment. The absorbance values of the MAb capture ELISA showed a good correlation to the SN titers ( r value > 0.85). On the other hand, serum samples from the birds which received the live vaccine twice (group IV) or were inoculated with a virulent ARV (group V) did not show antibody responses to sigmaNS, similar to those from the mock-infected birds (group VI), as the absorbance values maintained at a low level (below 0.5) throughout the length of the experiment. Similar results were obtained in the sera detected by MAb capture ELISA with crude esigmaNS antigens, except that the absorbance values in the sera from the birds in group III were gradually increased and later approximated a peak at 11 weeks of age and maintained this throughout the length of the experiments. The results suggest that MAb capture ELISAs can be readily used to detect antibody responses of the birds against ARV nonstructural protein sigmaNS which may reflect an immune status of a chicken flock, receiving ARV vaccine as long as including an inactivated vaccine.     

Development of an enzyme-linked immunosorbent assay for Bordetella avium

A Bordetella avium enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies in 1-day-old poults, experimentally infected turkeys, and naturally infected turkeys. The optimized procedure included use of a suspension of whole bacteria coated onto plastic microtiter plates, a 1:200 serum dilution, a 1:3200 dilution of commercially available goat anti-turkey IgG (heavy and light chain) conjugated with horseradish peroxidase, and 0.04% orthophenylenediamine as substrate. A sample/negative (S/N) ratio method of analysis was used to estimate antibody titer from absorbance values. The regression equation used to estimate antibody titers was derived from the testing of naturally infected turkey sera. The equation was derived by plotting the log10 titer of the sera against the S/N ratio at a 1:200 serum dilution. The ELISA was an effective method for detecting antibody to B. avium, and the procedure should prove useful for laboratories equipped for high-volume ELISA testing.     

小反刍兽疫病毒N蛋白抗体与H蛋白抗体在羊体内代谢消长规律的研究

为研究小反刍兽疫病毒N蛋白抗体与H蛋白抗体在羊体内的代谢消长规律,试验通过建立小反刍兽疫N蛋白双抗原夹心ELISA抗体检测方法、H蛋白阻断ELISA抗体检测方法与血清中和抗体试验方法,分别检测了羊免疫疫苗后体内N蛋白抗体与H蛋白抗体在每个免疫阶段的代谢消长变化。结果表明:羊免疫小反刍兽疫疫苗后,血清内的N蛋白抗体与H蛋白抗体在6~8周时血清抗体效价较高,对应的羊体内中和抗体效价为1∶512,且效价至少可以持续到10周以上,2种抗体具有一定消长代谢规律的相关性。该研究结果也为以N抗原与H抗原为基础建立相应的抗原捕获ELISA方法、竞争ELISA检测方法、间接ELISA检测方法以及双抗原夹心ELISA检测方法奠定了一定的理论依据。     

应用PPA－ELISA检测4头剂量三联苗增强猪瘟免疫效果

１９９０—１９９３年用４头剂量三联苗免疫２０—６０日龄仔猪３２４１７头，１９９３年应用ＰＰＡ－ＥＬＩＳＡ检测超剂量免疫后３０—６０天的猪血清８８份，结果血清中抗体能有效保护的占８１．９％，达不到保护的占１８．１％。     

猪流行性腹泻病毒N蛋白阻断ELISA抗体检测方法的建立及初步应用

旨在建立一种猪流行性腹泻病毒(PEDV)N蛋白阻断ELISA抗体检测方法.本研究将纯化的N蛋白作为包被抗原,通过棋盘滴定法优化ELISA反应条件,建立了检测PEDV抗体的阻断ELISA方法,并对其进行特异性、敏感性和重复性试验.对140份临床血清样品进行检测,并将检测结果与市售IDvet PEDV间接ELISA抗体检测...     

Measurement of antibody in serum and genital fluids of bulls by ELISA after vaccination and challenge with Tritrichomonas foetus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to Tritrichomonas foetus using both whole cell antigen (WCA) and membrane protein antigen (MPA). The test was used to detect specific antibody in serum, preputial washings and seminal plasma samples from 7 adult bulls which were vaccinated subcutaneously on 3 occasions with a membrane protein vaccine against T. foetus var brisbane in an oil adjuvant, and from 4 unvaccinated control animals. One month after administration of the third dose of vaccine, vaccinated and control bulls were repeatedly challenged with the live vaccine strain of the T. foetus. A steady increase in serum antibody titre was detected after each inoculation of vaccine when both antigens were used in the ELISA. However, MPA was more sensitive. After challenge, vaccinated bulls developed an increased titre. No specific antibody was detected in control bulls, except in one bull after challenge in which seroconversion was detected. The serum antibody titres of both groups of animals were also measured with the microagglutination test which proved less sensitive than the ELISA. Antibody titres to both antigens, although lower than in serum, were detected in the seminal plasma of vaccinated animals. The control bulls remained non-responsive. No antibody was detected by ELISA in preputial washings from either control or vaccinated bulls prior to challenge. Post-challenge, some of the vaccinated bulls were responsive with both antigens whereas the control bulls remained negative.