Cross-reaction between mouse and rat immunoglobulin G: does it matter in sandwich ELISA?

Abstract Background Sandwich ELISA is an ideal antigen detection and quantification assay. Recently, it was used as the basic concept for high technology diagnostics. The specificity of the assay depends on the exclusion of detection cross-reactivity which arises from using two antibodies developed in different species. Since mice and rats are the common laboratory animals used to develop antigen specific antibodies. Therefore, the questions we addressed here were (1) can one use antigen-specific antibodies raised in mice and rats in the same assay to specifically detect/quantify antigens? and (2) which antibodies of the two rodents should be placed for capturing and for detection in the antigen-detection sandwich? Results Direct ELISA assay was used to assess for the specific reaction of the HRP-conjugated antibody to the target serum. First reaction was to compare between either conjugate anti-rat IgG (homologous) or anti-mouse IgG (heterologous) for the detection of rat sera IgG. Following the dilution factor optimization, the O.D. were 0.744±0.051 and 0.604±0.05, respectively (p= .004). The difference in mean O.D. of 0.14 reflected an unaccepted non-specific reaction. The second reaction was to compare between either conjugate anti-mouse IgG (homologous) and anti-rat IgG (heterologous) for the detection of mouse sera IgG. The recorded O.D. were 0.9414±0.14 and 0.317 ±0.141, respectively (p= .0002). The improved difference in mean O.D. of 0.624 reflecting a minimized cross-reaction. Conclusions Our results suggest that it is possible to use both Swiss albino mice and albino rats in a single sandwich ELISA, given that the captured antibody species to be from the Swiss albino mice and the detection antibody to be from the albino rat. The described working details are limited to the source of the antibodies used in the study. However, the approach stresses on the importance of such optimization steps before making any interpretations based on the antigen detection. To our knowledge, this study is the first to cover the optimal order of the capturing and the detection antibodies in a sandwich ELISA assay. In addition to addressing the possible interfering cross-reactivity that result from using mouse and rat serum antibodies in a single assay. Graphical abstract

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Cross-Reactivity against Naja sumatrana (Black Spitting Cobra) Envenoming from the Haffkine Antivenom in a Mouse Model

ISRN Toxicology ◽

10.1155/2013/247645 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Gregory Cham ◽

Francis Lim ◽

Arul Earnest ◽

Ponnampalam Gopalakrishnakone

Keyword(s):

Mouse Model ◽

Standard Error ◽

Challenge Test ◽

Intraperitoneal Administration ◽

Cross Reactivity ◽

Naja Naja ◽

Swiss Albino Mice ◽

Tail Vein ◽

Albino Mice

Naja sumatrana is the dominant cobra species in Malaysia, Singapore, Borneo, and Sumatra, and it does not have specific antivenom. The Haffkine antivenom has been advocated instead. This study aims to determine the efficacy of this antivenom against Naja sumatrana envenoming using a mouse model. Methods. Male Swiss albino mice were used. Intravenous LD50 was first determined separately for Naja naja and Naja sumatrana venom. ED50 was determined by preincubating antivenom with each venom at 2.5 LD50 before administering the mixture into the tail vein. Validation was carried out using a challenge test. Each mouse received 111 µg of Naja sumatrana venom intramuscularly followed by intraperitoneal administration of dilute Haffkine antivenom. Survival was recorded 24 hours after envenoming. Results. The LD50 of Naja naja venom was 78.13 µg, standard error (SE) 13.3 µg. The ED50 of the Haffkine antivenom against Naja naja venom was 45.9 mg, SE 7.5 mg. The LD50 and ED50 of Naja sumatrana venom were 55.5 µg, SE 12.0 µg; and 73.9 mg, SE 12.0 mg, respectively. The intra-peritoneal ED50 against 111 µg intramuscular Naja sumatrana venom was 136.95 mg, SE 36.74 mg. Conclusion. The Haffkine polyvalent antivenom exhibited cross-neutralisation against Naja sumatrana venom when used at a higher dose.

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Cross-reaction of sera from COVID-19 patients with SARS-CoV assays

10.1101/2020.03.17.20034454 ◽

2020 ◽

Cited By ~ 4

Author(s):

Wei Yee Wan ◽

Eng Hong Seng ◽

Siew Hoon Lim

Keyword(s):

Indirect Immunofluorescence ◽

Early Phase ◽

Cross Reactivity ◽

Cross Reaction ◽

Contact Tracing ◽

Elisa Assay ◽

Diagnostic Assays ◽

Emerging Virus ◽

Interim Measure

AbstractBackgroundThe SARS-CoV-2 shares 74.5% genome identity with SARS-CoV, both exhibiting a similar well conserved structure. Therefore, antibodies produced in COVID-19 and SARS patients should not be that dissimilar. We evaluated SARS-CoV test assays to detect for the presence of antibodies to SARS-CoV-2 and tried to determine the timing of appearance of these antibodies by testing serial sera from these patients.MethodsTests were carried out using ELISA (total antibodies) and indirect immunofluorescence (IIFA) (IgM & IgG) methods on serial sera from patients confirmed with SARS-CoV-2 infection.ResultsCross-reactivity was seen in these two test assays with sera from COVID-19 patients and was detected in 6 out of 7 patients from 7 days after onset of symptoms. Five of the patients had detectable antibodies by the 3rd week into their illness and there was evidence of seroconversion in 4 patients. The IIFA method was marginally more sensitive compared to the ELISA assay, however the IIFA IgM test was not useful in the early phase of the illness with poor sensitivity.ConclusionsExisting diagnostic assays for SARS-CoV can detect antibodies in patients who were diagnosed with COVID-19. These assays maybe be utilized as an interim measure in epidemiological investigations for contact tracing and to determine the extent of community spread of this new emerging virus pending the availability of specific serology tests for SARS-CoV-2.

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Evaluation of Monoclonal Antibody (IgG2bMAb) for Detection of Coproantigen from Experimentally Infected Rats with Strongyloides ratti

Annual Research & Review in Biology ◽

10.9734/arrb/2019/v32i230082 ◽

2019 ◽

pp. 1-9

Author(s):

Noor Abduhaleem ◽

Aliyu Mamuda ◽

Tijjani Mustapha ◽

Roslaini Abd Majid ◽

Leslie Than Thian Lung ◽

...

Keyword(s):

Monoclonal Antibody ◽

Sandwich Elisa ◽

Cross Reactivity ◽

Cross Reaction ◽

Multiple Infections ◽

Soluble Antigen ◽

Heterologous Antigen ◽

Faecal Specimen ◽

Homologous Antigen ◽

Repeated Sampling

Background and Aim: Highly sensitive and specific diagnostic assay for the detection of Strongyloides is needed due to the intermittent and low concentration of eggs, larvae and adult worms that can be found in a faecal specimen. In some cases, repeated sampling of the faecal specimen is required to obtain satisfactory and reliable results. The aim of the study is to develop and evaluates monoclonal antibody-based Sandwich ELISA for the detection of coproantigen associated with Strongyloides infection using S. ratti as a model. Place and Duration of Study: Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, University Malaysia, Between September 2018 and March 2019. Methodology: The monoclonal antibody was raised against a soluble antigen of the infective filariform larvae (iL3) of S. ratti. The monoclonal antibody produced (IgG2bMAb) was evaluated for cross-reactivity against homologous and heterologous helminth antigens such as excretory-secretory (ES), infective larvae (iL3) and coproantigen of S. ratti, adult worms of A. caninum, A. suum, T. canis and T. cati. Results: An IgG2bMAb was observed to react with 30 kDa proteins associated with all homologous antigen from iL3, ES and coproantigen of S. ratti and cross-reacted with one heterologous antigen from adult worm of A. caninum at the same molecular weight. There was no cross-reaction observed with other heterologous antigens from adult worms of T. canis, T. cati and A. suum. The sensitivity of IgG2bMAb for the detection of S. ratti was 85% in Sandwich ELISA. Cross- reaction was observed with hookworm antigen that caused by A. caninum in Western immunoblotting. Conclusion: The results indicated that IgG2b have an immunodiagnostic property as IgG2bMAb and was able to detect antigens from coproantigen related to S. ratti with 85% sensitivity based on Sandwich ELISA) even though cross-reaction was observed with A. caninum. These findings will be very useful to tackle many cases of multiple worms’ infections such as both strongyloidiasis and hookworm. Therefore, we recommend that further evaluation and study in the human area where multiple infections can be common should be carried out.

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Protective Effects of Prunus Avium Extract on 10 Ghz Induced Damages in the Spleen of Swiss Albino Mice

Indian Journal Of Applied Research ◽

10.15373/2249555x/aug2013/219 ◽

2011 ◽

Vol 3 (8) ◽

pp. 680-684 ◽

Cited By ~ 2

Author(s):

Faiza Rifat ◽

◽

Archana Sharma ◽

Preeti Srivastava ◽

Shikha Patni ◽

...

Keyword(s):

Prunus Avium ◽

Protective Effects ◽

Swiss Albino Mice ◽

Albino Mice

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Imidacloprid Induced Histopathological Alterations in the Kidneys of Male Swiss Albino Mice

Indian Journal Of Applied Research ◽

10.15373/2249555x/feb2014/187 ◽

2011 ◽

Vol 4 (2) ◽

pp. 23-26

Author(s):

Naga Prasanna Meda ◽

◽

V. Viveka Vardhani

Keyword(s):

Swiss Albino Mice ◽

Histopathological Alterations ◽

Albino Mice

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Effects of 10-GHz Microwaves on Hematological Parameters in Swiss Albino Mice and Their Modulation by Prunus avium

Journal of Environmental Pathology Toxicology and Oncology ◽

10.1615/jenvironpatholtoxicoloncol.2013007891 ◽

2013 ◽

Vol 32 (3) ◽

pp. 205-217

Author(s):

Rashmi Sisodia ◽

Faiza Rifat ◽

Archana Sharma ◽

Preeti Srivastava ◽

K. V. Sharma

Keyword(s):

Prunus Avium ◽

Hematological Parameters ◽

Swiss Albino Mice ◽

Albino Mice

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PROTECTIVE EFFECT OF ROSEMARY (ROSMARINUS OFFICINALIS L.) EXTRACT OVER THE GENOTOXICITY INDUCED BY CYCLOPHOSPHAMIDE IN SWISS ALBINO MICE

10.21506/j.ponte.2016.9.24 ◽

2016 ◽

Vol 72 (9) ◽

Author(s):

Dr. Ayman Salah El-Seedy ◽

Hany George Shalaby ◽

Mohamed Ahmed El-Sehrigy ◽

Madiha Mohiy El-Dein Ghoneim

Keyword(s):

Protective Effect ◽

Rosmarinus Officinalis ◽

Swiss Albino Mice ◽

Albino Mice ◽

Rosmarinus Officinalis L

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HAEMATO-BIOCHEMICAL AND IMMUNOLOGICAL CHANGES IN EXPERIMENTALLY INDUCED ACETAMIPRID TOXICITY IN SWISS ALBINO MICE

THE INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY ◽

10.21887/tijovsab.v12i1.11235 ◽

2016 ◽

Vol 12 (1) ◽

Author(s):

D.T. Fefar ◽

Ankita N. Brahmbhatt ◽

B.P. Joshi ◽

D.J. Ghodasara

Keyword(s):

Dose Level ◽

Significant Rise ◽

High Dose ◽

Cell Mediated Immunity ◽

Swiss Albino Mice ◽

Mean Values ◽

Group Iii ◽

Treatment Groups ◽

Immunological Effects ◽

Albino Mice

A study was conducted on 5 weeks old 64 (32 male and 32 female) Swiss albino mice to assess the haemato-biochemical and immunological effects of acetamiprid. All the male and female mice were randomly divided into eight different groups. The groups I (male) and II (female) served as controls whereas remaining groups served as treatment groups and were administered acetamiprid at the daily dose rate of 20, 10, 5 mg/kg body weight in males(Group III, V, VII) and females (Group IV, VI,VIII),respectively for 28 days. After 28 days treatment, blood samples were collected for hematological, biochemical as well as immunological analysis. There was significant decrease in haematological parameters like Hb, TEC, TLC, neutrophils and lymphocytes count in high dose groups and revealed potential adversity of acetamiprid at rates of 20 mg/kg/day on haematopoetic system of mice. A dose dependent significant rise in mean values of AST and ALT was observed in treatment groups, whereas there was significant decrease in total protein and albumin and increase in BUN in high and mid dose treated groups, irrespective of sex of mice. Dinitroflurobenzene (DNFB) test conducted to assess the cell mediated immunity revealed the toxic effect of acetamiprid on cell mediated immunity of mice at dose level of 10 mg/kg/day. The mice of high dose group revealed a significant decrease in HA titer and indicated the immunotoxic potential of acetamiprid at dose level of 20 mg/kg/day.

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