IgG

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should comprise only ‘highly pure’ immunoglobulin fragments since Fc or other contaminating protein fragments or their aggregates may lead to side effects. The digestion of ovine antiserum and its purified IgG were investigated using pepsin and trypsin. Trypsin was effective at digesting purified IgG but unsuitable for the direct digestion of serum. In contrast, pepsin was highly effective at digesting all unwanted serum components to low molecular weight (≤13 kDa) fragments while leaving the ~100-kDa F(ab′)2 intact. The optimum pH for pepsin digestion was between 3.25 and 3.50. The effects of salt concentration and pH on the digestion products were investigated by size exclusion chromatography under various conditions, which revealed a pH-dependent aggregation of some of the low molecular weight Fc and non-IgG fragments. These high molecular weight aggregates were not shown by SDS-PAGE. Unwanted low molecular weight fragments could be removed simply by diafiltration with a 30-kDa nominal molecular weight cutoff membrane and piperazine buffer (containing 150 mM NaCl, pH 6), leaving an F(ab′)2 solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. These highly acidic contaminants were then removed easily using an anion exchange column and the F(ab′)2 produced following a subsequent concentration step was essentially free from pepsin and aggregates with a purity of over 96% and a yield of 19.3 g F(ab′)2/l serum. This novel, high yield method for processing serum to highly pure F(ab′)2 avoids salt precipitation and centrifugation and should be suitable for large-scale production.

The digestion of ovine antiserum under acidic conditions (pH 3.5) by pepsin is highly effective at reducing all unwanted serum components to low molecular weight (≤13 kDa) fragments while leaving the ~100-kDa F(ab’)2 intact. The pH is then raised to 6 to stop further digestion and the reaction mixture centrifuged or filtered to remove any insoluble contaminants. Next, unwanted low molecular weight fragments are removed by diafiltration with a 30-kDa nominal molecular weight cut-off membrane leaving an F(ab’)2 solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. Material of this purity is suitable for many applications but, since all the contaminants are highly acidic, they can be easily removed by passage down an anion-exchange column to yield F(ab’)2 that is essentially free from pepsin and aggregates with a typical purity of over 96% and yields of 16–19 g/l serum. When an antivenom was processed, ~78% of the original serum’s toxin neutralising capacity was recovered. This simple, high yield protocol for processing serum to highly purified F(ab’)2 avoids the need for an initial or any subsequent salt precipitation step and can be utilised for either bench or large scale production. If required, a mild reducing agent may be used finally to create Fab fragments.

Background: Different neurological disorders frequently display antibodies against several self-glycans. Increasing evidence supports their pathogenic role; however, far less is known about their origin. Meanwhile, antibodies recognizing non-self glycans appear in normal human serum during immune response to bacteria. Methods: Using high performance thin layer chromatography-immunostaining, we comparatively evaluated humoral immune response (IgG and IgM immunoreactivity) against glycolipids carrying self-glycans (GM3/GM2/ GM1/GD1a/GD1b/GD3/GT1b/GQ1b) and non-self glycans (Forssman/GA1/"A" blood group/Nt7) in sera from 383 patients with neurological disorders along with 87 healthy controls.

A cross-sectional study was conducted in Muong Lat town (Thanh Hoa province, North Vietnam), following the confirmed diagnosis of trichinellosis in six patients from that town who had eaten hunted wild boar meat during the Vietnamese lunar year celebration. All inhabitants who declared to have eaten undercooked or raw wild boar meat at the celebration and showed at least one clinical symptom compatible with trichinellosis were included in the study and blood sampled. Anti-TrichinellaIgG were determined by ELISA and Western Blot. Seropositive persons were given appropriate albendazole treatment and were followed up. A total of 100 inhabitants met the inclusion criteria. Among these, 30 (30%) had antibodies toTrichinella. Serologically confirmed cases had fever (90.0%), myalgia (86.7%), facial oedema (63.3%), diarrhoea (53.3%), and pain of the masseter muscles (43.3%). Eosinophilia was detected in 83.3% of these individuals. Clinical symptoms resolved in all patients during albendazol...

In the present investigation IgG from serum sample was purified by Ammonium sulfate ((NH 4) 2 SO 4) fractionation, Protein A and Thiophilic affinity chromatography in order to select a method that can be used for further studies. Among the three methods employed, Protein A affinity chromatography was the best method both in terms of purity and protein concentration. Presence of IgG in (NH 4) 2 SO 4) fractionation, Protein A and Thiophilic affinity chromatography fraction were confirmed by direct ELISA using goat anti human IgG-HRP conjugate. The molecular weights of (NH 4) 2 SO 4) fractionated IgG, Protein A and Thiophilic affinity chromatography purified IgG were found to be 28 and 52 kDa respectively. Index Terms— IgG, (NH 4) 2 SO 4) fractionation, Protein A affinity chromatography, Thiophilic affinity chromatography, ELISA, SDS-PAGE

S U M M A R Y The study was conducted to determine raw egg addition to milk at suckling period on growth performance, concentration of serum IgG and some blood parameters in calf. Sixteen, 1 day age male Holstein calves were used in this study. They were divided into two groups according to similar body weight. Calves in the Control group were fed normal cow milk during the 59 day of study period, at the rate of 10 % their body weight as two meal (morning 08:00, night 18:00). Calves in the Trial group were fed in the same way, but at the each meal, 1 liquid egg supplemented their milk. Body weights of the calves were determined biweekly interval. At 59 d of blood samples were collected by jugular vein for measurement of IgG and blood biochemistry parameters. There was no statistical difference on the body weight, weight gain, feed intake and feed conversion at the end of the study between the groups. Serum IgG in the liquid egg supplemented group was higher in the Trial group than those of the Control. There were no statistical differences on the serum total cholesterol, triglyceride, HDL, LDL, VLDL and total protein between the groups. In conclusion; it is concluded that liquid egg supplementation for calf milk did not affect on the growth performance, serum lipid fraction and total protein, while it fortified the immun system. Süt Emme Dönemindeki Buzağıların Sütüne Çiğ Yumurta İlavesinin Besi Performansı, Serum IgG Konsantrasyonu ve Bazı Kan Parametrelerine Etkisi ÖZ Bu araştırma süt emen buzağıların sütlerine çiğ yumurta ilavesinin besi performansı, serum IgG ve bazı kan parametrelerine etkilerini belirlemek amacıyla yapıldı. Araştırmada bir günlük yaşta 16 adet Holştayn ırkı erkek buzağı kullanıldı. Buzağılar canlı ağırlık ortalamaları birbirine yakın olacak şekilde iki gruba ayrıldı. Kontrol grubundaki buzağılar 59 gün süren araştırma süresince canlı ağırlıklarının % 10'u kadar normal inek sütü ile günde 2 öğün (sabah 08:00, akşam 18:00) halinde beslendi. Deneme grubundaki buzağılar kontrol grubu ile aynı şekilde beslendi, bunun yanında her öğünde sütlerine 1 adet çiğ yumurta ilave edildi. Buzağıların canlı ağırlıkları iki haftalık aralıklarla belirlendi. Serum IgG ve serum parametrelerini belirlemek amacıyla araştırma bitimi olan 59. günde kan örnekleri alındı. Araştırma sonu itibariyle; canlı ağırlık, canlı ağırlık artışı, yem tüketimi ve yemden yararlanma oranı bakımından gruplar arasında istatistiksel bir farklılık görülmedi. Deneme grubunun serum IgG konsantrasyonu kontrol grubundan önemli derecede yüksek bulundu (p<0.01). Serum total kolesterol, trigliserit, HDL, LDL, VLDL ve total protein konsantrasyonları gruplar arasında istatistiksel olarak farklılık göstermedi. Sonuç olarak; buzağı sütüne çiğ yumurta ilavesinin besi performansını, serum lipit metabolizması ürünlerini ve total protein miktarını değiştirmediği, buna karşın immun sistemi güçlendirdiği kanaatine ulaşıldı.

BACKGROUND: Enzyme immunoassay has been successfully used for the diagnosis of hypersensitivity pneumonitis (HP), but there are difficulties in standardising this method because of the difficulty in obtaining stable solid phases from a huge variety of heterogeneous antigenic sources. With this in mind, the aim of this work was to demonstrate the usefulness of the standard available fluoro-enzyme-immuno-assay (FEIA) to measure specific IgG to uncommon, commercially unavailable HP-causing antigens.

METHODS: Serum samples from patients suffering from HP caused by canary and pigeon protein exposure with positive immunoprecipitation values, as well as cystic fibrosis patients, patients with respiratory allergies, asymptomatic subjects exposed to canary proteins (canary breeders) and healthy unexposed individuals were evaluated by FEIA using biotinylated serum proteins coupled to streptavidin-activated solid phases. Statistical analyses were performed to compare the different groups and to evaluate the performance of this technique.

RESULTS: The group of patients with hypersensitivity pneumonitis caused by exposure to canary proteins showed the highest value of specific IgG (35.70 mg IgG/mL). ROC analysis demonstrated an optimal cut-off value of 8.44 mg IgG/mL with a sensitivity of 100% and a specificity of 91.76%. Comparisons with other groups revealed statistically significant differences. Comparison between ELISA and FEIA results revealed a strong positive relationship between values obtained in both assays.

CONCLUSION: The results demonstrate that Streptavidin ImmunoCAP is a sensitive, specific and efficient laboratory method for routine diagnostic testing of HP caused by protein antigens that are not commercially available.""

The objective of this experiment was to detect antibody (IgG) against enterotoxigenic Escherichia coli (ETEC) K99 in the blood of cows vaccinated by Escherichia coli polyvalent vaccine. Eight dry cows were injected subcutaneously by polyvalent Escherichia coli twice prior to parturition. Before vaccinated, the cows were given immunomodulator orally for 3 days. Blood samples were drawn from coccigeal vein prior to the 1st vaccination, two week following the 1st vaccination and at 1, 2, and 4 weeks after the 2nd vaccination. Blood samples were analyzed for IgG and ETEC K99 using indirect Enzyme Linked Immunosorbent Assay (ELISA) assay. Results of the experiment indicated that absorbance values of all vaccinated cows before the first vaccination until third week following the 2nd vaccination were below cut off values. The absorbance values then increased and were above cut off values at fourth week following the 2nd vaccination. In conclusion, antibody against ETEC K99, were detected i...

A cross-sectional study of Chlamydia trachomatis antibody seroprevalence was carried out among persons living with HIV in Enugu, Nigeria. One hundred and fifty persons diagnosed of HIV and receiving treatment at the University of Nigeria Teaching Hospital, Enugu were recruited for the study. Enzyme-linked immunosorbent assay kits were used to assess the seroprevalence of Chlamydia trachomatis antibodies among the participants. A structured PROFORMA was used to collect data on demographic factors and high-risk behaviours. The results showed a 60.7% (91/150) of Chlamydia trachomatis antibodies among the study participants. Among the participants with antibodies, the IgM prevalence was 51.6% (47/91), while IgG was 35.2% (32/91) and both IgM and IgG were 13.2% (12/91). Logistic regression showed that the risk of chlamydia infection was increased with the history of STD (OR = 1.4, (0.7 – 2.7)), history of multiple sexual partners (OR = 1.9, (0.9 – 3.7)), those currently sexually active (OR = 1.5, (0.7 – 3.1)), the use of unsterilized objects (OR = 4.9, (2.3 – 10.4)) and the irregular or non-usage of condoms (OR = 10.2 (4.6 – 22.7)). The findings of the study showed a high prevalence of C. trachomatis antibodies among the participants. Although these may not all represent active C. trachomatis infection, it can be inferred that a high percentage of the population has been exposed to the infection and many of them may still be harbouring this infection inadvertently. This may contribute to the high rate of spread of this infection as well as HIV in Nigeria.

Cerebral toxoplasmosis is one of the commonest opportunistic infection of the nervous
system in HIV patients. We present a case of gradual onset haemiparesis in an ART naïve
HIV patient with high CD4 count who was subsequently diagnosed to be a case of cerebral
toxoplasmosis based on radiological and serological investigations. The patient responded to a
regime of Cotrimoxazole and Pyrimethamine. His CD4 count at diagnosis was 299/μl. HAART
was started after completion of treatment of cerebral toxoplasmosis. Prophylaxis against toxoplasmosis
is recommended with cotrimoxazole if the CD4 count is below 200/μl. However, in
this case as the patient had developed toxoplasmosis at a CD4 count value above the cut-off
value for prophylaxis for cerebral toxoplasmosis, it may be worth considering to starting prophylaxis
at a higher CD4 count than 200/μl and continuing for a longer time than the current
guidelines.

The objective of this study was to investigate the presence of anti-T. gondii in goats raised on farms in the Jenin and Tulkarm districts in the north of Palestine. The investigation was conducted on 14 herds comprising of 280 goats. Blood samples were collected via the jugular veins of 151 goats during the period from January to December 2011. The indirect ELISA test was used for the detection of anti-T. gondii antibodies. Results showed that an anti-T.gondii IgG antibody was detected in 13.4 % of the samples. The presence of the anti-T. gondii antibody was influenced by the location of the goat herds. The highest incidence was in Jenin district (17.44 %) while it was 7.69 % in Tulkarm district. These results indicated the possible contamination of meat and milk of the goats with this parasite, which in turn negatively affects human health.

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti-inflammatory properties depend on sialylation of the N-glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti-inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.

Macrophages are pivotal cells of the immune system and play a key role in the host defence mechanism against pathogens. To date, the importance of macrophages and the role of humoral response in eliciting macrophage activity against Mycoplasma mycoides subsp. mycoides small colony (Mmm‑SC), the causative agent of contagious bovine pleuropneumonia (CBPP), have only been marginally elucidated or are almost unknown. The present study was undertaken to investigate the changes in surface morphology of macrophages after in vitro infection with Mmm‑SC in the presence of bovine immune serum. Morphological analysis was performed on macrophage cultures at 6 h post infection using the three-dimensional vision of scanning electron microscopy. Non-infected macrophages in the presence of negative or immune serum and macro phages infected with Mmm‑SC in the absence of serum showed only minor cell surface changes. In contrast, clear surface modifications, broad veils, fine philopodia highlighting cell activation and small aggregates of mycoplasma closely attached to the macrophage membrane, were observed in infected macrophage cultures in the presence of immune serum. Our results suggest that specific humoral response to Mmm‑SC may contribute and support phagocytic activity of macrophages.